Monthly Archives: August 2023

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Raman Spectroscopic Determination of Water in Glasses and Melt Inclusions: 25 Years after the Beginning

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DOI: 10.31038/GEMS.2023554

Abstract

The method of water determination in glasses and melt inclusions with Raman spectroscopy is shown. Furthermore, it is demonstrated that by the linearity of the calibration curve in a concentration range from 50 ppm to nearly 50%, the practicability of the method can be significantly simplified.

Keywords

Raman spectroscopy, H2O and D2O determination, Reference samples, Calibration

Introduction

Water is the most essential magmatic and pegmatitic volatile. Exact knowledge about the amount of water during magmatic and pegmatitic crystallization processes is critical in understanding the behavior of volatiles in silicate melts. Therefore a simple method for determining total water is necessary. The technique must be simple, fast, and cover a large concentration range. Because most information about the water in magmatic-pegmatitic systems comes from melt inclusions, the method must also have a high spatial resolution and sensitivity.

The author used micro-Raman spectroscopy from the beginning (1998) for this challenge. The author published the first data produced between 1998 and 1999, together with Webster and Heinrich [1]. The most significant problem was the acquisition of reference glasses with testified and independently determined water concentrations. The demand for a simple Raman spectroscopic method was in the air [2]. However, they are standing in this time only at the beginning.

Reference Samples

The prerequisite for developing such a method is the availability of good standards. In the first step, the author used synthetic glasses (of albite, granitic, or pegmatitic composition) with water concentrations determined with the Karl-Fischer method. These glasses come from different authors quoted in Thomas [3]. Water concentrations higher than 12% in silicate glasses are unstable over time. Therefore the author used for higher concentrations water-rich melt inclusions in pegmatite quartz. To obtain the necessary homogenous glass, the melt inclusions must be homogenized under pressure and subsequently fast-cooled using rapid quenching methods (see Thomas et al. [4,5] and the Electronic supplementary material – ESM). Generally, homogeneous, water-rich melt inclusions (>25%) are metastable and disintegrate after the first measurement into a water-bearing stable glass and a water-rich volatile sub-phase. Then the bulk-water concentration results from the water in the partial volumes. We used pure glasses as references with very low water concentrations below the ppm level. Thomas [6] determined the water content of these glasses with the self-calibrating proton-proton (pp) scattering method [7].

Methodology

For the measurements, we used primarily a Dilor XY Laser Raman Triple 800 mm spectrometer equipped with Olympus optical microscope and long-distance 80x and 100x objectives. The spectra were collected with a Peltier-cooled CCD detector [8]. We used the 514 and 488 nm lines of a Coherent Ar+ Laser Model Innova 70-3 and a power of 150 mW for the sample excitation. We obtained effectively interference-free Raman spectra of tiny melt inclusions embedded in the transparent quartz matrix with the confocal technique. The integral intensity in the 3100-3750 cm-1 frequency range was used for all measurements. Starting in 2006, we used a LabRam HR800 UV-VIS spectrometer for all further measurements. This exchange gave no problems because we could, using well-studied reference samples, transform the calibration data for the new device. Furthermore, starting in 2006 [6,9], we used the so-called “Comparator Technique,” justified by the fact that the calibration curve is strongly linear and goes practically through the zero point. However, note here that the exact zero point is for the integral intensity not defined. With this work in 2006, we could also determine the water content in homogenized melt inclusions deep in the quartz matrix by extrapolation to a deep of zero because the integral intensity increases linearly with the decrease of the surface layer (for example, by polishing). Another way is the use of different deep inclusions with the same composition. If on both sides, the polished sample is not too thick (~200 µm) and the inclusion is not precisely between both surfaces, then can, from two measurements, the water content adequate be determined or estimated. In 2006 we also showed that integral intensity between 2250 and 2900 cm-1 for D2OT forms a linear relationship with the concentration and can be used to determine D2O beside the water. We have made such determination (H2O and D2O) for water-rich melt inclusions in the Shaw meteorite [10]. Regarding the rare appearance of fluorescence in the frequency range around 3500 cm-1, we have used the weaker band at about 1630 cm-1 (see McMillan) [11] to quantify water.

Results

The primary aim was to complete a general calibration curve for an extensive concentration range. Also, the simplicity of the method was always a request. That was only possible because the research on melt inclusion was an essential target over a long time. Forty-eight different glasses or melt inclusions were used for the calibration curve plot. Each point represents at least 10 determinations [8]. For simplification, the ± 1s standard deviation is not shown (however. can be seen in Thomas) [8] (Figure 1).

The proof of a general calibration curve was the prerequisite for applying the Raman spectroscopy in the simplified form of the “Comparator Technique.” That means only one certified reference sample is necessary to determine water in glasses and melt inclusions.

fig 1

Figure 1: Generalized calibration curve for water in silicate glasses and melt inclusions in the concentrations range from about 50 ppm to nearly 50% – that is five orders of magnitude. The upper smaller diagram shows the concentration range from 50 ppm (determined with pp-scattering – see Reichard et al. (2004) and Thomas et al. (2008) to 1%.

Conclusions

The outlined Raman method for determining H2O and D2O can also be used in analogy for other components. Thomas (2002) [3] has shown that for boric acid [H3BO3] in fluid and melt inclusions and later also for sulfate [12], carbonate/hydrogen carbonate (see, e.g., Thomas et al. 2020) [13].

Acknowledgment

I thank many colleagues and coauthors who accompanied me for over 50 years in my research, mostly on melt inclusions. Furthermore, I think here first to Jim D. Webster (1955-2019), who was very interested in my work on melt inclusions in granites and pegmatites of the Variscan Erzgebirge. I am, above all, grateful to H. Behrens and F. Holtz (University of Hannover, Germany), Robert (Bob) Bodnar (Virginia Tech, Blacksburg), H.R. Westrich (Sandia National Laboratories, Albuquerque, New Mexico), and M. Leschik (University Clausthal, Germany) for providing glass standards with defined H2O and D2O concentrations. Bob Bodnar encouraged me at the ECROFI XV in 1999 to publish the first results on water determination very fast.

References

  1. Thomas R, Webster JD, Heinrich W (1999) Melt inclusions in pegmatite quartz: Complete miscibility between silicate melts and hydrous fluids. ECROFI Abstracts, Terra Nostra 99/6, 305-307.
  2. Chabiron A, Pfeiffert C, Pironon J, Cuney M (1999) Determination of water content in melt inclusions by Raman spectroscopy. ECROFI Abstracts, Terra Nostra 99/6, 68-69.
  3. Thomas R (2002) Determination of the H3BO3 concentration in fluid and melt inclusions in granite pegmatites by laser Raman microprobe spectroscopy. American Mineralogist 87: 56-68.
  4. Thomas R, Davidson P, Rhede D, Leh M (2009) The miarolitic pegmatites from the Königshain: a contribution to understanding the genesis of pegmatites. Comtrib. Mineral Petrol 157: 505-523.
  5. Thomas R, Davidson P (2016) Origin of miarolitic pegmatites in the Königshain granite/Lusatia. Lithos 260: 225-241.
  6. Thomas SM, Thomas R, Davidson P, Reichart P, Koch-Müller M, et al. (2008) Application of Raman spectroscopy to quantify trace water concentrations in glasses and garnets. American Mineralogist 93: 1550-1557.
  7. Reichart P, Datzmann G, Hauptner A, Hertenberger R, Wild C, et al. (2004) Three-dimensional hydrogen microscopy in diamond. Science 306: 1537-1540.
  8. Thomas R (2000) Determination of water contents of granite melt inclusions by confocal laser Raman microprobe spectroscopy. American Mineralogist 85: 868-872.
  9. Thomas R, Kamenetsky VS, Davidson P (2006) Laser Raman spectroscopic measurements of water in unexposed glass inclusions. American Mineralogist 91: 467-470.
  10. Thomas R, Davidson P (2019) Shaw meteorite: water-poor and water-rich melt inclusions in olivine and enstatite. Mineralogy and Petrology 113: 1-5.
  11. McMillan PF (1994) Water solubility and speciation models. In: Mineralogical Society of America. Reviews in Mineralogy 30: 131-156.
  12. Thomas R, Davidson P (2017) Hingganite-(Y) from a small aplite vein in granodiorite from Oppach, Lusatia Mts., E-Germany. Miner Petrol 111: 821-826.
  13. Thomas R, Davidson P, Rericha A (2020) Emerald from the Habachtal: new observations. Mineralogy and Petrology 114: 161-173.
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Macrophage Activation Syndrome in a Patient with Systemic Lupus Erythematosus Undergoing Cyclophosphamide Treatment: A Case Report

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DOI: 10.31038/JCRM.2023631

Summary

Macrophage Activation Syndrome (MAS) is a disorder related to hemophagocytic lymphohistiocytosis and is a life-threatening complication of rheumatic diseases. The diagnosis is challenging because MAS symptoms are quite similar to those of many active autoimmune diseases or severe sepsis. We describe the case of a female patient with systemic lupus erythematosus that presented with symptoms suggesting acute decompensation of autoimmune disease and sepsis. She was later diagnosed with MAS. Despite an aggressive immunosuppressive treatment, she developed a fatal outcome.

Keywords

Macrophage activation syndrome, Systemic lupus erythematosus, Hemophagocytic lymphohistiocytosis, Infection

Introduction

Macrophage Activation Syndrome (MAS) is a disorder related to hemophagocytic lymphohistiocytosis (HLH) and is a life-threatening complication of rheumatic diseases [1]. HLH is divided into primary and secondary. While primary (or familial) HLH is an inherited disease, secondary HLH is triggered by other diseases, including infections, malignancy, and autoimmune diseases [2]. MAS is a secondary HLH associated with autoimmune diseases. The most common is Systemic Juvenile Idiopathic Arthritis (SJIA), followed by Systemic Lupus Erythematosus (SLE) [3].

The diagnosis is challenging because MAS symptoms are quite similar to those of many active autoimmune diseases or severe sepsis. The typical signs and symptoms are persistent fever, hepatosplenomegaly, lymphadenopathy, and hemorrhagic manifestations. Abnormal results include cytopenia, coagulopathy, and hyperferritinemia. Since the mortality rate is up to 50% in adults, early recognition of MAS is important to improve prognosis [4].

We describe a case report of a SLE female patient that presented with symptoms suggesting acute decompensation of the auto-immune disease and sepsis. She was later diagnosed with MAS. Despite aggressive immunosuppressive treatment, she developed a fatal outcome.

Case Report

A 29-year-old female patient with a known diagnosis of SLE since 2013 was admitted to our Department with a 38.5°C fever, diffuse edema, and dyspnea with one-week duration. Her medical history was characterized by severe SLE with recent exacerbation of the disease while on maintenance treatment with azathioprine and oral low-dose corticosteroids. Therefore, she was started on treatment with cyclophosphamide. At admission, laboratory examination revealed anemia (hemoglobin of 4.3 mg/dL), leucocytes of 8,980/mm3 (35% immature forms), and low platelet count (83,000 per microliter of blood). There were signs of renal impairment function (creatinine of 1.97 mg/dl) and proteinuria (urinary protein-to-creatinine ratio of 1.8). The albumin concentration was low (0.8 g/dL). The complement levels were reduced (C3 of 70 mg/dL and C4 of 30 mg/dL). An abdominal ultrasound revealed splenomegaly.

At this point, cyclophosphamide was withdrawn due to the possibility of medication toxicity. She was kept on a high dose of prednisone. Given the possibility of systemic infection, the patient was started on large-spectrum antibiotics (piperacillin plus tazobactam). An upper Gastrointestinal (GI) endoscopy revealed signs of esophageal candidiasis, but no ulcer or gastritis. Therefore, oral fluconazole was also started. Blood and urine cultures posteriorly showed negative results.

Despite the treatment with antibiotics and antifungals and the suspension of cyclophosphamide, the patient presented an unfavorable evolution. After 3 days of blood and filtered platelet transfusion, she recurred with anemia (hemoglobin of 5.2 mg/dL) and low platelets (13,000 per microliter of blood). Additional blood exams also revealed leucopenia (5,120/mm³), normal fibrinogen (216 mg/dL), and high ferritin levels (10,508 ng/mL – reference of 13-150 ng/mL). The aspartate aminotransferase was elevated (65 units/L), as well as triglycerides (236 mg/dL). After a few days, the renal function continued to deteriorate (creatinine level of 2.86 mg/dL) with metabolic acidosis.

A bone marrow biopsy was performed after hematologic consultation, showing evidence of hemophagocytosis (Figure 1). At this point, treatment was switched to high dose of methylprednisolone (1000 mg per day) associated with intravenous immunoglobulin (30 g per day for 5 days). Despite this approach, on the third day, the patient presented with massive GI bleeding. A new endoscopy did not reveal any ulcer or severe gastritis. In a few days, respiratory failure treated with mechanical ventilation occurred, with the patient dying due to multiple organ failures.

fig 1

Figure 1: Bone marrow biopsy showing focal signs of hemophagocytosis (arrows). Hematoxylin and eosin stained, 1000× magnification.

Discussion

MAS is a secondary HLH related to rheumatic diseases, and it was first described as a complication of SJIA in 1985 by Hadchouel et al. [5]. It is a life-threatening condition characterized by cytopenia, high fever, liver insufficiency, andl coagulopathy.

Excessive activation and proliferation of T lymphocytes and macrophages or histiocytes lead to extensive hemophagocytosis in the bone marrow and cytokine storm [1]. The exact incidence of MAS in rheumatic diseases is still unknown. Although MAS is by far the most common disease in the pediatric population with SJIA, there have been increased reports of MAS related to SLE. The occurrence of MAS-associated SLE in adults is uncommon, ranging from 0.9 to 4.6%. It usually occurs in young female patients [3]. The clinical characteristics of MAS-associated SLE and active SLE are very similar. Both entities share clinical and laboratory features, which include fever, cytopenia, and splenomegaly. This makes the differential diagnosis very difficult. Hyperferritinemia is considered the best parameter to distinguish between MAS and SLE, with a sensitivity and specificity of about 100% favoring MAS [6]. Following multiple organ failures, if MAS is left untreated or even undetected, the mortality rate can rise to 42% in adults [4]. The index of suspicion for MAS is higher when an infection is ruled out or inflammation persists and does not respond to treatment of an underlying infection. In the present case, it could be useful to start an immunomodulatory therapy even in the face of infection. Systemic antibiotics were started as soon as the patient was admitted, even in the absence of a clear infection [7]. Although an identifiable precipitating factor is often not clearly identified, MAS has been related to numerous triggers, including among others a flare of the underlying disease, the toxicity of nonsteroidal antiinflammatory drugs, and viral infections [2]. Most cases are triggered by high activities of autoimmune diseases or infectious agents, resulting in a prolonged immune activation, predominantly by the cytotoxic T cells and the macrophages [3]. In a systematic review of the literature, Aziz et al. described the major risk factors that led to the development of MAS in SLE [8]. According to these authors, the most important precipitating factors were the lupus flare itself, its time of onset, and a high systemic lupus erythematosus disease activity index. Other factors identified were infections, drugs, underlying malignancy, and pregnancy [3,8]. Although there is no obvious cause of MAS occurrence in the present case, we considered the presence of systemic infection as the most probable cause. However, one should consider SLE decompensation or cyclophosphamide toxicity as potential triggers.

In 2014, Fardet et al. developed the HScore, a clinical tool that may be used to determine the probability of getting secondary HLH in adults [9]. This score encompasses 9 variables (known underlying immunosuppression, high temperature, organomegaly, triglyceride, ferritin, serum glutamic oxaloacetic transaminase, fibrinogen, cytopenia, and hemophagocitosys features on bone marrow aspirate). Each variable has a distinct weight, as reported by Fardet et al. [9] (Table 1).

Table 1: The HScore. Reproduced from Fardet et al. (2014) with permission of John Wiley and Sons.

Parameter

No. of points (criteria for scoring)
Known underlying immunosuppression 0 (no) or 18 (yes)
Temperature (°C) 0 (<38.4)

33 (38.4-39.4)

49 (>39.4)
Organomegaly 0 (no)

23 (hepatomegaly or splenomegaly) 38 (hepatomegaly and splenomegaly)
Number of cytopenias 0 (one lineage)

24 (two lineages)

34 (three lineages)
Ferritin (ng/mL) 0 (<2000)

35 (2000-6000)

50 (>6000)
Triglyceride (mmol/L) 0 (<1.5)

44 (1.5-4)

64 (>4)
Fibrinogen (g/L) 0 (>2.5)

30 (<2.5)
Aspartate Aminotransferase (U/L) 0 (<30)

19 (>30)
Hemophagocytosis on bone marrow 0 (no)

35 (yes)

According to the authors, the probability of having hemophagocytic syndrome ranged from <1% with an HScore of ≤90 to >99% with an HScore of ≥250 [9]. In the present case, the HScore was 263, suggesting a greater than 99% probability of having secondary HLH/MAS.

To date, several therapeutic options are available, including non-biologic and biologic treatments. The mainstay of MAS treatment is glucocorticoid therapy, usually with intravenous methylprednisolone 30 mg/Kg/dose (maximum 1 g) for 1 to 3 days. For the nonresponders, additional therapy with cyclosporin is recommended. For patients who are refractory to the high dosages of the above medications, alternative options like etoposide, cyclophosphamide, and plasma exchange can be useful, although no randomized trials are showing consistent results of these medications [3]. Recently, reports with biological agents in refractory cases have shown promising results, including infliximab, rituximab, and intravenous immunoglobulin [7].

In the present case, some factors may have contributed to the unfavorable outcome. Firstly, we must acknowledge that there was some delay in the proper diagnosis of MAS. Secondly, despite the recent use of cyclophosphamide and oral corticosteroid, none of these therapies were the first-line treatment for MAS; therefore, the institution of high-dose methylprednisolone contributed to an additional delay in correct treatment. Finally, our patient was treated in a public tertiary Brazilian institution that lacks the more recent options for the treatment of MAS refractory cases, like immunobiological agents. Consequently, we could only opt for intravenous immunoglobulin in the present case.

Conclusions

MAS is a challenging and life-threatening disorder related to HLH. The occurrence of MAS-associated SLE in adults is relatively uncommon. The clinical characteristics of MAS-associated SLE and active SLE are very similar. Both entities share clinical and laboratory features, which include fever, cytopenia, and splenomegaly. This makes the diagnosis very difficult. A high index of suspicion, associated with immediate treatment is essential for the achievement of better outcomes.

References

  1. Dhote R, Simon J, Papo T, Detournay B, Sailler L, et al. (2003) Reactive hemophagocytic syndrome in adult systemic disease: report of twenty-six cases and literature review. Arthritis Rheum 49: 633-639. [crossref]
  2. Ravelli A (2002) Macrophage activation syndrome. Curr Opin Rheumatol 14: 548-552.
  3. Lerkvaleekul B, Vilaiyuk S (2018) Macrophage activation syndrome: early diagnosis is Open Access Rheumatol 10: 117-128. [crossref]
  4. Crayne CB, Albeituni S, Nichols KE, Cron RQ (2019) The immunology of macrophage activation Front Immunol 10: 119. [crossref]
  5. Hadchouel M, Prieur AM, Griscelli C (1985) Acute hemorrhagic, hepatic, and neurologic manifestations in juvenile rheumatoid arthritis: possible relationship to drugs or infection. J Pediatr 106: 561-566. [crossref]
  6. Egües Dubuc CA, Uriarte Ecenarro M, Meneses Villalba C, Aldasoro Cáceres V, Hernando Rubio I, et al. (2014) Hemophagocytic syndrome as the initial manifestation of systemic lupus Reumatol Clin 10: 321-324. [crossref]
  7. Granata G, Didona D, Stifano G, Feola A, Granata M (2015) Macrophage activation syndrome as onset of systemic lupus erythematosus: a case report and a review of the Case Rep Med 2015: 294041. [crossref]
  8. Aziz A, Castaneda EE, Ahmad N, Veerapalli H, Rockferry AG, et al. (2021) Exploring macrophage activation syndrome secondary to systemic lupus erythematosus in adults: a systematic review of the literature. Cureus 13: e18822. [crossref]
  9. Fardet L, Galicier L, Lambotte O, Marzac C, Aumont C, et (2014) Development and validation of the HScore, a score for the diagnosis of reactive hemophagocytic syndrome. Arthritis Rheumatol 66: 2613-2620. [crossref]
FIG 2

An Intriguing Half Leaf from the Middle Jurassic of China

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DOI: 10.31038/GEMS.2023553

Abstract

Most leaves in angiosperms have reticulate venation, but not all leaves with reticulate venation belong to angiosperms. Although angiosperms flowers have been reported in the Jurassic, leaves similar to angiosperms are lacking in the Jurassic. Here we report an unnamed fossil leaf from the Middle Jurassic Yan’an Formation of Ningxia, China. Although its partial preservation does not allow us to determine the affinity of the fossil, its occurrence underscores the probablity that future digging may uncover angiosperm leaves in the Jurassic.

Keywords

Fossil, Middle Jurassic, China, Angiosperms, Leaf

The origin and early evolution of angiosperms, which have more than 300,000 species and account for more than 90% species diversity of land plants, have been foci of botanical debates for long time. Some palaeobotanists thought that angiosperms did not occur on the Earth until the Cretaceous. But recent years witnessed increasing reports of angiosperms in the pre-Cretaceous age. Among the reports, Schmeissneria [1,2] and Nanjinganthus [3,4], both from the Early Jurassic, are based on tens even hundreds of specimens, making a strong case that angiosperms have long existed in the pre-Cretaceous. Theoretically, angiosperm leaves have more potential to be preserved as fossils. But the fact is that angiosperm leaves are almost completely lacking in the Jurassic. This situation makes any trace of angiosperm-like leaves in the Jurassic especially badly wanted. Here we report a partially preserved leaf from the Yan’an Formation (the Middle Jurassic) of Lingwu, Ningxia, China (37°43’N, 106°26’E, Figure 1). The character assemblage of reticulate venation and intramarginal vein of this leaf are only seen in angiosperms hitherto, making it unique in the fossil record. This discovery makes future discovery of angiosperm leaves in the Jurassic more likely.

FIG 1

Figure 1: Geographical information of the fossil locality in Lingwu, Ningxia, China (37˚43’N, 106˚26’E). a. Fossil locality (black Square) in northwestern China. b. Detailed position of fossil locality (black square) in Lingwu, Ningxia, China.

The specimen (No. SGY007-16, deposited in Ningxia Geological Museum) was preserved as a compression with some coaly residue, uncovered from the Yan’an Formation (the Middle Jurassic), which is widely distributed in Northeastern China and has yielded various fossil plants [5-8]. The specimen is a grey siltstone slab 36 mm x 26 mm. The details were imaged using a Nikon SMZ1500 stereomicroscope equipped with a Nikon DS-Fi1 digital camera. All figures are organized using a Photoshop 7.0.

The leaf is incomplete, at least 15 mm long, 4 mm wide (Figure 2a). The leaf is smooth-margined, with an intramarginal vein (Figure 2a, 2b and 2d). Lateral veins parallel each other, branching from the midvein at an angle between 40° and 50° (Figure 2a and 2b). Lateral veins and transverse veins in between form angular meshes, which are 0.44-1.22 mm long and 0.2-0.45 mm wide (Figure 2a-2c). Lateral and transverse veins form right angles or acute angles, about 88 μm wide, with no obvious differentiation between lateral and transverse veins (Figure 2a-2c). Rarely, there is a freely-ending veinlet in an areole (Figure 2c).

FIG 2

Figure 2: The partially preserved leaf and its details. All scale bar=1 mm. a. The general view of the specimen, showing the partial lamina and partially preserved midvein (black arrow) and the freely ending veinlets (white arrows). b. Detailed view of the basal portion of the leaf, showing a branch in the background (white arrow) and vein (black arrow) after skeletonization. c. Vein meshes after skeletonization, showing the transverse veins (black arrows) between lateral veins. Note the freely ending veinlet (white arrow). d. Detailed view showing smooth margin with intramarginal vein (black arrow) and parallel lateral veins (white arrows).

Other than in angiosperms, reticulate venation has been in several fossil taxa, including ferns (Clathropteridaceae, Dipteridaceae, Polypodiaceae), uncertain group (Gigantopteridales), seed plants (Glossopteridales, Caytoniales, Ginkgoales, Cycadales, Gnetales, angiosperms, uncertain groups) (Table 1). Therefore the occurrence of reticulate venation does not ensure that a taxon with reticulate venation is an angiosperms [9-12].

Table 1: Comparison of our leaf and previously reported taxa with reticulate venation. Note that intramarginal vein is restricted to angiosperms.

Affinity

Margin

Reticulate venation

Intramarginal vein

Vein order

Midrib

Secondary vein

Freely ending veinlet

 Age

Ref
Clathropteris Clathropteridaceae toothed frequent absent 4+ present unbranched absent Mesozoic
Hausmannia Dipteridaceae toothed frequent absent 3 absent dichotomous absent Mesozoic 9
Polypodium Polypodiaceae toothed frequent absent 3 present unbranched absent extant 10
Woodwardia Polypodiaceae smooth frequent absent 3? present dichotomous absent extant 10
Onoclea Polypodiaceae smooth frequent absent 2 present ? absent extant 10
Linopteris Seed plants smooth frequent absent 1 present? dichotomous absent Palaeozoic 10
Reticulopteris Seed plants smooth frequent absent 2 present dichotomous absent Palaeozoic 10
Lonchopteris Seed plants smooth frequent absent 2 present dichotomous absent Palaeozoic 10
Ginkgo biloba Ginkgoales infrequent 1 absent absent extant 10
Stangeria Cycadales frequent 2 multi-strand dichotomous absent extant 10
Ctenis Cycadales infrequent 1 absent absent Mesozic 10
Dictyozamites Bennettitales frequent 1 absent absent Mesozoic 10
Drewria Gnetales frequent 2 absent unbranched absent Cretaceous 10
Welwitschia Gnetales frequent 2 absent unbranched absent extant 10
Gnetum Gnetales frequent 4 multi-strand dichotomous simple-branched extant 10
Glossopteris Glossopteridales smooth frequent 2 present dichotomous absent Permian 10
Gangamopteris Glossopteridales smooth frequent 1 multistrand dichotomous absent Permian 10
Gigantonoclea Gigantopteridales frequent 4 multi-strand unbranched absent, branched Permian 10
Delnortea Gigantopteridales frequent 4 multi-strand unbranched absent Permian 10
Sagenopteris Caytoniales frequent 2 present dichotomous absent Jurassic 10
Sanmiguelia Uncertain variable 4 absent variable absent Triassic 10
Marcouia Uncertain frequent 2 present dichotomous absent Triassic 10
Furcula Uncertain frequent 3–4 present excurrent/dichotomous present Triassic 10
Pannaulika Uncertain frequent 4 present excurrent present Triassic 10
Myrtophyllum geinitzii Angiosperms smooth frequent present 4 present dichotomous Cretaceous 11,12
Myrtophyllum angustum Angiosperms present 11
Grevilleophyllum constans Angiosperms present 11
Eucalyptophyllum oblongifolium Angiosperms present 11
Eucalyptolaurus depreii Angiosperms smooth frequent present 3 present brochidodromous absent Cretaceous 11
Eucalyptolaurus Angiosperms smooth frequent present 3 present brochidodromous absent Cretaceous
Callianthus Angiosperms smooth frequent present 1 absent dichotomous absent Cretaceous
Our leaf smooth frequent present 2? present unbranched rare Jurassic

Our survey of fossil and extant taxa with reticulate venation indicates that, besides the implication given by reticulate venation, the occurrence of intramarginal vein appears to be restricted to angiosperms (Table 1). Therefore the occurrence of intramarginal vein in our new leaf seems to underscore its possibility of an angiosperm. This inference is further strengthened by the occurrence of freely ending veinlet in areole, which, although not strictly restricted to angiosperms, is only seen in angiosperms, Gnetales, and fossil taxa of uncertain affinity. Taking all together, despite its Jurassic age and partial preservation, our new leaf with reticulate venation, intramarginal vein, and freely ending veinlet suggests that, unlike widely-believed, angiosperms are more likely to be a truthful existence in the Jurassic, in line with previous reports of Jurassic flowers  [1-4, 13] and implication given by molecular clock studies [14].

Acknowledgement

This research was supported by the National Natural Science Foundation of China (42288201, 41688103, 91514302), Strategic Priority Research Program (B) of Chinese Academy of Sciences (XDB26000000), and Natural Science Foundation of Ningxia (2021AAC03471).

References

  1. Wang X (2010) Schmeissneria: An angiosperm from the Early Jurassic. Journal of Systematics and Evolution 48: 326-335. [crossref]
  2. Wang X, Duan S, Geng B, Cui J, Yang Y (2007) Schmeissneria: A missing link to angiosperms? BMC Evolutionary Biology 7. [crossref]
  3. Fu Q, Jose BD, Mike P, Manuel GÁ, Zhong JL et al. (2018) An unexpected noncarpellate epigynous flower from the Jurassic of China. eLife 7. [crossref]
  4. Fu Q, Diez JB, Pole M, García ÁM, Wang X (2020) Nanjinganthus is an angiosperm, isn’t it?. China Geology 3: 359-361.
  5. Tanner LH, Wang X, Morabito AC (2012) Fossil charcoal from the Middle Jurassic of the Ordos Basin, China and its paleoatmospheric implications. Geoscience Frontiers 3: 493-502.
  6. Wang X (1995) Study on the middle Jurassic flora of Tongchuan, Shaanxi Province. Chinese Journal of Botany 37: 81-88.
  7. Deng S, Fang L, Lu Y, Fan R, Yuan X (2010) The Mesozoic stratigraphy of the Ordos Basin. (Shehong, Sichuan, China).
  8. Deng S, et al. (2003) Stratum introduction. Petroleum Industry Press, Beijing.
  9. Golovneva LB, Grabovskiy AA (2019) The genus Hausmannia (Dipteridaceae) in the Cretaceous of the North-East of Russia and its paleobiogeographic implications. Cretaceous Research 93: 22-32.
  10. Trivett ML, Pigg KB (1996) A survey of reticulate venation among fossil and living land plants, in ” Flowering plant origin, evolution and phylogeny ” (eds) Taylor DW, Hickey LJ, Ch. 2: 8-31 (Chapman and Hall).
  11. Coiffard C, Gomez B, Thiébaut M, Kvacek J, Thévenard F, et al. (2009) Intramarginal veined Lauracease leaves from the Albian-Cenomanian of Charente-Maritime (western France). Palaeontology 52: 323-336.
  12. Gall L (2022) Paleobotany Division, Yale Peabody Museum. Yale University Peabody Museum. Occurrence dataset.
  13. Han L, Zhao Y, Zhao M, Sun J, Sun B, Wang, X (2023) New fossil evidence suggests that angiosperms flourished in the Middle Jurassic. Life 13.
  14. Li H, et al. (2019) Origin of angiosperms and the puzzle of the Jurassic gap. Nature Plants 5: 461-470.
FIG 1

To the Geochemistry of Beryllium: The Other Side of the Coin

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DOI: 10.31038/GEMS.2023552

Abstract

Two very different examples of Be mineralizations show that Be can be enriched to very high values. This short paper tries to find the origin of such unusual behavior. Possibilities are discussed.

Keywords

Water-rich melt inclusions, Be-enrichment, Gaussian and Lorentzian element distribution, Supercritical phases, Mantle-crust connection

Introduction

There are a couple of comprehensive contributions to the chemistry, mineralogy, petrology, and geochemistry of beryllium: Gmelin [1] and the following issues up to 1997, Beus [2], Everest [3], Grew ES and coauthors (2002) [4], London D [5]. Considering such substantial work, it should be said all essential facts. However, careful microscopical and Raman spectroscopic studies of beryl mineralization tell us a different, maybe second, story. That is related to the supercritical melt and fluid state. While studying minerals from pegmatites related to the Variscan tin mineralization of Ehrenfriedersdorf in central Erzgebirge/Germany, we often found high concentrations of beryllium, which does not fit the expected ideas. In one melt inclusion (No. 61), Webster et al. [6] determined 1130 ppm Be with ion microprobe. This runaway value was the start-point for an intense search for higher Be values. And we were successful. In 2011 Thomas et al. [7] could, for the first time, present a more significant number of Be data for the Ehrenfriedersdorf pegmatite. Conspicuously, the data showed a strong dependence on the melt inclusions’ homogenization temperature and water concentration. As daughter minerals, we found in Ehrenfriedersdorf pegmatite beryllonite [NaBePO4] and hambergite [Be2BO3(OH, F)]. Beryllonite is related chiefly to water-rich melt inclusions in beryl of the beryl-quartz veins. In other beryl mineralizations, for example, Orlovka, Eastern Transbaikalia/Russia (Thomas et al. 2009 and unpublished data together with Badanina [8], and the Habachtal emerald deposit Thomas et al. 2020) [9], we found Be-carbonates as the main Be daughter phase. Many data and thoughts are presented in the concise papers by Thomas et al. [10,11]. Taleb’s book, “The Black Swan. The Impact of the Highly Improbable” [12], significantly influenced our thinking. Particularly the finding of water-rich melt inclusions, which depict a pseudobinary melt-water solvus in emerald, has stimulated our understanding of processes that are not so obvious. In this short contribution, we want to show that beryllium can enrich to very high concentrations – far away from all expectations. The first indications came from melt and fluid inclusions produced synthetically in morganite crystals from the Muiane pegmatite (Thomas et al., 2010) [13] during heating at 700°C, 1.0 kbar for 20 hours.

Samples

For this study, we used two different samples: well-transparent morganite crystals from the Muiane pegmatite [13] and beryl from a small beryl-quartz vein related to the Variscan tin deposit Ehrenfriedersdorf in the central Erzgebirge, Germany [14] – see Figure 1.

Other photos of such beryl-quartz samples from the Sauberg mine are in Thomas et al. [15,16].

FIG 1

Figure 1: Specimen from a beryl-rich quartz (Qtz) vein from the Sauberg mine near Ehrenfriedersdorf. The sample is from a vertically directed vein – is rotated by 90°. The dark minerals on the roof and the bottom are cassiterite, molybdenite, and other minor ore minerals.

Methods

Samples used in this study have been prepared over a long time, starting in 1996. For this, we used different high-pressure devices and analytical methods. A concise description of the methods used is summarized in Thomas et al. [17,18] and the ESMs. This study used a petrographic polarization microscope with a rotating stage coupled with the RamMics R532 Raman spectrometer working in the spectral range of 0-4000 cm-1 using a 60mW single mode 532nm laser. For Raman spectroscopic routine measurements, we used an Olympus long-distance LMPLN100x objective. Details are given in Thomas et al. [15,16]. In a paper published last year [19] used for the homogenization of melt inclusions the HDAC (Hydrothermal Diamond Anvil Cell) technique because water-rich melt inclusions show the tendency to leak or decrepitate during heating on the microscopic heating stage due to the growing pressure inside the inclusions. However, the HDAC method has the disadvantage that only a single or only a small couple of inclusions can be studied. Therefore we used different high-pressure devices coupled with rapid quenching because the obtained samples are larger and contain many inclusions which can be analyzed with other methods over the years. Of course, we also used the HDAC technique to see that our interpretations obtained from rapid quench experiments were realistic.

Results

Morganite from the Muiane Pegmatite, Mozambique

Morganite is the pale pink variety of beryl colored by Mn2+. Crystals from the Muiane pegmatite contain a lot of melt and daughter mineral-rich fluid inclusions. From first homogenizations at 700°C and 1.0 kbar for 20 hours, some melt inclusions homogenized totally, and during fast cooling, these inclusions heterogenized into a silicate melt and a fluid subphase containing hambergite [Be2BO3(OH,F)] and small bromelite [BeO] daughter crystals. According to Raman measurements, the silicate glass phase beside the water-rich phase has 7.0 ± 0.2% (n=36) H2O. After heating and quenching, a microscopic study of the morganite ships showed newly formed, very plane melt and fluid inclusions in newly formed halos around large melt inclusions produced during partial decrepitation under pressure. And to our surprise, these inclusions are rich in bromelite (Figure 2). These inclusions were not present before the heating procedure under constant CO2 pressure.

FIG 2

Figure 2: Bromelite [BeO]-rich inclusions in morganite, produced by heating to 700°C at a constant pressure of 1.0 kbar (20 hours) and fast quenching after the run.

That means that bromelite in water or water-rich melts are at 700°C highly mobile. This test tube-type experiment under pressure showed us Be’s high solubility and mobility. We obtained 14 to 17% Be in the H2O solution from well-formed inclusions. That is far from all ideas, for the natural examples given in Grew [4]. Another point is essential. There are two newly formed inclusion types: Melt- or glass-rich inclusions (Type-A melt inclusions) and water-rich inclusions with only a tiny part of the glass or melt (Type-B melt inclusions). Both inclusion types formed at the same time at 700°C. Furthermore, the observations show that the Be-content in the silicate glass with 7% water is very low and in the water-rich glass very high, higher than in the extreme water-rich inclusions.

Beryl-quartz Vein from the Sauberg Mine Near Ehrenfriedersdorf, Erzgebirge, Germany

This beryl contains a lot of fluid and melt inclusions. Many melt inclusions have beryllonite daughter crystals, which can be used to estimate the inclusions’ natural Be content [7]. Note here the inclusions are samples of the specific mineral-forming phases [20]. New studies [14] show that the beryl-quartz vein has a complicated history. This mineralization shows clear hints of the participation of supercritical melts or fluids – showing an interaction between mantle and crust [21]. The beryllium distribution of water-rich melt inclusions (Figure 3) around the solvus crest of a pseudobinary melt-water solvus shows this exceptionally well.

FIG 3

Figure 3: Gaussian distribution of Be (in ppm) in water-rich melt inclusions in beryl from the Sauberg mine near Ehrenfriedersdorf. The abscissa represents the measured inclusions’ determined bulk water content (H2O). All points are the mean of at least five determinations. The center of the peak is at 26.4% H2O, the width is 9.5% H2O, the height is 12075 ppm Be, and the offset is 214 ppm Be. The offset represents the general enrichment level of the mineralization in question.

Note, however, that the shown Gaussian curve represents average values produced by the limited number of analyses. The highest up-to-now measured Be value is 71500 ppm (from the volume of a well-formed beryllonite daughter crystal in a melt inclusion). By such values, the Gaussian curve degenerates to a Lorentzian curve type – typical for supercritical conditions. In normal beryl mineralization [5], such extreme enrichment is entirely out of the question. We must find an acceptable answer because the measured data are correct and always checked (starting with Webster et al.) – [6]. In the case of the beryl-quartz veins from the Sauberg mine near Ehrenfriedersdorf, enrichment in a miarolitic cavity is here not in question. The steady presence of HP and HT minerals (nanodiamond, moissanite, beryl-II, kumdykolite, and others) in the beryl-quartz mineralization makes it probable that supercritical phases from mantle depths participate in this mineralization. The finding of high-pressure and high-temperature minerals related to the Variscan granites and mineralizations supported this idea. To such phases belong spherical nanodiamond crystals, moissanite, stishovite, coesite, cristobalite-X-I, and beryl-II intergrown with moissanite and kumdykolite [15,16,21]. These minerals are all foreign crystals not in natural equilibrium with present surrounding mineralizations and rocks. Fundamental questions arise from the direct paragenetic relationship of beryl and moissanite at around 700°C and a pressure ≤ 2 kbar: (i) is the supercritical phase (melt, fluid?) primarily rich in beryllium? (ii) which role acts the spherical beryl-moissanite intergrow? (iii) what is the mechanism for the simultaneous growth of beryl and moissanite? (iv) Can such a mechanism be used for the technological crystallization of moissanite at significantly lower temperatures?.

Discussion

In cooperation with my coauthors, I found many element distributions showing such extreme enrichment, and such enrichment shows precise Gaussian or Lorentzian distribution curves [10,11]. For the Ehrenfriedersdorf deposit, we found Gaussian or Lorentzian distributed plots for the elements Li, Be, B, P, Cl, Zn, As, Sn, Cs, Ta, and W. A prerequisite for this analytical approach was developing a simple, destructions-free and fast analytical method for determining water in homogenized melt inclusions [22] on the base of the Raman spectroscopy. Different techniques were developed and used to determine the elements in question. Noteworthy was the Raman spectroscopic and electron microscopic determination of B, Be, and other elements [23,24], as well as the synchrotron radiation XRF (SXRF) method [25,26], the femtosecond LA-ICP-QMS microanalysis [27]. Why would up to now not similar distribution found? Is it a question of the direct availability of necessary experimental and analytical techniques? Or are such distributions related to the supercritical phases? Further studies on melt inclusions, for example, performed on mineralizations related to the Lusatian Block, made the last possibility highly plausible. More work is necessary!

Acknowledgment

I have written these lines in “we-form” because progress in the inclusion research would not be possible without the advisors and coauthors. I thank many colleagues who accompanied me for over 50 years in my research. Thanks go to Edwin (Ed) W. Roedder (1919-2006), which brought me on the right path. Furthermore, I think here first to Jim D. Webster (1955-2019), who was very interested in my work on melt inclusions in granites and pegmatites of the Variscan Erzgebirge, and I-Ming Chou and William (Bill) A. Bassett, who introduced me to work with the HDAC device and enabled me to judge this technique. Unforgotten is also the longstanding cooperation with Paul Davidson.

References

  1. Gmelin Handbuch der anorganischen Chemie (1930), System-Nr.26 – Beryllium. Verlag Chemie Berlin. Pg: 180.
  2. Beus AA (1960) Geochemistry of Beryllium and genetic types of Beryllium deposits. W.H. Freeman and Company, San Francisco and London. Pg: 401.
  3. Everest DA (1964) The Chemistry of Beryllium. Elsevier, Amsterdam, London, New York. Pg: 151.
  4. Grew ES, and coauthors (2002) Beryllium – Mineralogy, Petrology, and Geochemistry, Ed: ES Grew, Review in Mineralogy & Geochemistry 50.
  5. London D (2015) Reading pegmatites: What beryl says. Rock & Minerals 90: 138-149.
  6. Webster JD, Thomas R, Rhede D, Förster HJ, Seltmann R (1997) Melt inclusions in quartz from an evolved peraluminous pegmatite: Geochemical evidence for strong tin enrichment in fluorine-rich and phosphorus-rich residual liquids. Geochimica et Cosmochimica Acta 61: 2589-2604.
  7. Thomas R, Webster JD, Davidson P (2011) Be-daughter minerals in fluid and melt inclusions: implications for the enrichment of Be in granite-pegmatite systems. Mineralogy and Petrology 161: 483-495.
  8. Thomas R, Davidson P, Badanina E (2009) A melt and fluid inclusion assemblage in beryl from pegmatite in the Orlovka amazonite granite, East Transbaikalia, Russia: implications for pegmatite-forming melt systems. Petrol 96: 129-140.
  9. Thomas R, Davidson P, Rericha A (2020) Emerald from the Habachtal: new observations. Mineralogy and Petrology, 114, 161-173.
  10. Thomas R, Davidson P, Appel K (2019) The enhanced element enrichment in the supercritical states of granite-pegmatite systems. Acta Geochim. 38: 335-349.
  11. Thomas R, Davidson P, Rericha A, Voznyak DK (2022) Water-Rich Melt Inclusion as “Frozen” Samples of the Supercritical State in Granites and Pegmatites Reveal Extreme Element Enrichment Resulting Under Non-Equilibrium Conditions. Mineralogical Journal (Ukraine), 44: 3-15.
  12. Taleb NN (2018) Der Schwarze Schwan. Die Macht höchst unwahrscheinlicher Ereignisse. Pantheon. 624.
  13. Thomas R, Davidson P (2010) Hambergite-rich melt inclusions in morganite crystals from the Muiane pegmatite, Mozambique and some remarks on the paragenesis of hambergite. Mineralogy and Petrology, 100: 227-239.
  14. Thomas R (2023a) Growth of SiC whiskers in beryl by a natural supercritical VLS process. Aspects in Mining & Mineral Science. 11: 1292-1297.
  15. Thomas R, Davidson P, Rericha A, Recknagel U (2023) Ultrahigh-pressure mineral inclusions in a crustal granite. Evidence for a novel transcrustal transport mechanism. Geosciences 13: 1-13.
  16. Thomas R, Recknagel U, Rericha A (2023) A moissanite-diamond-graphite paragenesis in a small beryl-quartz vein related to the Variscan tin-mineralization of the Ehrenfriedersdorf deposit, Germany. Aspects in Mining & Mineral Science 11: 1310-1319.
  17. Thomas R, Davidson P, Rhede D, Leh M (2009) The miarolitic pegmatites from the Königshain: a contribution to understanding the genesis of pegmatites. Contribution to mineralogy and Petrology 157: 505-523.
  18. Thomas R, Davidson P (2016) Origin of miarolitic pegmatites in the Königshain granite/Lusatia. Lithos 260: 225-241.
  19. Deng J, Li J. Zhang D, Chou IM, Yan Q, Xiong X (2022) Origin of pegmatitic melts from granitic magmas in the formation of the Jiajika lithium deposit in the eastern Tibetan Plateau. Journal of Asian Earth Sciences 229: 1-10.
  20. Roedder E (1984) Fluid inclusions. Reviews in mineralogy. In: Ribbe PH (Eds). Mineralogical Society of America 12: pg: 644.
  21. Thomas R (2023b) Ultrahigh-pressure and -temperature mineral inclusions in more crustal mineralizations: The role of supercritical fluids. Geology, Earth and Marine Sciences 5: 1-2.
  22. Thomas R (2000) Determination of water contents of granite melt inclusions by confocal laser Raman microprobe spectroscopy. American Mineralogist 85: 868-872.
  23. Thomas R (2002) Determination of the H3BO3 concentration in fluid and melt inclusions in granite pegmatites by laser Raman microprobe spectroscopy. American Mineralogist 87: 56-68.
  24. Thomas R, Davidson P (2017) Hingganite-(Y) from a small aplite vein in granodiorite from Oppach, Lusatian Mts., E-Germany. Miner. Petrol 111: 821-826.
  25. Rickers K, Thomas R, Heinrich W (2004) Trace-element analysis of individual synthetic and natural fluid inclusions with synchrotron radiation XRF using Monte Carlo simulation for quantification. J. Mineral. 16: 23-35.
  26. Rickers K, Thomas R, Heinrich W (2006) The behavior of trace elements during the chemical evolution of the H2O-, B-, and F-rich granite–pegmatite–hydrothermal system at Ehrenfriedersdorf, Germany: a SXRF study of melt and fluid inclusions. Mineralium Deposita 41: 229-245.
  27. Borisova AY, Thomas R, Salvi S, Candaudap F, Lanzanova A, et al. (2012) Tin and associated metal and metalloid geochemistry by femtosecond LA-ICP-QMS microanalysis of pegmatite-leucogranite melt and fluid inclusions: new evidence for melt-melt-fluid immiscibility. Mineralogical Magazine 76: 91-113.
fig 1

The Role of the Nucleotide-Excision Repair (NER) Pathway in Soft Tissue Sarcomas: A Review and a Focus on Its Potential as a Therapeutic Target

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DOI: 10.31038/CST.2023824

Simple Summary

The nucleotide excision repair (NER) pathway involves more than thirty protein-protein interactions and removes DNA adducts caused by chemotherapy drugs. The key genes of NER are often over-expressed in cancer cells and alterations of this pathway are responsible for increased or decreased sensitivity to specific therapeutic agents. This is particularly relevant in soft tissue sarcomas (STS), rare mesenchymal-originated tumors whose underlying mechanisms still lack understanding. Altogether the NER pathway components can be potential therapeutic targets in STS. The subtle regulation of NER activity may be clinically relevant as a surrogate prognostic marker or to predict sensitivity to chemotherapy agents. Further prospective evaluation of NER should be performed to address this question.

Abstract

Soft tissue sarcomas (STS) are low-incidence, mesenchymal-derived tumors represented by more than 50 his to types. Despite the latest developments, the rates of patients developing recurrent and metastatic disease are high. Many of the mechanisms underlying STS are still unknown, but there is evidence of the possible role of DNA damage response (DDR) pathways. DDR pathways include a variety of pathways used by cells to repair DNA damage of various kinds; they also have roles in protecting cancer cells from exogenous agents that target DNA, for these reasons are one of the main targets of potential anticancer therapeutic strategies. Nucleotide excision repair (NER) is one of the key repair pathways that can remove various bulky DNA lesions, often given by UV light, and is the main repair mechanism of DNA damage caused by carcinogens and chemotherapeutic drugs. Defects in NER are often the cause of several autosomal recessive genetic diseases. Variations in NER pathway actors can lead to a NER proficiency or NER deficiency condition, and this can be a risk, prognostic, and treatment response factor in cancer. This review focuses on the association between variations in the NER pathway in STS and is intended to point to NER as a pathway to focus on in the next future to optimize the treatments in use and improve the possibilities of personalizing therapies in STS patients in clinical practice.

Keywords

DNA damage response (DDR) mechanism, Nucleotide excision repair (NER), Soft tissue sarcoma (STS), Chemosensitivity

Introduction

Soft tissue sarcomas (STS) are rare tumors with more than 100 different histological subtypes. The scientific community has focused over the years on the search for biomarkers in STS [1] and the identification of variations at the genomic [2], expression [3], and protein [4] level.

Maintaining the integrity of genetic material is critical for the survival of all cell lines, yet various factors, both endogenous and exogenous, can compromise DNA stability. DNA damage repair (DDR) mechanisms are necessary for the maintenance of genome integrity. This is particularly important in cancer cells, in which mechanisms of resistance and sensitivity to radio- and chemotherapeutic cytotoxic agents are directly controlled by DDR pathways. For these reasons, DDR pathways are one of the main targets of potential anticancer therapeutic strategies [5]. The main DDR pathways are direct repair (DR), base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), non-homologous end joining (NHEJ), and homologous recombination repair (HRR) [6]. The absence or deficiency of a specific DDR mechanism can result in genomic instability and tumor progression. Certain modifications of specific genes of a DDR pathway are typical, with some frequency, of some specific cancers [7]. Transcriptomic profiling of tumor tissues suggested codependences between DDR pathways, indicating a potential benefit of combination therapies, which were confirmed by in vitro studies. Somatic alterations in the NER pathway, especially in ERCC genes, are common in various types of cancer. In a 2007 study, Castro et al showed that in cancer cells, NER and apoptosis pathways are the most impaired, with a high diversity of gene expression profiles in comparison to normal cells [8]. Other studies have shown how a deficiency in the NER pathway correlates with increased sensitivity to irofulven and cisplatin [9,10] and decreased sensitivity to trabectedin [11]. Moreover, inhibiting NER has been shown to increase sensitivity to alkylating agents in multiple myeloma cases [12]. DDR pathway alterations are present in numerous histologic subtypes of sarcoma. In a study conducted on STS specimens, at least one pathogenic mutation of the DDR pathway was detected in 15.9% of the patients, the most altered gene was ATRX (10%) and furthermore mutations were observed in 25 sarcoma subtypes. [13]. Recent studies have analyzed the genotype and expression profile of NER genes in STS patients, showing a correlation between ERCC1 and ERCC2 specific single nucleotide polymorphisms (SNPs) and a higher expression of both genes [14].

Nucleotide Excision Repair (NER) Pathway

NER mechanism recognizes and repairs various types of DNA damage caused by UV irradiation, cisplatin, and other damaging agents. NER pathways can be classified as either global genome repair (GGR), which repairs DNA damage anywhere in the genome, or transcription-coupled repair (TCR), which specifically restores DNA strands that are being transcribed (Figure 1). NER mechanisms rely on a series of reactions: recognition of DNA damage, unwinding double-strand DNA in the neighborhood of the damage, excision of the damaged nucleotides, and filling of the single-stranded gap by DNA synthesis. In GGR, DNA damage is recognized by XPC/Rad23 (xeroderma pigmentosum, C/Rad23 complementation group) or UV-DDB (UV-damaged DNA binding protein) [15] while in TC-NER, DNA damage blocks RNA polymerase II (RNAPII) interacting with CSB (ERCC excision repair 6, chromatin remodeling factor) and CSA (ERCC excision repair 8, subunit of CSA ubiquitin ligase complex)-CSB. After damage recognition, in both pathways, RNA polymerase II H transcription initiation factor (TFIIH) is recruited. It is subsequently recruited to the XPG (ERCC excision repair 5, endonuclease) complex, a single-stranded DNA-specific endonuclease. TFIIH unwinds DNA in the vicinity of damage, XPD (ERCC excision repair 2, helicase subunit of the TFIIH core complex), XPB (ERCC excision repair 3, helicase subunit of the TFIIH core complex) and XPA (DNA damage recognition and repair factor) are in charge of recognizing and verifying the damage. XPA binds to the chemically altered nucleotides in a single strand of DNA and recruits the XPF (ERCC excision repair 4, endonuclease catalytic subunit)-ERCC1 (ERCC excision repair 1, endonuclease non-catalytic subunit) catalytic subunit, which makes a cut on the damaged strand of 5′ to extract the damage. Next, XPG makes a 3′ cut that leads to the excision of a single-stranded DNA fragment containing the damage. Then thanks to PCNA (proliferating cell nuclear antigen) and DNA polymerase δ or ε new DNA is synthesized, finally DNA ligase 1 or 3 seals the DNA [16]. Defects in the NER pathway can be attributed to several inherited human diseases, including xeroderma pigmentosum (XP), an autosomal recessive genetic disease characterized by increased sensitivity to UV radiation [17] (Figure 1).

fig 1

Figure 1: NER proficient and NER deficient tumoral cells. Effects of variations in the NER pathway leading to a NER proficient condition (left) with correct repair of DNA damage or NER deficient (right) with DNA damage persisting. The NER deficient condition can be reversible, resulting in DNA repair, or irreversible, resulting in permanent DNA damage that can lead to cellular damage.

ERCC1

The product of this gene is required for the repair of DNA lesions such as those induced by UV light or formed by electrophilic compounds including cisplatin. The encoded protein forms a heterodimer with the XPF endonuclease, and the heterodimeric endonuclease catalyzes the 5′ incision in the process of excising the DNA lesion. The heterodimeric endonuclease is also involved in HRR and in the repair of inter-strand crosslinks [18]. Overexpression of ERCC1 is correlated with better progression-free survival (PFS) in patients treated with doxorubicin plus trabectedin [19] and favorable overall survival (OS) [19]. Additionally, high expression of ERCC1, and BRCA1 haplotype were associated with the improved progression-free rate (PFR), PFS, and OS in STS [20]. Increased ERCC1 and XPF expression were associated with improved disease-free survival (DFS) and distant disease-free survival (DDFS) in STS [21]. A study carried out on STS patients showed that regarding the SNP rs11615, the alternative allele has a higher germline frequency than the general population and ERCC1 is overexpressed in 75% of STS samples analyzed compared to healthy corresponding tissue and its expression varies according to the genotype [14].

ERCC2 (XPD)

ERCC2 is part of the BTF2/TFIIH complex, which is essential in TCR. The translated protein has ATP-dependent DNA helicase activity and belongs to the RAD3/XPD subfamily of helicases. Defects in this gene are related to Cockayne syndrome, XP cancer-prone complementation group D syndrome, and trichothiodystrophy. [22]. ERCC2 gene is overexpressed in STS and its expression varies according to the genotyping of rs13181 and rs1799793, in addition, these SNPs have a higher frequency than the general population [14]. Additionally, ERCC2 is mutated in 3% of epithelioid sarcoma and 6.5% in perivascular epithelioid cell tumors and is significantly associated with increased homologous recombination deficiency (HRD) scores [13].

ERCC3 (XPB)

This gene encodes an ATP-dependent DNA helicase that is a subunit of basal transcription factor 2 (TFIIH) and, therefore, also functions in class II transcription. Mutations in this gene are associated with XP B, Cockayne’s syndrome, and trichothiodystrophy [23] ERCC3 overexpression is associated with disease progression in STS patients treated with trabectedin [24]. A study analyzing the genetic background, by whole-exome analysis, of a family with a 4-year-old child who has a Li-Fraumeni tumor (often associated with STS) hypothesized that ERCC3 may be a potential TP53-related modifier candidate responsible for accelerated tumor onset by the proband compared with the mother, who carries the same TP53 mutation [25].

ERCC4 (XPF)

XPF forms a complex with ERCC1 by playing a role in the 5′ incision made during NER. This complex is a DNA repair-specific endonuclease that interacts with meiotic structure-specific essential endonuclease 1 (EME1). Variations in this gene can underlie xeroderma pigmentosum complementation group F (XP-F), or xeroderma pigmentosum VI (XP6) [26]. In a study that performed targeted genomic sequencing within an Asian cohort of sarcoma patients, a truncating mutation in ERCC4 (p.Cys723*) was found in two patients with sarcoma diagnosed under 25 years of age [27]. In a retrospective study on angiosarcoma, ERCC4 was found mutated in 6% of patients [28].

ERCC5 (XPG)

This gene encodes a single-stranded DNA-specific endonuclease that makes the 3′ incision in DNA excision repair. XPG also plays a role in RNAPII transcription. Variations in this gene can cause xeroderma pigmentosum complementation group G (XP-G) and Cockayne syndrome [29]. A study of 113 STS samples showed a correlation between high expression of the common allele (aspartic acid at codon 1104) and better PFR, PFS, and OS [20]. A translational study showed that an overexpression of ERCC5 correlates with trabectedin activity and is associated with longer PFS in advanced STS treated with trabectidine [30]. Furthermore, in a cohort of STS, the frequency of SNP rs1047768 is the same as that of the general population, while the frequency of SNP rs2296147 is lower than that of the general population; the gene is overexpressed in 42% of the STSs analyzed and its expression correlates with that of the ERCC2 gene. Finally, the effect of SNP rs1047768 in protein structure was hypothesized for the first time in this study, suggesting a possible effect in ssDNA binding [14]. A meta-analysis associated variations on the ERCC5 gene with an increased risk of STS [31].

ERCC6 (CSB)

The encoded protein has ATP-stimulated ATPase activity, interacts with several transcription and excision repair proteins, and may promote complex formation at DNA repair sites. CSB interacts with RNAPII at the damaged site, and by direct interaction it recruits CSA [32], forming a complex responsible for the association and stabilization of UV-stimulated scaffold protein A (UVSSA), which stimulates TC-NER [33]. Mutations in this gene are associated with Cockayne syndrome type B and cerebro-oculo-facio-skeletal syndrome (COFS) [34]. On dbSNP are reported 68 clinically significant pathogenic variants of ERCC6 [35]. A retrospective translational study on STS showed that ERCC6 was underexpressed in L-sarcomas, compared with other STS subtypes [24].

ERCC8 (CSA)

CSA, encoded by ERCC8 (chr 10), is part of an E3-ubiquitin-ligase complex. CSA, in TC-NER, is required for recovery of DNA synthesis after repair is responsible for ubiquitination and proteasomal degradation of CSB, is required for recovery of DNA synthesis after repair [36] and interacts with CSB and with p44, a subunit of TFIIH. Mutations in this gene have been identified in patients with the hereditary disease of Cockayne syndrome (CS). [34], however genetic polymorphisms are shown to increase breast [37], gastric [38] and oral [39] cancer risk. On dbSNP are reported 32 clinically significant pathogenic variants of ERCC8 [35]. There are no data at present on the correlation of ERCC8 and STS.

XPA

The XPA protein is a zinc finger protein that plays a central role in NER by interacting with DNA and other proteins, forming the structure required to assemble the NER etching complex [40]. A retrospective translational study on STS showed that high levels of XPA expression correlated with better efficacy of trabectedin. [24].

XPC

It is a key component of the XPC complex, which plays an important role in the early stages of GG-NER. It has higher affinity for single-stranded DNA, and is important for damage detection [41]. At present, there are no experimental data on the role of XPC in STS.

TFIIH

TFIIH is a 10-subunit protein complex involved in both transcription and DNA repair, highly conserved in the entire eukaryotic domain. It can be divided in a 7-subunit CORE complex, consisting of XPB, XPD, p62, p44, p34, p52 and p8, and a CAK module (Cyclin Activated Kinase), comprised of CDK7, cyclin H and MAT1 [42]. XPB and XPD are both ATP-dependent DNA helicase and they catalyze the ATP-dependent opening of the DNA at the transcription starting site or at the damaged site. XPB, encoded by ERCC3 in chromosome 2, unwinds the DNA helix in the 3′-5′ direction and can also function as a 5′-3′ DNA traslocase [43], while XPD, encoded by ERCC2 in chromosome 19, acts in 5′-3′ direction and it’s responsible for recruiting the CAK complex [44]. The remaining 5 subunits (p62, p44, p34, p52 and p8) are encoded respectively by GTF2H1 in chromosome 11, GTF2H2 in chromosome 5, GTF2H3 in chromosome 12, GTF2H4 and GTF2H5 in chromosome 6; they carry out structural and ATPase regulation roles [45]. The three-subunit detachable CAK module has a fundamental role as a regulator of both transcription and damage repair pathways; in particular it is necessary for transcriptional activation, but its presence inhibits damage-repair functions [46]. Of the seven genes encoding the CORE components of TFIIH, mutations in ERCC3 and ERCC2 affect both RNA transcription and DNA repair pathway, causing severe disorders such as Xeroderma Pigmentosum, Cockayne Syndrome and Trichothiodystrophy [47]. NCBI dbSNP reports 14 clinically significant pathogenic or likely-pathogenic variants of ERCC3 [35]; in addition, various ERCC3 polymorphisms have been linked to increased risk of lung cancer [48] and osteosarcoma [49]. Regarding ERCC2, dbSNP reports 41 pathogenic or likely-pathogenic variants and polymorphisms in this gene have been associated with a higher risk of lung [50] and colorectal cancer [51]. Furthermore, a significant link has been reported between specific ERCC2 and ERCC3 SNPs and their predisposition to specific types of sarcomas [52]. The other 5 genes of the CORE complex are less affected by clinically significant polymorphisms, although it has been reported that mutations in p52, p8, and p44 are associated with developmental disorders [45]. Regarding the CAK module, high expression of cyclin H has been associated with trabectedin sensitivity in STS [24].

Ubiquitylation in NER Pathway

Ubiquitin is a 76-amino acid protein used for labeling targeted proteins, regulating their stability and function. Ubiquitylation is a sequential process that involves the action of E1 ubiquitin activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligating enzyme [53]. There are 2 E1, 40 E2 and about 600 E3 enzymes in the human genome [54], which emphasizes the specificity of the ligation process [55]. The initial ubiquitin is attached to the target protein in a lysine (K) residue in the C-terminal portion of the target. Subsequent ubiquitin molecules are sequentially attached to lysine residues of the previous molecule. Poly-ubiquitin chains can have different functions depending on the lysine residue to which the molecules link. The K48-linked chains signal proteasomal degradation of the target protein, whereas K63-linked chains regulate target protein function [56]. Ubiquitylation is reversible by the action of deubiquitinating enzymes (DUBs) [57]. Complex processes such as NER require similarly complex regulation through easily inducible and reversible post-translational modifications. Ubiquitylation has been shown to play a key role in this pathway [58].

GG-NER Regulation by Ubiquitylation

In the presence of UV damage to DNA, the COP9 signalosome is released from the CRL4DDB2 complex, an E3 ubiquitin ligase comprising CUL4, ROC1, and DDB2. In normal conditions the COP9 signalosome inhibits the CRL4DDB2 complex activity, in the absence of this inhibition CUL4 can be neddylated by NEDD8, leading to the activation of the E3 complex [59]. At this stage, recognition of DNA damage by XPC and DDB2 occurs [60]. The CRL4DDB2 complex carries out the action of E3 ubiquitin-ligase on histones H2A, H2B, H3, and H4, weakening the histones-DNA interactions in the damaged area [61]. The complex auto-ubiquitinates DDB2, decreasing its affinity to damaged DNA [62]. This process competes with the presence of XPC at the damaged site, which stabilizes DDB2 [63], and with PARylation of DDB2 by PARP1, which inhibits its ubiquitination [64]. Deubiquitinase BAP1 also appears to be involved in this regulation process [65]. DDB2 is extracted from the complex by VCP/p97 and targeted to the proteasome. The removal of DDB2 from DNA increases the binding affinity of XPC to TFIIH, which is recruited at the damaged site. TFIIH promotes, through its p62 subunit, DDB2 extraction from the complex. XPC-TFIIH-XPA complex formation allows the initiation of the DNA damage verification process [66]. Simultaneously with DDB2 ubiquitylation, the CRL4DDB2 complex also ubiquitinates XPC, not resulting in degradative signaling, but increasing its affinity to DNA [67]. XPC then undergoes SUMOylation, induced by UV damage to DNA, which results in the recruitment of RNF111/Arkadia (SUMO-targeted ubiquitin ligase), responsible for XPC ubiquitylation that leads to its removal from damaged DNA [68]. Extraction of XPC by VCP/p97 allows the other factors of GG-NER to be recruited.

TC-NER Regulation by Ubiquitylation

In the presence of DNA damage, RNA-Pol II is interrupted, recruiting CSA and CSB, which in turn recruit UVSSA. CSA is part of CRL4CSA E3 ubiquitin-ligase complex, comprising CUL4, ROC1 and CSA. As in the GG-NER regulation, this complex is regulated by the COP9 signalosome, which detaches in the presence of DNA damage and allows the neddylation of CUL4, thereby activating the E3 complex [59]. CSB undergoes modification by multiple factors, as it is ubiquitylated by CRL4CSA complex and BRCA1-BARD [69] and deubiquitylated by the deubiquitylating enzyme USP7 [70], which is recruited by UVSSA [71]. This fine-tuned regulation controls the stability of CSB before its extraction by VCP/p97, allowing the recruitment of the other damage repair factors. CRL4CSA complex also mono-ubiquitylates UVSSA, allowing recruitment of TFIIH, which is then linked to RBP1 [60]. If the damage is not repaired, as in the case of mutations in CSA or CSB, the RBP1 subunit is ubiquitylated by NEDD4 [72] and the Elongin A ubiquitin ligase complex [73], inducing its extraction by VCP/p97 and subsequent proteasome degradation.

Role of Chromatin in NER Pathway

Activation of the NER pathway requires DNA not wrapped around histones, as various proteins need to access the double helix. UV damage to DNA provides the signal that leads to post-translational modifications of histones or ATP-dependent chromatin remodeling. These mechanisms allow the relaxation of chromatin around histones and increase the efficiency of the NER pathway [74,75].

Histone Modifications

Acetylation is mediated by histone acetyltransferases (HATs) and the reverse process, deacetylation, is catalyzed by deacetylases (HDACs). Histone acetylation promotes chromatin relaxation and activation, promoting DNA transcription [76]. Histone acetylation stimulates NER after UV damage [65]. Recent studies report that DDB2 interacts with HBO1 (HAT) in a UV-dependent manner leading to acetylation of H3 and H4, which increases chromatin accessibility [77]. DDB2 appears to be responsible for deacetylation of H3 and H4 through HDACs, leading to the stimulation of XPC recruitment [78]. Methylation acts differently on chromatin state depending on the number of methyl groups and the residues on which they are added [79]. Recent work has shown that DDB2 recruits the methyltransferase ASH1L to damaged DNA regions, leading to tri-methylation of histone H3K4. This process induces XPC binding to nucleosomes [80]. UV irradiation also stimulates tri-methylation of H3K79 by DOT1L, which also correlates with XPC recruitment. Deletion of DOT1L is present in many cases of melanoma [81]. Finally, another histone modification is phosphorylation which leads to a more relaxed state of chromatin and serves as a checkpoint in several processes, including DNA repair [82]. Phosphorylation of specific histones does not appear to affect the NER pathway; rather, it is the NER pathway that induces phosphorylation of histone H2AX via single-strand DNA production [83].

ATP-dependent Chromatin Remodeling

Chromatin remodelers are enzymes with an ATPase domain, which use ATP energy to modify the structure of nucleosomes. They are divided into 4 families: SWI/SNF, CHD, INO80, ISWI [84]. During NER, the SWI/SNF complex catalyzes the relaxation of chromatin, making it more accessible. CSB, which plays a key role in the NER pathway, belongs to this group [85]. An additional role of SWI/SNF is its association with XPC, which promotes the recruitment of subsequent repair factors [86]. The remodeler INO80 interacts at the level of damaged sites with DDB1, suggesting a role in XPC recruitment [87]. CHD is recruited following UV damage and mediates XPC binding to TFIIH [88].

Histone Chaperones

Proteins are involved in the transport and mobilization of histones at the chromatin level [89]. CAF-1 and HIRA are associated with NER, as they are recruited in the late stages of the pathway and are involved in the deposition of neo-synthesis histones after damage repair [90,91].

Conclusions

DDR pathways, including NER, play a key role in the formation of various tumor types and their sensitivity or resistance to treatment. The NER pathway is composed of a variety of processes that are finely regulated by each other and integrated with many other cellular pathways and alterations in this pathway play an important role in many tumor types, including STS. Recent studies contribute to the notion that NER pathway deficiencies constitute potential cancer therapeutic targets. It has been found that putative damaging germline and somatic alterations in NER genes were present in STS [14]. Moreover, recent findings provide novel insights into a synthetic lethal relationship between clinically observed NER gene deficiencies and sensitivity to irofulven and its potential synergistic combination with other drugs [9]. There is still little evidence on the association between cancer risk and variations in NER genes; in fact, there are no FDA-approved targeted therapies that target germline or somatic mutations in NER pathway genes. However, mutations in NER genes can have multiple roles as biomarkers: they can act as predictive biomarkers, indicating an increased risk of developing cancer, being useful for early cancer detection by subjecting the population to higher levels of screening; at the same time, they can also be used as prognostic biomarkers, giving precise indications to physicians about the degree of sensitivity to drugs targeting DNA repair deficiencies, if variations in NER genes are present. Recent studies focus instead on the therapeutic role of NER inhibitors, such as spironolactone [92] well as triptolide, which inhibits NER by affecting XPB and transcription. NER inhibition has been shown to reverse acquired resistance to alkylating agents in multiple myeloma cells [12]. Hence, it may be another adjunct target to be considered in combination therapies. Their further investigation in these tumor types is necessary for the identification of new biomarkers or therapeutic targets (Figures 1 and 2).

fig 2

Figure 2: Nucleotide excision repair pathway (NER). NER pathways can be classified as either global genome repair (GGR) and transcription-coupled repair (TCR). The NER pathway consists of a series of reactions: recognition of DNA damage, unwinding double-stranded DNA in the neighborhood of the damage, excision of the damaged nucleotides, and filling the gap by DNA synthesis and ligation.

Author Contributions

Conceptualization, S.P.; writing—original draft preparation, F.S., A.P., A.B., E.C, S.F., O.C., E.G. and S.P; writing—review and editing, I.P, A.B. and G.S.; supervision, A.L. and S.P.; funding acquisition, I.P., D.A.C, S.P.; figures preparation: S.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Regione Toscana Bando Salute 2018, (Research project CUP n. D78D20000870002), grant number D78D20000870002.

Acknowledgments

Our special memory goes to Alessio Cerasola, an unforgettable guy who fights the disease with his ever-present smile.

Conflicts of Interest

The authors declare no conflict of interest

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Alpha-Fetoprotein-derived Segments as Integrin Peptidomimetics for Potential Cancer Cell Targeting and Therapy: A Review and Commentary

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DOI: 10.31038/CST.2023823

Abstract

Integrins constitute a group of dimeric polypeptide chains that function as natural agonists of cell surface receptor-dependent cell activities. The integrins themselves comprise a superfamily of hetero-dimeric (alpha and beta chains) transmembrane cell surface receptors whose functions include cell adhesion, growth, migration, and angiogenesis. In comparison, the integrin-like peptides (ILP) comprise groups of protein derived segments, namely, short peptides derived from naturally occurring proteins from intrinsic subdomain fragments or short motifs present on larger proteins or enzymes. Certain ILPs can bind or compete for amino acid sequence sites located on integrin beta-1 and beta-3 chains of heterocomplex receptors. Binding at major sites or allosteric minor sites can inhibit or block cell migration, angiogenesis, metastasis, and platelet aggregation. Recently, a small integrin-like peptide derived from naturally occurring alpha-fetoprotein (AFP), similar to a disintegrin, has been reported to inhibit growth and adhesion functions associated with integrin-dependent cell activities. The present report describes an example of an AFP integrin-like peptide and lends credence to support its proposed use in adjunct cancer therapies.

Keywords

Alpha-fetoprotein, Integrins, Cell adhesion, Migration, Cell-to-cell contact, Breast cancer

Introduction

A) General

Integrins comprise a superfamily of hetero-dimeric (alpha and beta chain) transmembrane receptors present on multiple cell types including tumor cells [1]. The numerous functions that integrins mediate include cell-to-cell and cell-to-extracellular matrix (ECM) adhesion, cell growth, migration and spreading, metastases, angiogenesis, cytoskeletal-induced locomotion, and platelet aggregation [2,3]. Naturally occurring antagonists of the integrin receptor are termed disintegrins (DTs) which block or inhibit integrin cell functions [4,5]. While the integrins comprise 23 or more different alpha and beta chain combinations, the DTs constitute families of only two types of molecules [6,7]. Integrin-like peptides function in a similar manner to the disintegrins which are derived from metalloproteinases. It has been demonstrated in previous publications, that small peptides, derived from naturally occurring serum-related proteins, can mimic portions of the integrin polypeptide chains [8,9]. Such an action could interfere, compete, interrupt, or block signal transduction in the integrin receptors. Thus, small integrin-like peptides (ILP) are being proposed that can inhibit or compete with adhesion functions associated with metastasis, cell migration, cell-to-cell contact, and cell spreading. Since integrins show promise as potential molecular targets for cancer, the integrin-like peptides could possibly serve as formidable anticancer therapeutic agents for cell migration and metastatic targets.

B) Objectives and Aims

The objectives in the present report comprise several in number. First, the integrin biologic functions and activities are described. Second, the types and family members of the heterodimeric cell-adhesion integrin molecules are discussed on an overview fashion. Third, naturally occurring peptides derived from “mother” proteins will be addressed as integrin-like peptidomimetics. Finally, a prime example of an integrin-like non-toxic peptide mimetic is described which displays activities such as inhibition of platelet aggregation, suppression of cell-to-matrix adhesion, cell migration/spreading, and cell-to-cell contact activities.

C) The Integrin Cell Surface Receptors

The integrin superfamily of cell surface receptors consists of hetero-dimeric (alpha and beta chains) transmembrane glycoproteins that mediate cell-to-extracellular matrix (ECM), adhesion, and cell-to-cell contact interactions [10,11]. The integrins are integral cell surface single pass-transmembrane receptors consisting of two paired chains of non-covalently linked alpha and beta polypeptide chains. Both integrins and the ECM molecules play important roles in ontogenetic development, maintenance of adult cell physiology, tissue repair, hyperplastic growth, hemostasis, and tumor oncogenesis [12-14]. The dimeric hetero-complexed integrins further serve as cell membrane receptors capable of forming focal adhesion contact linkages to the cytoskeleton; such links are located on the inner layer of cell membranes. Integrins can further bind to multiple ECM ligand proteins such as: fibronectin, laminin, vitronectin, collagen, thrombospondin, entactin, fibrinogen, talin, the intracellular adhesion molecule (ICAM), and the vascular cell adhesion molecular (VCAM) [15-17]. Studies have further linked integrin signaling to cytoplasmic cytoskeletal filament-associated proteins such as vinculin, talin, α-actinin, paxillin, and divalent cation-dependent proteins such as calreticulin [18,19]. The integrins further play a major role in cell adhesion activities in the immune system [20-22].

Each integrin subfamily is characterized by a combination of a small number of β-chains associated with a large number of α-chains. To date, eight different β-chains and 14 different α-chains have been described, accounting for at least 20 combinatorial variations of the two heterodimeric receptors [9]. Both the α- and β- subunits are integral membrane glycoproteins containing variable long-lengths of extracellular domain chains linked to short intracellular chains [10]. The α-chains exhibit four repeat amino acid segments which bind calcium (Ca++) and other divalent cations such as Mg++ and Mn++ [18,19]. The β-subunits display at least four cysteine-rich repeats in linear juxtaposition; these repeats stabilize the chains of the extracellular amino terminal loops [9,20]. In overview, both chains contribute to the formation of an interface which forms the ligand binding pocket. In contrast to their extracellular domains, the intracellular domains of both the α- and β- chain constitute short amino acid segments capable of binding to cytoskeletal-associated proteins that can link the integrins to G-proteins, actin, and calreticulin, a Ca++ influx regulator involved in cell migration [12,13].

D) The Integrins-ECM Interaction and Signal Transduction

Studies of ECM interaction with cells via their integrin receptors have shown that integrins function as bidirectional transducers of extra- and intracellular signals. The two-way (bidirectional) signaling can occur from “outside-to-inside” and from “inside-to-outside” the cell [21,22]. The regulation of cell proliferation, differentiation, survival, and immediate gene expression is influenced by integrin mediation of cell interaction associated with the ECM. The disruption of epithelial and endothelial cell interactions with the ECM can induce programmed cell death, while fibroblast-integrin adhesion can affect cell cycle activities by influencing cyclin-A and D expressions [23,24]. In addition to signal transduction with the actin cytoskeleton, the cytoplasmic domains of the integrins interact in a cascade fashion with protein kinases, calcium-binding proteins, focal adhesion kinases, Na+/H+ antiporters, tyrosine MAP kinases, and transcription nuclear factors such as NFkB and AP1 [25-27].

The integrins must be activated in order to undergo adhesion and binding to the ECM. Activation of integrins occurs through local soluble mediators such as hormones, cytokines, growth factors, or by interfaces with the ECM. Thus, cell activation is known to involve adhesion to clusters of stimulated integrins which culminate in signals triggered by local events in the cellular environment, such as thrombogenic agonists, antigen stimulation/processing, and T-cell activities [20,28,29]. In contrast, integrin activation can be blocked by ILP and/or disintegrins to disallow cell adhesion and ECM binding at inopportune times and locations. Untimely adhesions can lead to unwanted thrombosis and inflammation, while already adhered cells may need to detach in order to undergo mitosis and cell migration [21,27]. As previously reported, both the disintegrins and ILPs can effectively contribute to blocking, inhibiting, reducing, and dysregulation of integrin function.

Protein-Encrypted Peptides; Growth Inhibitory Peptide (GIP)

The inclusion of a class of growth regulatory factors, extracellular ligands, and angiogenic peptide fragments encrypted within a polypeptide chain of a full-length protein is known but is not widely recognized [30]. However, some of the most potent growth inhibitors are derived from short peptide fragments (segments) already existent in naturally occurring mammalian full length proteins. Such intrinsic segments themselves can affect cell growth and proliferation in an opposite function from that of the “mother” protein [31,32]. This less recognized concept of a protein-derived body reserve containing peptide growth inhibitor fragments is becoming a recurring theme in the field of growth regulation, intracellular signaling, and crosstalk among and between signal transduction pathways. Classical examples of such occult (cryptic) peptides derived from proteins include the following examples,

  1. Tenacin binding peptide derived from fibronectin;
  2. Angiostatin from plasmin;
  3. Endostatin from type XVIII collagen;
  4. Vasostatin from calreticulin; and
  5. Constatin from type IV collagen.

Such cryptic hidden peptide sites can be exposed following a conformational change on a protein or can be revealed following proteolytic cleavage from a larger protein [33,34]. Such peptides can also be chemically synthesized as single fragments of 20-45 amino acids. A well-published example of a peptide site revealed following a conformational transition change on a full-length protein is an encrypted “growth inhibitory” site on the alpha-fetoprotein (AFP) molecule [35]. The AFP protein is normally a growth promoting molecule, but can be temporarily converted to a growth inhibitory molecule.

The encrypted peptide segment on AFP, termed the growth inhibitory peptide (GIP), is a 34 amino acid segment concealed in a hydrophobic cleft of the tertiary folded AFP molecule. The GIP-34 site is revealed following protein unfolding in chemical environments consisting of high ligand concentrations of estrogens, fatty acids, and growth factors [32,35]. The exposed transitory GIP site converts the usually growth-enhancing AFP molecule into a temporary growth-inhibiting molecule. This conversion occurs via protein unfolding via a conformational change resulting in a denatured intermediate state that reflects a molten globular form (MGF) of the AFP protein [36]. Since the MGF of AFP is a transitory intermediate form, AFP can refold back to its native tertiary fold following removal of excess ligands (agents) in the microenvironment [37]. Because the AFP-MGF form is unstable, the GIP-34 amino acid segment alone has been synthesized, purified, and characterized as a free and distinct 34-mer synthetic peptide segment [33-35]. Thus, 34-mer GIP fragment can inhibit growth factor, fatty acid, and estrogen-induced growth in a concentration-dependent manner in addition to blocking metastatic and cell migration-associated activities.

GIP-34 Physicochemical Properties

GIP-34 has been synthesized by classical F-MOC (9-fluronylemethoxy-carbonxyl)- protected solid phase synthesis, as previously described [38]. Following peptide syntheses, the lyophilized peptide was purified by reverse-phase high-performance liquid chromatography (HPLC), producing a peptide whose major peak displayed a molecular mass of 3573 (34-mer) as determined by electrospray ionization mass spectroscopy. Cyclization of GIP-34-mer can be accomplished by addition of reducing agents to form a disulfide bridge construct at the time of the linear peptide synthesis. Circular dichroism (CD) analyzed in the UV wavelength for GIP-34 displayed a negative maximum at approximately 201nm. Computer modeling and analysis of the GIP-34 CD spectrum revealed a secondary structure comprising 45% β-sheets and turns, 45% random coli (disordered), and 10% α-helix structure [35].

Amino Acid Sequence Matches

The GIP-34 AA sequence was subjected to a FASTA search in the Genbank (GCG Wisconsin Program) database, as described [32,33,35]. The GCG search found identity/similarity sequence matches to receptor-binding proteins, such as the fibroblast growth factor (FGF) receptor, insulin growth factor II receptor (IGFIIR), transforming growth factor-β (TGF-β), and the dopamine (DOPA) receptor [39]. Other Genbank matches revealed transcription-associated proteins, including homeodomain proteins and FTZ-F1 (the AFP transcription factor), which have been previously reported [40-42]. These AA matches provide evidence that the GIP fragments contain short recognition cassettes for multiple and varied receptor involvement and interactions. Matches with cell-adhesion related proteins were also found; these included collagen XIII, collagen IV, laminin, fibrinogen, and fibronectin [41,42], (Table 1). Finally, identities/similarities were identified with transcription-associated factors, such as Hox, c-myc, forkhead, and Pax. GIP-34 matches were further found with integrin-associated proteins, the ECM proteins, cell mitosis proteins, and other adhesion proteins (Tables 1 and 3). Further identities were found with the integrin α/β chain proteins such as α11bβ3, α1β3, and αvβ1. Such integrins can serve as receptors for ECM proteins and are known to participate in cell-to-cell activities such as cell adhesion and migration (spreading) activities. Finally, matches were also made with ECM-associated proteins, such as the Von-Willebrand Factor, VLA-1, and PG-IIIa proteins, which are involved in cell adhesion, aggregation, and the action of metalloproteinases (i.e., the Adams Family) (Table 3). Thus, GIP-34 shows an identity/similarity matches to integrins, basement membrane proteins, and ECM proteins, all of which are involved in cell-to-cell and cell-to-ECM interactions. A comparison of the properties and traits of integrins versus GIP are displayed on Table 2.

Table 1: Growth Inhibitory Peptide (GIP) Amino Acid Sequences * were matched in the Genbank to Various Integrin Alpha/Beta Chain Complexes and the Compared to their Extracellular Matrix (ECM) Adhesion Inhibition by GIP. Note that many of the Integrins are expressed on a variety of tumor cells.

Integrin Subunits

*GIP Amino Acid Sequence

AA Identity%

ECM Binding Ligand

Tumor to ECM Adhesion% Inhibition

Cell/Tissue and Tumor Distribution

α

β
αVβ3A LSEDKLLACGEGAAD,

SEDKLLACG

100(9)

47(15)

 FIB, VTN, FBN, TSP

40-50

 Melanomas and angiogenic cell
αMβ2 (Mac) SEDKLLACG,

LACGEGAADI

66.7(9)

43(10)

 FBN, C3bi, I CAM

50

 Immune, Inflammatory cells
αVβ6 SEDKLLA

100(7)

50(12)

 FBN

50

 Carcinoma cells virus associated fusion
α6β1 GEGAADIII

78(9)

75(8)

 LAM-1

10-45

 NSCL carcinoma
αVβ1 SEDKLLA-CGEG

100(7)

75(4)

 VTN, FBN

40-50

 Analytic tumors
α1β1 CGEGAADIIIGH

43(12)

75(8)

 LAM COLL

10-45

 Breast carcinoma
αLβ2 (LFA-1) CGEGAADIIIG

80(11)

43(10)

 FBN, C3i

50

 Myeloid cells, Leucocytes
α4β7 GEGAADIII

MTPVNPGV

78(9)

56(9)

 FBN, VCAM MADCAM

50

 Endothelial mucosal cells
α3β1 DKLLACGEGAADIIICGEG

43(14)

75(4)

 FBN, COLL LAM

30-55

 Many tumor cells
αVβ8 IRHEMTPVNPG

67(12)

50(12)

 Not reported

not done

 Reproductive tissues
αVβ5 CGEGAADIIIGHLCIRHEM-TPBNPGVGQ

67(12)

80(25)

 VTN, FBN

45-50

 Epithelium carcinoma cells
α6β4 IRHEMTPVPVNPGV

78(8)

50(12)

 LAM-1, LAM-2

10-45

 Keratinocyte malignancy
α2β1 IIGHLCIRHE

MTPVNPGV

53(17)

75(8)

 COLL, LAM

10-55

 Epithelium, endothelium leucocytes

Table 2: Comparison of properties shared by integrin-related components and the AFP-derived Growth Inhibitory Peptide (GIP).

Activity and/or Property

Integrin-related Properties

GIP Peptide Related Properties
Cell Toxicity Non-toxic Non-toxic (cytostatic)
Working Range Nanogram concentrations Nanogram concentrations
Platelet Physiology Activate platelets for aggregation Inhibits platelet aggregation
Cell Type Localization Most body cells, platelets, uterus, breast cancer cells Platelet, uterus, breast cancer cells
Ligand Binding Extra-cellular matrix proteins. (fibromectin, virtomectin, etc.) Extra-cellular matrix protein interaction
Protein Homology C3b complement & C2 component, Factor B Von Willebrand factor, Mac-1 Von Willebrand factor, fibronectin precursor
Aggregation Form dimers, receptor aggregation (clustering) Forms dimers, trimers & oligomers
Adhesion Cell-to-cell, cell-to-ECM Cell-to-cell &cell-to ECM
Cellular Internalization Soluble ligand/integrin internalization Apparent cellular internalization
Secondary Structure Beta sheets & turns in extracellular subunits Mainly beta sheets & turns in soluble peptide
Distinctive Amino Acid Presence Cysteine relative to aspartic acid spacing Display 2 cysteine with aspartic acid spacing
Ligand Binding Region N-terminal half of α and β subunits Short sequence homologies to α chain component
Cellular Localization Cell surface transmembrane peptides extending into cytoplasm Fluorescence localization at cell surface and intercytoplasmic sites
Ligand Recognition Specificity  Controlled by the α subunit AFP-peptide more homologous to α chain subunit
Influence of Estradial Estradiol suppresses integrin ligand regulation of α2 subunit Peptide suppresses estrogen-sensitive growth
Integrin α (I-domain) Homology Similar to collagen binding domain of Von Willebrand Factor Similar to collagen binding domain of Von Willebrand Factor

Table 3: Integrin-associated Protein (IAP) amino acid sequences (left column) are matched to Growth Inhibitory Peptide (GIP) amino acid sequence stretches (middle column). Numbers to the left of the single letter amino acid code of GIP signify the amino acid number located on the full-length alpha-fetoprotein polypeptide.

I. Mitosis-associated Proteins

Protein (IAP) Name

Growth Inhibitory Peptide Amino Acid Sequences

Biological Activity or Function Affected by GIP
Contactin-associated Proteins 481 IGHLCIRH Cell adhesion
Neurotropic Tyrosine Kinase Receptor-3 461 CCQLSEDK Cell migration and invasion
Matrix metalloproteinase-13 497 ADIIIGHL

485 CIRHEMTP

 Collagenases (ADAM-13)
ADAM-22, Integrin α2β1 481 IGHLCIRH Cell-to-Cell contact, cell migration, cell adhesion
Integrin α6 (IGAG) linked to Beta chain (VLA-6) 485 CIRHEMTP Cell-to-cell contact, cell migration, cell adhesion

II. Extracellular Matrix Proteins

Protein Name

Alpha-fetoprotein Growth Inhibitory Peptide Sequence Matches

Biological Analysis or Function Affected by GIP
Receptor for Peptin-54 (G-coupled receptor) 481 IGHCIRH G-coupled receptor for signal transduction
Fibroblast Growth Factor receptor-4 497 ADIIIGHL Regulates growth and proliferation, blood vessel angiogenesis
Ephrin Receptor 2B 481 IGHCIRH Regulates bidirectional signaling related to tumor growth/metastasis
Met Oncogene Hepatocyte Factor Receptor (C-Met) 481 IGHCIRH Tyrosine Kinase Receptor, axon guidance, cell segmentation, angiogeneis

III. Growth Factor Associated Proteins

Protein Name

Alpha-fetoprotein Growth Inhibitory Peptide Sequence Matches

Biological Activity or Function Affected by GIP
Vascular Endothelial Growth Factor 477 ADIIIGHL Stimulates vascular permeability
P53 Protein Cell Tumor Antigen 477 ADIIIGHL Prevents cancer growth, a tumor suppressor
Tyrosine Phosphate Non-Receptor-7 477 ADIIIGHL Tyrosine kinase related
Cell Growth Regulator 477 ADIIIGHL Enzyme that regulates cell growth/proliferation
NF-KB Signal Factor 477 ADIIIGHL Signal transduction factor regulating phosphorylation

Cell Adhesion Assays with the AFP-derived Peptide

AFP-derived GIP has been subjected to cell adhesion studies involving many of the ECM ligand proteins known in the literature and discussed herein [32,33]. Various ECM proteins were coated on microtiter plates to serve as solid attachment surfaces for two breast cancer cell types: the human MCF-7 and the murine mammary 6WI-1 cell culture lines (Table 1). The adhesion of MCF-1 and 6WI-1 tumor cells either in the presence of AFP peptide or in peptide-free medium were assayed on ECM-coated microtiter plates with soluble GIP used as a competitive inhibitor. GIP-34 was capable of inhibiting cell adhesion of the ECM ligand proteins in both tumor cell lines which spanned inhibition of 30-50%. Inhibition of the mouse and human tumor cell adhesion was roughly equivalent on microtiter plates coated with either collagen IV, fibrinogen, fibronectin, or thrombospondin and slightly less for laminin, collagen-I, and vitronectin in the two cell types. Human MCF-7 breast cancer cells, in the presence of GIP-34, further displayed substantial inhibition of vitronectin-induced adhesion, while mouse 6WI-1 cells demonstrated similar peptide inhibition of laminin coated adhesion [34,35]. Overall, the AFP peptide was found to competitively inhibit both MCF-7 and 6WI-1 cell-to-ligand attachments which ranged from 40-60%. Finally, it was found that rabbit anti-GIP antibodies could also block the ligand adhesion inhibition effects, similar to the GIP fragment itself.

Inhibition of Cell Migration Spreading and Metastasis by GIP

Cell adhesion receptors and their ligands (i.e., ECM proteins), provide the traction and stimulus for the migration and spreading of tumor cells [28,41,43] (Table 3). In general, most cells including tumor cells, use adhesion molecules to execute cell migration, which is termed cell spreading in cell culture. The integrins initiate migration of adherent cells such as fibroblasts, epithelial cells, and tumor cells upon the ECM surfaces. Cell migration requires multivalent binding of integrins to matrix bound ligands such as collagen, laminin, and fibronectin [15,16,27]. Analysis of coverslip cell migration assays revealed that the GIP inhibited more than 60% of the MCF-7 cancer cells’ spreading and migration on the surface of coverslips [34,35]. The MCF-7 cells that exhibited migration displayed distorted morphology such as star-shaped configurations, cytoplasmic spiking, surface spiny spheres, membrane ruffling, and extensions of cytoplasmic processes, all coupled with low cell viability. In cancer movements, it is noteworthy that cell migration and spreading constitute the initial steps in the metastatic process; furthermore, GIP has been reported to inhibit metastases in vivo in animal models [32,33,36].

Tumor Cell Adhesion to the Extracellular Matrix

Tumor cell adhesion to the ECM is an essential step in the tumor cell migration and metastases process, providing a means for migrating cancer cells to transiently attach to the connective tissue substratum while spreading [41]. A tumor cell adhesion ECM assay was utilized to assess whether the AFP derived GIP-34 could influence or modify tumor cell attachment to a protein substratum or matrix [38]. Various ECM proteins were absorbed to the walls of microtiter plates and screen for their ability to serve as a substratum for enhanced tumor cell adhesion, as compared to non-ECM protein-coated microtiter plates [32,33]. Using 6WI-1 mouse mammary tumor cells, substantial cell attachment was observed with vitronectin, laminin, fibrinogen, fibronectin, and collagens I and IV after 2.0 hours of incubation at 37°C. GIP-34 was then tested for its ability to compete with tumor cell adhesion to the ECM substratum. GIP-34 was capable of inhibiting many of the ECM proteins spanning from 40% to 60% [32,33,43,45].

Cross-talk signaling between the ECM and the tumor cell membrane is known to occur. Overall, GIP-34 was capable of inhibiting both the attachment of tumor cells to the substratum and the subsequent growth of remaining tumor cells on that particular ECM substratum. Based on the ECM adhesion data, tables of integrin-association inhibition with GIP fragments are presently presented, which exemplify integrin α- and β- chain to ECM interactions (Tables 1 and 3, and above references).

Additional Activities of Integrin-like Peptides (GIP)

It is germane to this discourse that additional insight and perspectives be addressed regarding the use of integrin-like peptides (ILPs) in cancer therapies. For example, short ILPs can be structurally altered and modified to produce more potent forms of such inhibitors. Recombinant and chimeric forms of ILPs and AFP subdomains have been synthesized for use in studies of integrin inhibition/competition of tumor growth, proliferation, adhesion, migration, and angiogenesis of cancers such as liver, breast, lung, melanoma, and others [32,33,46,47]. In addition, ILPs such as GIP have been reported to induce apoptosis in radio-sensitized cultured lymphocytes [33]. Moreover, it has been reported that ADAM-22, a disintegrin-like metalloproteinase, is an active participant in the development of breast cancer resistance during endocrine hormone therapy in women [48-50]. With regard to this report, GIP administered to cultured MCF-7 human breast cancer cells was shown to down-regulate the expression of ADAM-22 by 30-fold as determined by a global RNA microarray analysis [44]. These data would suggest that GIP treatment not only could down-regulate the expression of ADAM-22, but could also block the development of hormone-resistance in breast cancer. In a further study, GIP was reported to further suppress the growth of MCF-7 human breast cancer cells in vitro and in vivo [51].

Concluding Remarks

It now seems plausible that interference with integrin signaling could provide a rational basis for the development of aids in the therapeutic treatments for cancer growth, progression, and metastases. Anti-integrin antibodies, disintegrins and ILPs all predict promise in future anti-cancer therapy studies. Integrin interruption of the adhesive interaction of tumor-to-tumor cells and platelets to tumor cells should be capable of serving to arrest or impede cancer cell migration and metastasis [41,45]. The observations that different integrins are expressed on various tumor types and are differentially expressed during tumor transformation, progression, and metastasis suggest that integrins might also serve as prognostic biomarkers [10]. Integrin-like mimetic agents that block or interfere with the initial attachment of integrins to ECM components, can also blunt signal transduction events thus inhibiting proliferation, cell migration/invasion, and platelet aggregation. Such agents could constitute a formidable armamentarium of non-toxic anti-cancer agents. Such anti-adhesive agents might further find potential application in the treatment of the five major classes of human disorders, namely; neoplasia, inflammation, trauma, wound healing, and infection.

Since integrin dysfunction frequently results in cancer pathology, integrins represent an appealing array of targets for anti-tumor therapy. Because ILPs specifically bind or compete with integrins, they serve to interfere with and/or block functions such as cancer cell growth and proliferation, and the migration activities described herein. All such activities described above suggest that integrins might have the potential to serve as prominent candidates for molecular cancer targets and as such, make integrin-like peptides promising non-toxic therapeutic adjunct agents to treat cancers.

Acknowledgment

The author extends his thanks and gratitude to Ms. Sarah Andres for her commitment and time expenditure in the skilled typing and processing of the manuscript, references, tables of this report.

Abbreviations

Coll: Collagen; FBG: Fibrinogen; FIB: Fibrin; LAM: Laminin; TSP: Thrombospondin; VTN: Vitronectin; VWF: Von Willebrand Factor; *: Amino Acid Single Letter Code; C3i: Complement Factor-3 inhibited. Integrin data obtained from References 34 and 35.
ECM: Extracellular Matrix; C: Complement Protein; C3b: Complement Subunit; AFP: Alpha-Fetoprotein; GIP: Growth Inhibitory Peptide-34.

Disclosures

Financial

None; no U.S. federal grants were used in the preparation of this paper.

Interest

The author declares that there are no known conflicts of interest in the preparation of this manuscript.

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fig 3

TRG-High/ATM-Expressed/SIRT1-Absent Profile as Negative Prognostic Marker after Neoadjuvant Chemotherapy for Gastric Cancer

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DOI: 10.31038/CST.2023822

Abstract

Gastric cancer (GC) is one of the three most deadly among cancers. Although several new drugs have been introduced for metastatic disease, the median overall survival (OS) remains 11-14 months. Perioperative chemotherapy (CT) is the current standard of care for resectable cT2-4 and/or N+ GC, and FLOT (5-fluorouracil, oxaliplatin, docetaxel) is the treatment of choice. To date, no predictive factor of response has been identified. Considering their synergism in DNA repair, ataxia telangiectasia mutated (ATM) and sirtuin-1 (SIRT1) merit investigation for prognostic stratification.

We evaluated by immunohistochemistry the expression levels of ATM and SIRT1 in the surgical specimens from 42 patients with a resectable GC or gastroesophageal junction adenocarcinoma treated with neoadjuvant CT and surgery. In the entire population, median DFS was 22.2 months (95%CI 14.9 – NR) and median OS was not reached (95%CI 26.9 – NR). DFS was significantly longer in patients achieving tumor regression grade (TRG) 2-3 compared with those achieving TRG 4-5 (median DFS not reached vs. 14.9 months; HR=0,36 CI=0,14-0,97; p=0.034), and a trend toward a better OS was also observed across the two subgroups (p=0.068). The proportion of patients who obtained a major/medium pathological regression was higher in the ATM-absent group than in the ATM-expressed group (69% vs. 50%; X-squared=6.05; p=0.1). In the overall population, OS and DFS did not have a significantly different distribution according to ATM and SIRT1 expressions. In contrast, in the TRG 4-5 subgroup, the ATM expression seems to be associated with inferior DFS (7.7 months vs. 32.1 months; p=0.055), particularly when combined with absence of SIRT1. In conclusion, TRG has been confirmed a surrogate of survival, ATM expression correlates with TRG and TRG-high/ATM-expressed/SIRT1-absent profile should be studied as a prognostic marker in prospective trials.

Keywords

Gastric cancer, Prognostic biomarker, Predictive biomarker, Epigenetic, DNA damage repair, ATM, SIRT1, TRG, DNA double-strand break

Key Points

  1. No prognostic markers are available in gastric cancer patients after neoadjuvant chemotherapy.
  2. TRG is a surrogate of survival outcomes.
  3. TRG-high/ATM-expressed/SIRT1-absent profile should be prospectively investigated as a marker for prognostic stratification.

Introduction

Gastric cancer (GC) is one of the three most deadly among cancers, and the fifth most commonly diagnosed tumor worldwide, with about 950,000 new cases per year [1]. Although several new drugs have been introduced for metastatic disease, the median overall survival (OS) remains 11-14 months [2,3]. In recent years, perioperative chemotherapy (CT) has been considered the standard of care for the vast majority of patients with resectable GC, and FLOT (5-fluorouracil, oxaliplatin, docetaxel) is the treatment of choice in patients fit for intensive CT. Doublets containing fluoropyrimidine and platinum are considered feasible for frail, comorbid or elderly patients [4-6]. To date, no reliable predictive factor of benefit from perioperative treatments has been identified and a tailored strategy is yet to be applied [7]. An appropriate patient selection for perioperative therapy is challenging, since only a few features have been associated with tumor regression and survival outcome after FLOT treatment. In the FLOT4-AIO trial, diffuse histotype has been associated with a reduced rate of pathological complete response (pCR) compared to the intestinal one (3% vs. 23%, respectively). Among the tumor-centered endpoints, pCR is currently considered of great interest as it could be a surrogate for survival outcomes [8]. Unfortunately, in resectable disease, only limited evidence is available regarding a molecular biomarker-based patient selection [7]. Although several molecular subgroups have been identified as potentially associated with a prognostic or predictive effect, stratification in prospective trials is still needed. Among the putative biomarkers of interest, microsatellite instability (MSI) seems to be the best candidate to drive treatment choice in the near future [7,9,10]. Consistent with previous evidence, a recent international meta-analysis conducted by Pietrantonio et al. confirmed MSI-high (MSI-H) status as a favorable prognostic factor in patients with resected GC. The 5-year disease-free survival (DFS) and overall survival (OS) rates were higher in patients with MSI-H GC than in those with microsatellite stable (MSS) tumor (5-year DFS: 71.8% vs. 52.3%, respectively; 5-year OS: 77.5% vs. 59.3%) [9]. In addition, the benefit from neoadjuvant CT in resectable MSI-H GC seems to be limited, and that ongoing studies are evaluating immune checkpoint inhibitors (ICIs) as neoadjuvant therapy or potentially curative strategy in patients achieving a complete clinical-pathological-molecular response [10]. Although evidence is limited, ataxia telangiectasia mutated (ATM) protein and sirtuin-1 (SIRT1) have demonstrated a deep synergism of action and could be considered for prognostic stratification, since they are involved in the repair of DNA double-strand breaks (DSB) and epigenetic regulation [11-13].

The ATM gene is located on chromosome 11 and encodes a serine/threonine protein kinase that contributes to maintaining genomic integrity transducing a DSB repair signal to effectors. ATM protein levels are decreased in GC compared to normal samples and low levels of phosphorylated ATM are associated with poor differentiation, lymph node metastasis and poor prognosis [14]. GC cells with defective ATM (expression or activity) determining homologous recombination deficiency are more sensitive to therapies that cause the accumulation of DNA DSBs [15]. In particular, in GC cell lines, ATM overexpression is associated with cisplatin-resistance and its inhibition, using the ATM inhibitor CP466722 or siRNA, induces the reversion of epithelial-to-mesenchymal transition [11]. Therefore, mediating platinum-resistance, we supposed that ATM expression could influence tumor regression in GC patients treated with platinum-based neoadjuvant CT.SIRT1 is a NAD+-dependent class-III histone deacetylase (HDAC) involved in several cell functions including DNA repair. Contributing to the identification of DNA damage sites and access of DNA repair proteins, SIRT1 has a crucial role in the epigenetic regulation of cell homeostasis by deacetylating both histone and non-histone proteins. SIRT1 acts both as tumor suppressor and tumor promoter, depending on location (nucleus vs. cytoplasm) and tissue type. DNA damage is a trigger for SIRT1 dissociation and re localization to DSB. In the SIRT1-DNA repair interplay, ATM preserves the efficient recruitment of SIRT1 to DSBs by signaling DNA damage. Simultaneously, SIRT1 stabilizes ATM at DSB sites and stimulates its autophosphorylation and activity [12,13].

The aim of this study was to investigate the prognostic role of ATM and SIRT1 expression in a cohort of patients who underwent neoadjuvant CT for resectable GC.

Materials and methods

Study Design and Population

This was a single-institution, observational, retrospective and prospective study, which enrolled patients with a resectable GC or gastroesophageal junction adenocarcinoma treated with neoadjuvant CT and surgery from September 2017 to April 2022.

Being an observational study, treatments were performed according to clinical practice and national and international guidelines, regardless of the inclusion in the study.

All patients had a histological diagnosis of GC or gastroesophageal junction adenocarcinoma and availability of tissue samples of primary tumor for translational analysis. Clinical stage was assessed in a multidisciplinary board according to the 7th edition of the International Union Against Cancer Tumour–Node–Metastasis classification [16]. A CT scan, FDG-PET scan, US-endoscopy and, wherever indicated, diagnostic laparoscopy, were routinely performed for staging. Patients with metastatic disease, squamous cell carcinoma, pure neuroendocrine carcinoma or esophageal cancer were excluded.

The primary objective was to describe the expression of ATM and SIRT1 in a cohort of patients with resectable GC who had received neoadjuvant CT. Secondary objectives included the description of clinical characteristics associated with specific patterns of expression and the identification of clinical and/or pathological characteristics that may be related to prognosis.

This study was approved by the Institutional review board of Azienda Ospedaliero-Universitaria Careggi (Comitato Etico Regionale for clinical experimentation of Toscana Region – Italy – Area Vasta Centro – 22070_BIO). Informed consent from each patient enrolled in the study was obtained.

Histopathological Evaluation and Immunohistochemical Staining

Immunohistochemistry (IHC) was performed on formalin-fixed paraffin-embedded (FFPE) tumor sections of GC, 3µm thick. Tissue sections were processed by fully automated detection and staining techniques through Discovery Ultra immunostainer (Ventana Medical Systems, AZ, USA). Slide sections were incubated with the following primary antibodies: anti-SIRT1 (#ab104833; mouse monoclonal, clone 1F3, 1:500, Abcam, Cambridge, UK) and anti-ATM (#ab32420; rabbit monoclonal, clone Y170, 1:100, Abcam, Cambridge, UK). Anti-SIRT1 signals were developed with UltraMap anti-mouse HRP, and anti-ATM with UltraMap anti-Rabbit HRP (Ventana Medical Systems, AZ, USA). The bound antibodies were visualized using Discovery ChromoMap DAB Kit (Ventana Medical Systems, AZ, USA). Finally, sections counterstaining was performed with Haematoxylin II ready-to-use; Ventana, AZ, USA). Healthy human colon for SIRT1 and testis for ATM were used as positive controls. Negative control was performed by replacing the primary antibodies with Mouse IgG1 and Rabbit IgG Isotype Control (Invitrogen). The negative and positive control sections were treated in parallel with the samples in the same run Immunohistochemical expression of SIRT1 was evaluated according to An et al. [17].

The expression score was assessed by combining staining intensity score and the positive percentages. The expression was scored as follows: <10%, 10-24%, 25-49%, 50-74%, and ≥75%. Immunohistochemical expression of ATM was evaluated according to Miller et al. [18]. The staining was evaluated based on nuclear DAB signal, and the intensity score was assessed as: 0 to 3, scaled in 0.25-point increments (0=totally negative; +/3=weak positive; ++/3=moderate positive; +++/3=strongly positive).

Statistical Analysis

Demographic and clinical data, molecular alterations, disease and treatment characteristics were analyzed using descriptive statistics. Statistical comparisons for categorical variables were performed using X2 test. Time-to-event endpoints were described by Kaplan-Mayer curves. Survival distributions for specific subgroups of patients were tested with the log-rank test. A p-value of 0.05 or lower was considered statistically significant. All the analyses were corrected for multiple testing when appropriate and challenged with comprehensive multivariate modeling.

Results

Between September 2017 and April 2022, a total of 42 patients were prospectively and retrospectively enrolled. Twenty-nine were men (69%) and 13 were women (31%) with a median age of 68 years (range 49-79). The primary tumor location was corpus-antrum in 79% of cases and gastroesophageal junction in 21%. A vast majority of the patients (90%) had an optimal performance status (ECOG PS0) at the time of the initial diagnosis. Among the most common presenting symptoms, weight loss more than or equal to 5 kg was reported in 19%. As neoadjuvant treatment, 79% (n=33) of patients had received a taxane-based triplet, while 21% (n=9) received a taxane-free doublet. Postoperative CT was administered in only 50% of cases, due to suboptimal recovery from surgery or postoperative complications, and 85% of them required a dose reduction due to toxicities. In the surgical specimens, lymph node involvement was reported in 50% (n=21) of patients and pT3-4 in 64% (n =27). Clinico-pathological characteristics are summarized in Table 1.

Table 1: Clinical and pathological characteristics of patients

Patients N (%)
Sex

Male

29 (69%)

Female

13 (31%)
Age

Median

68 (range 49-79)
Location

Corpus antrum

33 (79%)

gastroesophageal junction

9 (21%)

Baseline weight loss

No

24 (57%)

< 5Kg

10 (24%)

> 5Kg

8 (19%)
ECOG PS

0

38 (90%)

1

3 (7%)

2

1 (3%)
Type of Surgery

Ivor Lewis

7 (17%)

Partial Gastrectomy

17 (40%)

Total Gastrectomy

18 (43%)
ypN

0

21 (50%)

1

10 (24%)

2

5 (12%)

3

6 (14%)
ypT

0

0 (0%)

1

9 (21%)

2

6 (15%)

3

24 (57%)

4

3 (7%)
Intraoperative metastases

No

39 (93%)

Yes

3 (7%)
Pre-operative Treatment

Platinum-based doublet

9 (21%)

Taxane-based triplets

33 (79%)
Post-operative Treatment

Fluoropirimidine single agent

3 (7%)

Platinum-based doublet

9 (21%)

Taxane-based triplets

21 (50%)

No

9 (21%)
Grading

G1

2 (5%)

G2

23 (55%)

G3

15 (35%)

NA

2 (5%)
TRG Mandard

2

10 (24%)

3

14 (33%)

4

8 (19%)

5

8 (19%)

NA

2 (4%)
Recurrence

No

25 (60%)

Yes

17 (40%)

At the time of the analysis, 16 patients were deceased and 26 patients were still living. Disease recurrence occurred in 40% of patients (n=17). Median DFS was 22.2 months (95%CI 14.9 – NR) and median OS was not reached (95%CI 26.9 – NR). Among the other clinico-pathological factors, univariate analysis showed that weight loss at diagnosis (p=0.03), pathological nodal involvement (p=0.002) and number of neoadjuvant cycles (p=0.03) were significantly associated with DFS. In addition, ypT (p=0.05), ypN (p=0.007), number of neoadjuvant cycles (p=0.0018) and number of adjuvant cycles (p=0.008) were significantly associated with OS. The multivariate analysis confirmed the association between the number of adjuvant cycles and DFS (p=0.018) and OS (0.023) and between the number of neoadjuvant cycles and OS (p=0.042).

A histopathological review was performed by dedicated pathologists focusing on the assessment of tumor regression grade (TRG) according to Mandard. As reported in Table 1, TRG 2 was reported in 24% of cases (n=10) and TGR 3 was reported in 33,3% (n=14), while both TGR 4 and TGR 5 were described in 19% of cases (n=8). Then, we assessed whether TRG was associated with survival outcomes. Consistent with the literature, DFS was significantly longer in patients achieving TRG 2-3 compared with those achieving TRG 4-5 (median DFS not reached vs. 14.9 months; HR=0,36 CI=0,14-0,97; p=0.034) and a trend toward a better OS was also observed across the two subgroups (median OS not reached vs. 26.9 months; HR=0,39; CI=0,14-1,11; p=0.068) (Figures 1 and 2).

fig 1

Figure 1: DFS in GC patients achieving TRG 2-3 vs TRG 4-5

fig 2

Figure 2: OS in GC patients achieving TRG 2-3 vs TRG 4-5

We evaluated the expression of ATM and SIRT1 in the surgical specimens by IHC. Although optimal ATM staining cutoffs were controversial, according to Kim et al. the criteria for negative cases were set as less than 10% of cells stained as weak positive (+/3) or higher intensity, that is, more than 90% of cells showing totally negative (0) or equivocal staining (±) [15]. For example, if more than 90% of tumor cells showed equivocal (±) or negative (0) staining and less than 10% showed any positive (+, ++ or +++/3) staining, a case was defined as negative (Figure 3).

fig 3

Figure 3: Representative H&E images and IHC of ATM and SIRT-1 expression in GC specimens. Representative images of GC patient with 0 ATM score, 3 SIRT1 score, and TRG of 2 (A); representative images of GC with 3+ ATM score, 0 SIRT1 score, and TRG of 5 (B); representative images of GC with ATM and SIRT1 score 0, and TRG of 5 (C); representative images of GC with 0 ATM score, 1 SIRT1 score, and TRG of 4 (D); (Magnification ×20, inset ×40; scale bar 100 μm, 50 μm, respectively).

Accordingly, as reported in Table 2, ATM score was 0 in 16/42 cases (38%), 1+ in 16/42 (38%), 2+ in 7/42 (17%) and 3+ in 3/42 (7%).

Table 2: ATM and SIRT1 expression

Patients N (%)
ATM

0

16 (38%)

+/3

16 (38%)

++/3

7 (17%)

+++/3

3 (7%)
SIRT1

<10%

31 (74%)

10-24%

3 (7%)

25-49%

4 (10%)

50-74%

3 (7%)

≥75%

1 (2%)

SIRT1 expression was <10% in 74% of cases, 10-24% in 7%, 25-49% in 10%, 50-74% in 7% and ≥75% in 2% [12]. We then we evaluated the correlations between the expression of ATM and SIRT1 and TRG. Of note, the proportion of patients who obtained a major/medium pathological regression (TRG 2-3) was higher in the ATM-absent subgroup than in the ATM-expressed subgroup (69% vs. 50%; X-squared=6.05; p=0.1) (Figure 4).

fig 4

Figure 4: Mosaic plot including TRG and ATM as variables

In the overall population, OS and DFS did not have a significantly different distribution according to ATM and SIRT1 expressions (OS p=0.4 and p=0.2, respectively; DFS p=0.56 and p=0.81, respectively).

In contrast, in the subgroup of patients with TRG 4-5, usually characterized by poor prognosis, the absence of ATM expression seemed to be a positive prognostic factor. The median DFS in patients whose tumor had TRG 4-5 and absent ATM expression was 32.1 months compared to 7.7 months in those with TRG 4-5 and positive ATM expression (p=0.055). Although not statistically significant, the median OS was numerically higher in the subgroup with ATM-absent than in the subgroup with ATM expression (55.0 months vs. 26.9 months, respectively; p=0.6) (Figures 5 and 6).

fig 5

Figure 5: DFS distribution in patients whose tumor had TRG 4-5 according to ATM expression

fig 6

Figure 6: OS distribution in patients whose tumor had TRG 4-5 according to ATM expression

Furthermore, among patients with TRG-high and ATM-expressed, those with SIRT1 <10% had a median DFS of 12.7 months, which was significantly inferior if compared with the entire population (median DFS not reached; HR=0.31; CI=0.11-0.91; p=0.024) (Figure 7).

fig 7

Figure 7: DFS distribution between TRG-high/ATM-expressed/SIRT1-absent profile vs other profiles

This difference was partially sustained by the positive prognosis of TRG-low patients. Therefore, excluding patients with TRG-low, the TRG-high/ATM-expressed/SIRT1-absent profile was associated with a trend toward a lower DFS compared with other TRG-high patients (median DFS 12.7 months vs. 32.1 months; p=0.32) (Figure 8).

fig 8

Figure 8: DFS distribution between TRG-high/ATM-expressed/SIRT1-absent profile vs TRG-high subgroup

Discussion

Since the perioperative treatments have become the standard of care for a vast majority of patients with cT2 or higher and/or nodal-positive resectable GC, the identification of new prognostic and predictive biomarkers is an urgent need. ATM expression has been extensively studied with conflicting results [19-21]. Kelmpner et al. did not show any association between ATM expression and clinico-pathological factors and any impact on prognosis from ATM profiles in a cohort of patients who were treated with first-line XELOX for advanced GC [20]. In contrast, in a study of Kim et al., a low ATM expression was associated with older age, advanced stage, MSI, and lower DFS and OS in patients who underwent radical surgery for resectable GC. In this study, the worst prognosis was exhibited by the subgroup which had low ATM expression and MSS [21]. Although the setting was similar to our study, the patient population of Kim and colleagues received upfront surgery followed by adjuvant CT in 50% of cases, while all our patients received neoadjuvant CT followed by surgery, and this may have contributed to a different ATM expression and prognosis. In our study, patients with ATM-expressed cancer after neoadjuvant CT were more frequently associated with TRG 4-5 and, consequently, had a worse prognosis. Although the real reason remains largely unknown, we can suppose that high expression of ATM, playing a crucial role in the repair of DSBs, may offer a highly efficient mechanism of repair from damages induced by CT, radiation, oxidative stress, and stochastic events [11]. As previously described, repair of DSBs involves an extensive network of signals, including a synergism between ATM and SIRT1 with epigenetic implications [11-13]. To date, the role of epigenetic alterations and changes during CT is debated. An extensive knowledge of epigenetic mechanisms underlying prognosis and treatment response could produce new promising epigenetic strategies for GC treatment [22]. SIRT1 contributes to several processes involving GC development, invasion, and metastatic spread. In preclinical studies, knockdown of SIRT1 promoted GC cell migration and invasion in vitro and metastasis in vivo. Among genes downregulated by SIRT1, ARHGAP5 has been identified as an independent prognostic marker of GC [23-25]. As previously described, SIRT1 has an ambiguous role acting both as tumor suppressor and tumor promoter [12,13]. An et al. explored the role of SIRT1 expression in chemoresistance of GC both in vitro and in vivo. They showed that SIRT1 had inhibitory activity on chemoresistance and eliminated cancer stem cell properties [17]. Several retrospective studies suggested a negative prognostic impact of SIRT1-high profile than SIRT1-low, but the prognostic role of SIRT1 expression remains unclear. In a study by Noguchi et al, patients with SIRT1-high GC had a shorter cancer-specific survival than patients with SIRT1-low GC [26,27]. Similarly, Zhang et al. showed that low SIRT1 expression was associated with better outcomes both in patients with advanced GC and in those with early-stage GC [28]. In contrast, Kang et al. reported a positive prognostic effect from SIRT1 expression in a cohort of 452 patients who received surgery for GC. In this study, SIRT1-high profile was associated with more favorable clinicopathological features, including intestinal histotype, lower grade, and lower pT and pN stage [29]. In our study, among patients with poor prognosis (TRG-high and ATM-expressed), a numerically lower DFS was observed in the SIRT1 <10% group than in the SIRT1 ≥10% group. Although statistical significance was not reached, we can speculate that a complete loss of SIRT1 expression could be associated with inhibition in reversing chemoresistance and amplification of the negative prognostic effect of ATM-high profile which efficiently repairs chemotherapy-induced DNA damage. This hypothesis is consistent with a study by An et al. [17], in which silencing of SIRT1 facilitated resistance to 5-fluorouracil and cisplatin.

Regarding limitations, our study included only patients of European origin and the comparison with studies carried out in Asia might be precluded by the geographic heterogeneity in pathological features of GC. In addition, the limited sample size could have induced us to underestimate small differences or subgroup effects. Availability of pretreatment biopsies would have contributed to a better interpretation of the results.

In conclusion, the role of ATM and SIRT1 expression in resected GC patients has not been thoroughly explored and could be of interest for the generation of hypotheses that warrant a future prospective validation in larger clinical trials. This study confirms TRG as a surrogate of survival and suggests an association between ATM expression and TRG. The TRG-high/ATM-expressed/SIRT1-absent profile tends to be associated with a poor prognosis and merits study as a stratification marker after neoadjuvant CT.

Funding

No financial funding was received.

Availability of Data and Materials

All data generated or analyzed during this study are included in this published article.

Competing Interests

The authors declare that they have no competing interests.

Abbreviations

ATM: Ataxia Telangiectasia Mutated; CT: Chemotherapy; DFS: Disease-Free Survival; FFPE: Formalin Fixed Paraffin-Embedded; FLOT: 5-Fluorouracil, Oxaliplatin, Docetaxel; GC: Gastric Cancer; HDAC: Histone Deacetylase; IHC: Immunohistochemistry; MSI: Microsatellite Instability; MSI-H: Microsatellite Instability-High; MSS: Microsatellite Stable; OS: Overall Survival; pCR: Pathological Complete Response; SIRT1: Sirtuin-1; TRG: Tumor Regression Grade

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  17. An Y, Wang B, Wang X, et al. SIRT1 inhibits chemoresistance and cancer stemness of gastric cancer by initiating an AMPK/FOXO3 positive feedback loop. Cell Death Dis. [crossref]
  18. Miller RM, Nworu C, McKee L, et al. Development of an Immunohistochemical Assay to Detect the Ataxia-Telangiectasia Mutated (ATM) Protein in Gastric Carcinoma. Appl Immunohistochem Mol Morphol. [crossref]
  19. Bhangoo MS, Luu HY, Kim ST, et al. Low ATM expression is associated with improved progression-free and overall survival in advanced gastric cancer patients treated with platinum-based chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5543.
  20. Klempner SJ, Bhangoo MS, Luu HY, et al. Low ATM expression and progression-free and overall survival in advanced gastric cancer patients treated with first-line XELOX chemotherapy. J Gastrointest Oncol. [crossref]
  21. Kim JW, Im SA, Kim MA, et al. Ataxia-telangiectasia-mutated protein expression with microsatellite instability in gastric cancer as prognostic marker. Int J Cancer. [crossref]
  22. Sato H, Niimi A, Yasuhara T, et al. DNA double-strand break repair pathway regulates PD-L1 expression in cancer cells. Nat Commun. [crossref]
  23. Gigek C, Wisnieski F. Epigenetic mechanisms in gastric cancer. Epigenomics (2012) 4(3), 279–294.
  24. Canale M, Casadei-Gardini A, Ulivi P, et al. Epigenetic Mechanisms in Gastric Cancer: Potential New Therapeutic Opportunities. Int J Mol Sci. [crossref]
  25. Vaziri H, Dessain S, Eaton EN, et al. hSIR2(Sirt1) functions as an NAD-dependent p53 deacetylase. 2021. Cell 107(2): 149-59. Doi: 10.1016/S0092-8674(01)00527-X.
  26. Dong G, Wang B, An Y, et al. SIRT1 suppresses the migration and invasion of gastric cancer by regulating ARHGAP5 expression. Cell Death Dis 9, 977 (2018). https://doi.org/10.1038/s41419-018-1033-8.
  27. Noguchi A, Kikuchi K, Zheng H, et al. SIRT1 expression is associated with a poor prognosis, whereas DBC1 is associated with favorable outcomes in gastric cancer. Cancer Med. [crossref]
  28. Zhang S, Huang S, Deng C, et al. Co-ordinated overexpression of SIRT1 and STAT3 is associated with poor survival outcome in gastric cancer patients. [crossref]
  29. Kang Y, Jung WY, Lee H, et al. Expression of SIRT1 and DBC1 in Gastric Adenocarcinoma. Korean J Pathol. [crossref]

The Effects of Unprofessional Conduct in Daily Work and How We Can Correct Them

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DOI: 10.31038/IJNM.2023434

 
 

Each individual selects a domain to work in, according to their intellectual capacity, preferences, and ability to perform the duties imposed by chosen areas. Attaining expertise in that field, the art of transferring knowledge in practice, skills in collaborating with colleagues, partners, customers and their families, and other community members, and building a respectful, trustful, and merciful relationship with them are fundamental factors for a successful professional and social life, ensuring societal development and public health and define professionalism. The evidence in clinical practice highlights unprofessionalism’s adverse effects on people’s health, and a proposal for corrective actions to attenuate and prevent them is necessary. Sometimes, individuals with various backgrounds were assigned to positions that did not align with their preparation, except for a medical stance. Untrained for their new role, and misunderstanding and applying it in practice has generated errors, their product defect determining dissatisfied end-users, a decrease of imports /exports of goods or services prepared for, and a decline of investment return, finally affecting practice efficiency, personal reputation, and collaborator’s health. Therefore, additional training and flexibility in a modern environment are necessary. It’s important to have versatile, creative, and optimistic individuals who embrace novelty for job improvement. Otherwise, there may be personal and social disorders during the production process and risks to people’s health. The inflexibility in public relations has a detrimental effect on professional work effectiveness, altering the emotional and social life of collaborators. The mistakes are common, but they must be quantified and their impact on the individual and community members analysed. Professionalism in dealing with intentional or unintentional errors or abuse in daily work is also required.

Research in clinical practice has shown that improper communications between business partners can hurt vulnerable individuals involved in such affairs; usually, aggressive, inappropriate words’ energy use modifies an individual’s energetic picture in motion, finally altering blood flow through the vessels and cells’ function in fragile areas. Repetitive exposure to such unsafe interactions can cause sleep disruption, depression, dyslipidaemia, hyperglycaemia, high blood pressure, and even brain haemorrhage in people at risk. Therefore, the promotion of practical communication skills for the sake of public health is mandatory. Continuous education, prompt and precise responses in clinical practice, working in a comforting atmosphere, building confidence in social interactions, cultivating positive habits, and upholding professional conduct that aligns with social norms ensure a successful career and a prosperous personal, professional, and social life.

Business continuity and development in a competitive and dominant digital world are desired; in the digital age, personal info quickly spreads, offering alternative options for collaborators if disappointed by a supplier.

Professionalism is essential for enhancing practice efficiency, promoting social progress, and improving public health. Teaching and applying it across all global industries is at the forefront of individual life quality improvement. By acting professionally, one can become an admired licensed person who receives people’s gratitude and respect from the community they serve. Attaining excellence in job performance, as an ideal, soothes the mind and heart outlook transitioning a relative and perceptible –audible and visible world.

Community and Home Based Palliative Care Services: The Key to Equitable Access to Healthcare Services

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DOI: 10.31038/IDT.2023412

Abstract

Community and home based palliative care services are gaining more recognition especially now where primary health care is important for equitable access to healthcare services. Hospice Africa Uganda has exemplified the use of community and homebased care services to reach to the underprivileged populations and this approach is being adopted by a community initiative called, Lweza Community Health Program to promote primary health care. Through this approach, patients with serious illness including those with infectious diseases have been identified, initiated on treatment and are being followed up in the community. This approach has helped to bridge the divide between the rich and the poor but also helped to address other social-cultural factors which influence the health of individuals and communities. It is hoped that the community initiative will become a model for many communities in Africa to improve equitable access to healthcare services in communities.

Introduction

As a clinical discipline, palliative care relies on the collaboration of professionals, such as nurses, physicians and social workers, who have a fundamental role in running the team. Furthermore, primary care personnel – which includes family physicians, medical specialists, paediatricians, geriatricians, general practitioners, nurses from a variety of settings (home care, community care), social workers, pharmacists, physiotherapists, occupational therapists and volunteers – have a central function in this arena [1].

For communities, this requires a cooperative approach between the primary care and palliative care practitioners, both within the community and among experts in the hospital. It is these primary care professionals, who have core competencies in palliative care and access to specialist palliative care teams, who should manage the care of patients and families.

To date, most physicians in the community feel ill-prepared to deal with patients with palliative care requirements, due to their lack of training in pain and symptom management as well as the psychological issues that befall patients and their family caregivers. Thus, the most urgent need is to train physicians, nurses and social workers in the community to be able to practice basic palliation care principles [2].

One critical element that must be made available in order to support caregivers is the access to skilled homecare nursing, 24/7 [1]. Utilizing other providers, such as paramedics trained in palliative care, could fill some of the voids where round-the-clock home visits are not feasible.

Palliative care nurses have a pivotal role in community palliative care services. Not only do they treat, advise and help with ongoing treatment, they also assist in coordinating appropriate and timely care in the various institutions that are able to provide the appropriate treatment. In straight-forward situations, the palliative care nurses may simply advise the primary care physicians after consulting with a palliative care specialist. Unfortunately, the recommendations for the staffing of a palliative care team in the community are not yet applicable for the developing world. It is the profession’s obligation to ensure equitable availability of palliative care for rural and other vulnerable populations [3]. Let us not forget that the goal of palliative care is not just to improve the quality of life, but even more so to alleviate suffering for all patients with a serious illness be it an infectious disease. Thus, alleviating the suffering, be it physical or emotional, should guide us throughout the continuum of care.

The initial step in alleviating patients’ suffering is pain control. All those working in the field have experienced how unrelenting, agonizing pain impacts individuals and families and the magnitude of the hardship it engenders.

By and large, there is still very limited access to medication for moderate and severe pain in most low- and middle-income countries, worldwide. Under-treatment of severe pain is reported in more than 150 countries, accounting for about 75 percent of the world’s population, thereby creating profound global health inequity [4].

As already mentioned, in the greater part of the world there is an urgent need for basic palliative care services in the community; one important reason for this is the insufficient awareness of palliative care among primary healthcare providers as well as the extreme shortage of palliative care specialists in each region.

More recently, an innovative intervention – Project Extension for Community Healthcare Outcome – Palliative Care in Africa (ECHO-PACA) – was conducted in several sub-Saharan countries. One of the important things learned was the necessity to reach out to the primary care providers in the community and to develop a curriculum and guidelines for best practices, based on local palliative care requirements [5].

Our story describes the gradual development of community-home care services in Uganda. It tells, first-hand, of the arduous, yet successful and praiseworthy palliative care experiences owing to the endless devotion, endurance, goodwill and compassion of a dedicated physician and her nurse.

Our Personal Story

Having worked in Palliative Care for over 28 years, Dr. Anne Merriman introduced Palliative Care (PC) to Uganda through Hospice Africa Uganda (HAU), its goal being to reach “all in need of palliative care in Africa”. Hospice Africa Uganda is now a model for Palliative Care services in all of Africa. During her 40 years in Africa, Dr. Merriman has travelled through many African countries, primarily LMIC’s, and realizes that each country has its unique traditions.

Although Uganda was one of the poorest countries in Africa in the early 1990s and was just out of a long war, the people were so caring. The country had one of the lowest rates of corruption. Now, 30 years later, many things have changed. The divide between the rich and poor has increased, the population has doubled and, unfortunately, the caring spirit for each other (traditionally arising from “ubuntu”, meaning “humanity”) has declined. Those who have been blessed with greater financial wealth often have the attitude of caring only for oneself and lesser values when it comes to caring for the less fortunate. This is the situation in many African countries today.

With billions of people in the world falling under the poverty line, access to good quality health care services is becoming impossible despite the great advances in medicine. Quality, yet genuine, healthcare services are only available to the few lucky ones who can afford it. Most patients have to travel miles and miles to reach these services. The poor have been left to suffer in the hands of “quacks”. The little savings they are able to accrue goes to out-of-pocket payment for medical bills, yet the services received are often substandard and predisposes them to more ill health. It is for such reasons that the poor are now shunning hospital-based care and are seeking cheaper, friendlier and more accessible interventions in the community. Community health systems must therefore prioritize the needs of the poor.

As a Sister in charge of a gynaecology ward, Rose (the first HAU nurse, in 1993) had witnessed the unbearable suffering of patients with advanced cancer disease. Patients were in pain and some had a foul smell stemming from the fungating tumour. In such agony, doctors on ward rounds discharged these patients to go home telling them, “Nothing more can be done.” Rose wondered, what then happens to these patients when they go home in such agony? The needs of these suffering patients were as paramount then as they are today.

Rose left a promising Hospital Nursing career and, with much opposition, in 1993 joined the new Hospice Africa Uganda, travelling daily to local communities and providing professional palliative care within the homes. She was always mindful to adapt her care to the specific culture, economy and the tribe to which they belonged, while also respecting the spiritual and traditional beliefs of each family.

Although Dr. Anne had already worked in Nigeria for 10 years, the Ugandan culture was so different and there was so much to learn from Rose. So while Rose was learning from Anne, Anne was learning from Rose!

In Uganda, where 95% of those who are stricken with cancer cannot access treatment, there is an especially heavy burden on poorer families. These families are left abandoned as they watch a loved one writhing in pain, many times suffering in isolation due to the unpleasant smells that result from disease and decay. Simply attempting to attain a cancer diagnosis may throw the family into further poverty, no longer able to afford food; children stop attending school, as every penny is put toward reaching a cancer cure. The few who are able to pay for a biopsy then find that they cannot afford the cost of treatment, and are often sent home.

Community health funding is based on statistics – but are the cancer statistics realistic when they are often based on the number of biopsies and diagnoses which are unaffordable for most? When birth and death certificates are not mandatory in many parts of Africa?

By 1993, a new disease known locally as “Slim” due to a resistant fungal infection in the oesophagus which prevented eating due to pain on swallowing, was obvious wherever Dr. Anne and Rose went. This was HIV, and the pain associated with many opportunistic infections was often severe and uncontrolled. Over 30% of Uganda’s population was infected by HIV and the incidence of cancer had doubled.

What could be done to alleviate the suffering of these patients, as well as those in the community? Dr. Anne introduced the affordable formula for oral morphine for home use. With support from then the presiding Minister of Health, Dr. James Makumbi, the importation of morphine into Uganda for reconstitution in the pharmacy was endorsed. Dr. James said, “My people are suffering… you are welcome. Please come immediately.” Patients with moderate to severe pain have since been given the green liquid oral morphine, many of whom now call it the “magic” medicine as it has greatly relieved unnecessary suffering. Patients are now free of pain and “living until they die”, counting it as a true miracle. This drug has since modified pain control throughout Africa, albeit only in those countries that have overcome the myths and fears perceived by governmental officials and senior doctors who have never heard of palliative care and who equate using oral morphine with euthanasia. Beginning with only three countries in 1993, 37 African countries have been supported by HAU and are currently offering palliative care, besides 11 of them are now using the Merriman formula to reconstitute their own oral liquid morphine.

Looking at these advances, how can our medical students understand their own country’s needs, so distant from Western standards where health services reach nearly all, when they are trained in hospitals that use Western recommendations? How can international organisations, sometimes staffed by those who have never lived or worked in African countries, understand the local culture and make vital decisions affecting those communities on a daily basis? These are questions that must be considered.

In 1994, Dr. Anne, Rose and a medical student from UK studied pain in HIV and applied the treatment methods tried and proven for cancer to HIV pain management. They were careful to withdraw the pain treatment when the infection was under control. Realizing their success, they began teaching ‘pain management’ to health professionals, undergraduates and postgraduates of University Medical and Hospital Nursing schools.

Rose has now retired after 27 years in palliative care. She and her daughter, Dianah (currently a Nurse Trainer at Hospice Africa Uganda and a Lecturer at Uganda Christian University) are exemplifying the use of a community-based care approach to reach out to patients in their community by riding on the “ubuntu,” a spirit that has been in existence in Uganda for many years. The two have started a Community-Based Organization called Lweza Community Health Program (LCHP) with a vision towards a healthy, informed and productive community. The LCHP was established in April 2020, at a time when the country was under a total lock-down as a result of COVID-19 restrictions, and has registered a number of successes in its short existence. The organization has adopted some ideas from the community-based approach of HAU and tailored them to the needs of its community by strengthening Primary Healthcare to ensure that even the poorest patients can access quality medical healthcare services. This approach is destined to be a model for other areas within Uganda and, possibly, throughout Africa and beyond, but it is still in its early days.

Rose and other LCHP members have mobilized community volunteers and health workers living in the village to attend to the needs of patients. The healthcare providers work with community volunteers who go door-to-door, identifying patients in the community and offering them appropriate support.

During the home visits, the LCHP team has learnt that some patients attribute their illness to witchcraft or traditional or spiritual beliefs and, thus, do not see the benefit of modern medicine. As a result of these beliefs and the costs attached to diagnosis, some patients have not had the opportunity to get a proper diagnosis. The interface with the LCHP health worker is sometimes the first opportunity for them to consult a qualified health worker.

It was also evident that it is not only diseases that affect the health of members in Lweza community. There are a number of elements that determine the well-being of individuals, ranging from poverty, overcrowding, especially where large families are cramped into tiny dwellings without adequate facilities for privacy or protection for teenagers and women, mental illness as a result of substance abuse, child abuse, gender-based violence, which is not surprising in a traditionally male-dominated society, as well as other social problems. With all these issues and many more, where does one start? The solution was to get the backing of the community and work with the local leaders.

In 2021, Dr. Anne attended one of the medical health camps organized by Lweza Community Health Program. The camp was funded primarily by the local community and was hosted in a resident’s compound. It was a three-day camp and thirteen governmental and private health institutions provided Free services to everybody who sought them. Services included screening for; tuberculosis, heart disease, diabetes, HIV, cancers, including breast, cervix and prostate, eye problems, hepatitis B and vaccination. Other services provided were: family planning, blood donation, orthopaedic assessment, nutritional education, general counselling, legal health services, spiritual counselling and palliative care sensitization.

The camp attracted over six hundred people from not only Lweza, but also from neighbouring villages. A number of people were identified with health issues, especially hypertension, which they were not aware of previously. Those who were identified with health issues were advised on where to get health services; the LCHP team continues to guide, encourage and liaise with the health care workers in various health facilities to ensure that they get the necessary services. This has now become an annual activity for LCHP and the number of participants and service providers to the event have gradually increased. The LCHP has been contacted by other communities and has since inception been privileged to support in organizing three other community health camps in other regions of Uganda.

Dr. Anne was privileged to be the only m’sungu (white person) in the 2020 LCHP health camp. She attended the camp with a child survivor of Hospice Africa Uganda, Cathy, who had suffered a terrible tumour that, in spite of being classified histologically as non-malignant, had invaded her spine and, on several occasions, almost cost her life. Furthermore, the steep health care expenses that began to accrue six years prior, when Cathy was diagnosed at 14 years of age, had also cost the family all of their properties and personal income and caused the interruption of education for her two siblings. Despite being extremely disabled, Cathy shared a heart-rending speech in the local language to the attendees, inspiring others with her hope for her own future as well as theirs. She encouraged the attendees to embrace early screening for diseases and she touched many hearts present.

While treating diseases, LCHP members find it paramount to also address the issues that promote ill health in the community as these are major determinants to the success of any intervention. The LCHP performed a needs assessment and the main issues affecting the community identified were found to be: poor sanitation; domestic violence, child abuse/neglect, substance abuse and youth unemployment.

The LCHP, working with the local council leaders of the village, has continued to address these issues. The first undertaking was to keep the surroundings clean in order to prevent diseases that arise from poor sanitation. The community members are mobilized, sensitized and motivated through communal participation and empowered to keep their surroundings clean through bi-weekly community cleaning known locally as Bulungi Bwansi, when every person takes part in cleaning public areas and shares responsibility for maintaining a healthy and clean environment.

Some of the victims of substance abuse were identified and are now undergoing rehabilitation. A few cases of child abuse were identified and referred to legal aid services and other supportive organizations, in addition to ensuring that they are reunited with their parents.

Although there is still so much to be done to realize LCHP’s vision, the results so far are very promising. The good traditional values of the local community such as “ubuntu” are regaining ground and need to be preserved if the community health care approach is to be successful.

Local community health workers must be recognized as the experts for designing culturally appropriate community-based health care programs and should be empowered to own them.

Discussion

Palliative care is gaining ground globally and is endorsed in high-level policy commitments, however service provision, supporting policies, education and funding are incommensurate with rapidly growing needs. Uganda, along with only three other African countries (Kenya, South Africa and Zimbabwe), have reached level 4a, indicating that hospice-palliative care services reached the stage of preliminary integration into mainstream service provision [6]. Hence, Uganda has succeeded in developing a critical mass of palliative care activism in a number of locations; awareness of palliative care on the part of health professionals and local communities; the availability of morphine; the provision of a substantial number of training and education initiatives by a wide range of organizations; and the existence of a national palliative association.

In Uganda, like many developing countries, cultures have expressly prohibited informing patients of their diagnosis and prognosis at all stages of the disease (cancer, HIV) [7]. With time and training, views have been changing to the point where there is now some acceptance of idea that patients should be informed of their real condition and, accordingly, be given the opportunity to take a more active role in decisions related to their treatment planning.

The global COVID-19 pandemic was an eye opener for the increased need for palliative care services and resilient health care systems. The number of lives lost throughout the world to the coronavirus is a harsh reminder of the necessity to ensure that care, especially at the end of life, is a priority for healthcare providers, regardless of their geographical location and health system [8].

Unfortunately, inequities still remain in the provision of palliative care, both among and within countries, especially in the community. The majority of those needing palliative care, worldwide, continue to be those with non-malignant conditions. While traditionally associated with the care of people with cancer, palliative care should be available to all who need it, regardless of their diagnosis, particularly children and the elderly [9,10].

Let us reiterate that compassionate communities play an important role in access to palliative care, wherever they may be, by providing the required care, e.g. volunteer community caregivers and family members. It is especially important to understand and address the particularly gendered nature of caregiving, which falls upon women and girls in communities and families, without recompense, support or equipment, all contributing to ongoing gender inequalities [11].

In our efforts to treat individuals with cancer and to understand how to provide the best possible care to underserved populations, we must first acknowledge cultural diversity. Each culture is comprised of language, religion, social norms, history, tradition and spirituality. By recognizing each patient’s particular spiritual and cultural beliefs, and by utilizing all available approaches, we can help to improve care outcomes and, in turn, patients’ access and adherence to treatment [12].

References

  1. Henderson JD, Boyle A, Herx L, Alexiadis A, Barwich D, et al. (2019) Staffing a Specialist Palliative Care Service, a Team-Based Approach: Expert Consensus White Paper. J Palliat Med. [crossref]
  2. Silbermann M (2021) Preface: In Palliative Care for Chronic Cancer Patients in the Community: Global Approaches and Future Applications (Silbermann, M. ed.) Springer-Nature, Switzerland.
  3. Khan CP, Parver S, Lesch JK, DiGioia K, Gaglio B, et al. (2019) Comparative Clinical Effectiveness Research Focused on Community-Based Delivery of Palliative Care: Overview of the Patient-Centered Outcomes Research Institute’s Funding Initiative. J Palliat Med. [crossref]
  4. Knaul FM, Farmer PE, Krakauer EL, et al. (2018) Alleviating the access abyss in palliative care and pain relief-an imperative of universal health coverage: the Lancet Commission report. The Lancet. [crossref]
  5. Yennurajalingam S, Amos CE, Weru J, Opare-Lokko E, Arthur JA, et al. (2019) Extension for Community Healthcare Outcomes-Palliative Care in Africa Program: Improving Access to Quality Palliative Care. J Global Oncol. [crossref]
  6. Clark D, Centeno C, Clelland D, Garralda E. Lopez-Fidalgo J, et al. (2020) How are palliative care services developing worldwide to address the unmet need for care? In: Global Atlas of Palliative Care (Connor S. ed.) 2nd Edition, London, UK, pp. 45-57.
  7. Brant J, Silbermann M (2021) Global perspectives in palliative care for cancer patients. Not all countries are the same. Current Oncology Reports. [crossref]
  8. Silbermann M, Berger A (2020) The need for a universal language during the COVID-19 pandemic: Lessons learned from the Middle East Cancer Consortium (MECC) Palliative & Supportive Care. [crossref]
  9. Kebudi R, Cakir FB, Büyükkapu SB (2021) Palliative Care in high and low resource countries. Current Pediatric Reviews. [crossref]
  10. Ling J (2020) Forward in: Global Atlas of Palliative Care (Connor S., ed.) 2nd Edition, London, UK. p. 11.
  11. Morris C, Davies H (2020) What is the way forward? In: Global Atlas of Palliative Care (Connor S., ed.) 2nd Edition, London, UK, pp. 92-95.
  12. Silbermann M, Berger A. Preface in: Global Perspectives in Cancer Care: Religion, Spirituality and Cultural Diversity in Health and Healing (Silbermann M and Berger A eds.) Oxford University Press, UK (in press).

Speech-Language Therapy to Improve the Speech- Language Prosody of Clients with Autism

DOI: 10.31038/JCRM.2023624

Abstract

According to the National Autism Association, autism is a bio-neurological developmental disability that generally appears before 3 years of age. This disorder affects normal development of the brain in communication skills and cognitive function. Individuals with autism usually exhibit difficulties in verbal and non-verbal communication, social interactions, as well as leisure and play activities. These individuals may also exhibit other difficulties (e.g., allergies, asthma, sensory integration dysfunction, sleeping disorders, feeding disorders, epilepsy, sleep). “Autism is diagnosed four times more often in boys than girls.” Its prevalence is not affected by race, region, or socioeconomic status.” According to research, mortality risk among individuals with autism is twice as high as the general population, often related to accidents (e.g., drowning, and other accidents). At present, there is no cure for autism, but with early intervention and treatment, the diverse symptoms can be greatly reduced. According to Autism Parenting literature, the individual with autism may struggle with tone, speech rhythm and pitch, and the message or intention related to their words may be misunderstood (11/2/21).

Sentences with Rising Inflections (Raise Pitch at the End of the Sentence)

  1. Do you want to go? 1
  2. Do you like pie? 1
  3. Is your name Michael? 1
  4. Can you ride a bike? 1
  5. Do you have money? 1
  6. Are you going to the movies? 1
  7. Can you help me with math? 1
  8. Did you see that picture? 1
  9. Can you come over tonight? 1
  10. Do you want to go to college? 1

Sentences with Falling Inflections (Lower Pitch at the ends of Sentences)

  1. I’ll never do that again. 2
  2. She really didn’t like the food. 2
  3. I think I’ll go to the movies tonight 2
  4. I’ll never do that again. 2
  5. I’m sorry I bought that. 2
  6. I can’t come to the party next week. 2
  7. We can all enjoy the dessert. 2
  8. I do my exercises every night. 2
  9. The party was not fun. 2
  10. That story is very sad. 2

Below is a Portion of the Poem “A Little Seed” by Mabel Watts

3

Sequencing Pictures to be Described

The couple went into the restaurant.
The waiter brought the menu.
The couple chose their meal.
The waiter brought the food, and the couple ate their meal.
The waiter then brought dessert.
The couple ate the dessert and then left the restaurant.
When the client can produce the content with appropriate prosody, the clinician’s models may be removed, and the client can be presented with verbal material using role playing.

Relate a Short Familiar Story on material which is conducive to pitch variation (e.g., “The Three Bears). For older children, the content can be more mature with material conducive to pitch variation. Once the client is able to use appropriate prosody on content provided by the clinician, the client may relate an experience of his/her own, incorporating the techniques learned. A tape recorder can be used to illustrate to the client positive aspects of his content and to obtain his/her input of what may need improvement.

***A page from the below book was used during therapy with the client and together, the client and the therapist, would gauge where to put the intonations. ***

SOURCE: “President George Washington” by David A. Adler 2005 A Holiday House Book

George Washington was born on February 22, 1732, in a small virginia farmhouse. Virginia was an English Colony, and the People Were Loyal to King George Ii of England. The Washingtons Grew Tobacco, Fruit, And Vegetables on their Farm. Most of the Work Was Done By African-American Slaves. When George Was Seven, He Learned To Read And Write. He Studied Arithmetic Too. It Was His Favorite Subject. He Loved To Fish, Swim, And Hunt. Most Of All He Loved To Ride His Horse. When George Was Eleven, His Father Died, And He Was Now Needed To Help His Mother On The Farm. He Also Helped With His Younger Sister And Brothers. In 1751, George Was 19 Years Old. He Joined The Virginia Army And Became Soldier. The 13 Americ An Colonies Were At War With England Because They Wanted To Be Free Of English Rule. In May 1775, Leaders Of The Colonies Met And Talked About Their Fight With The English. The Leaders Chose Washington To Lead The Fight Against England. Washington’s Army Won Battles In New York And Philadelphia. In 1778, The French Joined The Fight Against England. Washington’s Army Won The Battles In Boston And Trenton. In 1778 M, The French Joined The Fight Against England. There Were Other Battles Too. The Americans And French Beat The English, And The Americans Would Be Free Of English Rule. In September 1783, The Americans And English Agreed To End The Fighting, And A Nation Was Born. The 13 Colonies Became The First 13 States Of The United States of America. President Washington Kept The New Nation At Peace. He Led It For 8 Years Until 1797. Then He Went Home To Virginia. In December 1799, George Washington Became Ill, And He Died That Night. People Everywhere Mourned The Death Of George Washington. It Was Said That George Washington Was “First In War, First In Peace, And First In The Hearts Of His Fellow Citizens.”

Conclusion

The literature on autism confirmed a number of the characteristics displayed by the autistic client about whom the present article was written: The client is an 11-year-old male, diagnosed with autism and recommended for speech therapy, secondary to problems with prosody related to autism: He presented with an excessively slow rate of speech, which incorporated excessive pausing and lack of pitch variation. His articulation, receptive and expressive language were within normal limits for a child of his age. The plan of therapy was to improve his prosody which included appropriate speech rate, speech rhythm, and pitch variation. Therapy began with the use of phrases and then sentences with arrows on words accompanied by clinician’s models in terms of where to raise and lower his pitch appropriately. The clinician first modelled the content for the client and then had him produce the material independently. This article contains the content on which the client practiced, accompanied by indications of where to raise or lower his pitch. Once the client could read these sentences aloud with the appropriate pitch variation, he was given short poems with arrows to depict where to raise or lower his pitch. He was eventually able to read the content without a model and incorporated appropriate pitch variation, speech rate and rhythm, and pausing appropriately. It was reported that he eventually gave a talk at school (one he practiced at home) and was applauded for his presentation. Although the client made a good deal of progress, further practice will continue in terms of using appropriate prosody during spontaneous speech and other speaking activities.