Investigating the therapeutic potential of Allium cepa extract in combating pesticide exposure induced ocular damage.

The ocular surface is subject to a range of potentially hazardous environmental factors and substances, owing to its anatomical location, sensitivity, and physiological makeup. Xenobiotic stress exerted by chronic pesticide exposure on the cornea is primarily responsible for ocular irritation, excessive tear production (hyper-lacrimation), corneal abrasions and decreased visual acuity. Traditional medicine hails the humble onion (Allium cepa) for its multi-faceted properties including but not limited to anti-microbial, antioxidant, anti-inflammatory and wound healing. However, there is a lacuna regarding its impact on the ocular surface. Thereby, the current study investigated whether topical application of crude extract of Allium cepa aided in mitigating pesticide-induced damage to the ocular surface. The deleterious effects of pesticide exposure and their mitigation through the topical application of herbal extract of Allium cepa were analysed initially through in vitro evaluation on cell lines and then on the ocular surface via various in-vivo and ex-vivo techniques. Pathophysiological alterations to the ocular surface that impacted vision were explored through detailed neurophysiological screening with special emphasis on visual acuity wherein it was observed that the murine group treated with topical application of Allium cepa extract had comparable visual capacity to the non-pesticide exposed group. Additionally, SOD2 was utilized as an oxidative stress marker along with the expression of cellular apoptotic markers such as Bcl-xL to analyse the impact of pesticide exposure and subsequent herbal intervention on oxidative stress-induced corneal damage. The impact on the corneal epithelial progenitor cell population (ABCG2 and TERT positive cells) was also flowcytometrically analysed. Therefore, from our observations, it can be postulated that the topical application of Allium cepa extract might serve as an effective strategy to alleviate pesticide exposure related ocular damage.

Environmental pollutant N-N'ethylnitrosourea-induced leukemic NLRP3 inflammasome activation and its amelioration by Eclipta prostrata and its active compound wedelolactone.

Environmental exposure of N-nitroso compounds (NOCs) from various sources like tobacco smoke, pesticides, smoked meat, and rubber manufacturing industries has been an alarming cause of carcinogenesis. Neonatal exposure to the carcinogenic N-N'ethylnitrosourea (ENU), a NOC has been established to cause leukemogenesis. Our world is constantly battling against cancer with consistent investigations of new anti-cancer therapeutics. Plant derived compounds have grasped worldwide attention of researchers for their promising anti-cancer potentials. Eclipta prostrata is one such ayurvedic herb, renowned for its anti-inflammatory properties. Currently, it has been explored in various cancer cell lines to establish its anti-cancer effect, but rarely in in-vivo cancer models. Wedelolactone (WDL), the major coumestan of E. prostrata is recognized as an inhibitor of IKK, a master regulator of the NF-kB inflammatory pathway. As persistent inflammation and activated inflammasome contribute to leukemogenesis, we tried to observe anti-leukemogenic efficacy of E. prostrata and its active compound WDL on the marrow cells of ENU induced experimental leukemic mice. Treatment groups were administered an oral gavage at a dose of 1200 mg/kg and 50 mg/kg b.w of crude extract and WDL respectively for 4 weeks. Various parameters like hemogram, survivability, cytological and histological investigations, migration assay, cell culture, flowcytometry and confocal microscopy were taken into consideration pre- and post-treatment. Interestingly, the plant concoction portrayed maximum effects in comparison to WDL alone. The study suggests E. prostrata and WDL as vital complementary adjuncts for anti-inflammasome mechanism in ENU-induced leukemia.

Environmental pollutant ENU induced leukemic NF-kB signaling amelioration by Eclipta alba in murine model.

Exposure to N-nitroso compounds (NOCs) in our environment via pesticides, tobacco, and smoked meat can be potentially carcinogenic. The induction of N-N' ethylnitrosourea (ENU), a genotoxic NOC, leads to leukemogenesis. The study aimed to explore the ameliorating effect of the Ayurvedic herb Eclipta alba on the bone marrow cells of ENU-induced leukemic mice. Eclipta alba is investigated for its anti-cancer effect on various cell lines, but never on haematological malignant models. Theefficacy of the extract was explored on leukemia by changes in body weight, survivability, peripheral blood hemogram, bone marrow cytological, histological, and cell culture studies pre-and post-treatment. The treated group revealed significant immunomodulation of the expressional profile of NF-kB family and IL-1ß in marrow cells, by flow-cytometry, and immunofluorescence study. Through our experimental endeavour we depicted the cellular mechanism, signaling modality and tried to establish the anti-cancer potency of Eclipta alba on ENU-induced leukemia.

The functional interplay of transcription factors and cell adhesion molecules in experimental myelodysplasia including hematopoietic stem progenitor compartment.

Myelodysplastic syndrome is a heterogenous group of disorder with clonal dysregulated hematopoiesis characterized by bone marrow failure, cytogenetic and molecular abnormalities and variable risk of progression to acute myeloid leukemia (AML). The bone marrow niche plays a major role in maintaining the homeostasis and is often injured by the chemotherapeutic drugs leading to catastrophic consequences like myelodysplastic syndrome. In the present study, we made an attempt to find out the osteoblastic niche related alterations in the myelodysplastic bone marrow through mainly flowcytometric and fluorescent microscopic studies. We have also checked the condition of the myelodysplastic bone through micro computed tomography. The results revealed that the affected osteoblasts of the myelodysplastic bone marrow compelled the hematopoietic stem cell to come out of quiescence and become actively proliferating, and in this scenario the decline in expression of cell adhesion molecules like N-Cadherin, Intercellular adhesion molecule 1 (ICAM) and upregulated focal adhesion kinase (FAK) played a major role. The hike in number of osteoclasts in myelodysplastic cases than control also shattered the balance between bone formation and resorption ratio. We have recorded a dysregulated expression of transcription factors GATA2 and CEBPα (CCAAT-enhancer-binding-protein) in the hematopoietic stem progenitor compartment of the myelodysplastic bone marrow, the main reason behind the presence of abnormal myeloblasts in myelodysplastic cases. Collectively, we can say the coordinated perturbations in the osteoblastic niche, cell adhesion molecules together with the transcription factors has resulted in the uncontrolled proliferation of hematopoietic stem cell, dysregulated myelopoiesis, early trafficking of hematopoietic progenitors to blood compartment and at the same time pancytopenic peripheral blood conditions during the progression of N-Ethyl N Nitroso Urea (ENU) induced myelodysplasia.

Quercetin induces autophagy in myelodysplastic bone marrow including hematopoietic stem/progenitor compartment.

Myelodysplastic syndrome (MDS) is regarded as a spectrum of bone marrow failure disorders that share hemato-pathological state of cellular dysplasia and cytopenia. The modern treatment of cancers like chemotherapy and radiation therapy sometimes severely pounce on the basic hematopoietic stem/progenitor cellular (HSPC) compartment which gradually disclose the clinical symptoms of MDS. The present study involves flowcytometric protein expression analysis of insulin growth factor receptor (IGFR), PI3K-Akt-mTOR pathway, the autophagy related proteins (ATG's), the status of antioxidative molecules SOD2 and SDF1 and apoptosis profiling in ethyl-nitroso-urea induced myelodysplasia. The redox status that is, reactive oxygen species was estimated with dihydroetidium and the status of mitochondria and lysosomes were checked by Janus green B and neutral red staining respectively, pre and post quercetin treatment in MDS bone marrow. The results revealed the activated IGFR/PI3K/Akt axis in MDS bone marrow but unconventionally both p-mTOR and autophagy (p-ATG1, p-AT6, ATG7, ATG12) was downregulated. Interestingly, post quercetin treatment an upregulation of basal autophagocytosis, reversal of oxidative damage and proper functionality of mitochondria and lysosome was recorded. Taken together, the study hinted that the PI3K-Akt-mTOR pathway does not rule over the process of autophagocytosis in HSPC's of MDS bone marrow and the isoflavanoid quercetin remarkably restored autophagocytosis and hematopoietic oxidative status toward normalcy during the progression of myelodysplasia.

Morphogen signaling by Wnt/ß-catenin pathway and microenvironmental alteration in the bone marrow of agricultural pesticide exposure-induced experimental aplastic anemia.

The etiologic link between pesticide toxicity and aplastic anemia in agricultural and agro-industrial setting has been frequently reported in epidemiological studies conducted worldwide. Chronic pesticide toxicity causes long-term bone marrow injury and perturbs the normal hematopoietic physiology, including survival of hematopoietic progenitor cells and bone marrow's blood cell forming ability. The purpose of this study is to understand the mechanism of pesticide toxicity-mediated bone marrow aplasia by studying Wnt/ß-catenin signaling pathway and microenvironmental stromal components. An agricultural pesticide formulation comprising of cypermethrin, chlorpyriphos, and hexaconazole was used to induce bone marrow aplasia in inbred Swiss albino mice. Marrow failure followed by the onset of aplastic condition was confirmed by pancytopenic peripheral blood and hypocellular bone marrow filled with adipocytes. Significant downregulation of canonical Wnt/ß-catenin signaling was identified by expression analysis of Wnt3a, ß-catenin, and telomerase reverse transcriptase in the aplastic bone marrow hematopoietic stem/progenitor compartment. Along with signaling deregulation, disruption in both the osteoblastic and vascular stromal components was observed in the pesticide-exposed bone marrow microenvironment when compared to control. In this study, we tried to establish the correlation among disease pathophysiology, signaling deregulation in the hematopoietic cells, and bone marrow microenvironmental alteration during environmental exposure-mediated aplastic hematopoietic catastrophe, which may shed light on the unexplored mechanistic perspective of this fatal blood disease.

Ocular surface and chronic pesticide exposure: Evaluating the alterations in corneal cellular turnover concerning cell cycle and apoptosis.

The consequences of chronic pesticide exposure on the ocular surface are not yet fully known and lacunae exist regarding the repercussions of this xenobiotic insult on cellular turnover. The present work aims to establish the mechanistic relationship between ocular morbidity and chronic pesticide exposure by analyzing the impact on key regulators responsible for cell cycle and death. Vital components of cell cycle and death were primarily explored in this study by mimicking the on-field scenario regarding chronic pesticide exposure in a murine model. Various cellular aspects were taken into consideration through culture analyses, flowcytometric evaluation, fluorescence microscopic studies etc. We observed downregulation of key players of the cell-cycle at different stages (viz. Cyclin-D1, CDK4, pRb, PCNA, PP1, PP2A, p-cdc 25c and Aurora kinase A) with a corresponding increase in the expression of cell-cycle inhibitors like p18 and p21, which lead to hypoproliferation of corneal epithelial cells post pesticide exposure. The expression of GSK 3ß, a master-molecule involved in both cell cycle and apoptotic pathways corresponded well with the scientific theme and indicated towards cellular hypoproliferation and increase of apoptosis. Key players of both the intrinsic (viz. Bax/Bcl2, JNK) and extrinsic (viz. CD 95) apoptotic pathways were found to be activated leading to enhanced cleaved Caspase 3 expression and corresponding cell death. We tried to highlight the mechanistic correlation between the alterations in cellular turnover as the reason behind the heightened ocular morbidity due to 'chronic pesticide exposure'- the xenobiotic stress exerted by these 'farmers' friends'.

Chronic pesticide exposure and consequential keratectasia & corneal neovascularisation.

Ocular toxicity as a consequence of chronic pesticide exposure is one of the health hazards caused due to extended exposure to pesticides. The cornea, due to its position as the outer ocular layer and its role in protecting the internal layers of the eye; is gravely affected by this xenobiotic insult to the eye, leading to ocular irritation and damage to normal vision. The deleterious effects of chronic pesticide exposure on the various corneal layers and the ocular risks involved therein, were explored by mimicking the on-field scenario. Cytological, histological and flowcytometric parameters were taken into consideration to determine the enhanced risk of corneal neovascularisation and keratectasia, specifically, keratoconus. Chronic exposure to pesticides leads to heightened ocular morbidity wherein there were visible pathophysiological changes to the ocular surface. The cornea was found to be adversely affected with visible protuberance in a cone-like shape, characteristic of keratoconus in a majority of the experimental animals. Further analyses revealed a detrimental impact on all the corneal layers and an amplified expression of inflammation markers such as TNF-α, VCAM-1 and ICAM-1. Additionally, it was found that post pesticide exposure, the corneal surface developed hypoxia, leading to a significant increase of angiogenesis promoting factors and consequential neovascularisation. Apart from ocular toxicity, chronic exposure to pesticides significantly increases the risks of keratectasia and corneal neovascularisation; disorders which lead to diminished vision and if untreated, blindness.

Differential expression of mitotic regulators and tumor microenvironment influences the regional growth pattern of solid sarcoma along the cranio-caudal axis.

Soft tissue sarcomas are relatively rare, unusual, anatomically diverse group of malignancies. According to the recent literature and medical bulletins, tumor growth and aggressiveness immensely relies on its anatomical locations. However, it is unclear whether the cranio-caudal anatomical axis of the mammalian body can influence sarcoma development and the underlying molecular mechanisms are not yet deciphered. Here, we investigated the growth pattern of solid sarcoma implanted into the murine cranial and caudal anatomical locations and tried to explore the location specific expression pattern of crucial mammalian mitotic regulators such as Aurora kinase A, Histone H3 and c-Myc in the cranio-caudally originated solid tumors. In addition, the influence of local tumor microenvironment on regional sarcoma growth was also taken into consideration. We found that solid sarcoma developed differentially when implanted into two different anatomical locations and most notably, enhanced tumor growth was observed in case of cranially implanted sarcoma than the caudal sarcoma. Interestingly, Aurora kinase A and c-Myc expression and histone H3 phosphorylation level were comparatively higher in the cranial tumor than the caudal. In addition, variation of tumor stroma in a location specific manner also facilitated tumor growth. Cranial sarcoma microenvironment was well vascularized than the caudal one and consequently, a significantly higher microvessel density count was observed which was parallel with low hypoxic response with sign of local tumor inflammation in this region. Taken together, our findings suggest that differential gradient of mitotic regulators together with varied angiogenic response and local tumor microenvironment largely controls solid sarcoma growth along the cranio-caudal anatomical axis.

Deregulation of vital mitotic kinase-phosphatase signaling in hematopoietic stem/progenitor compartment leads to cellular catastrophe in experimental aplastic anemia.

Aplastic anemia, the paradigm of bone marrow failure, is characterized by pancytopenic peripheral blood and hypoplastic bone marrow. Among various etiologies, inappropriate use of DNA alkylating drugs like cyclophosphamide and busulfan often causes the manifestation of the dreadful disease. Cell cycle impairment in marrow hematopoietic stem/progenitor compartment together with cellular apoptosis has been recognized as culpable factors behind aplastic pathophysiologies. However, the intricate molecular mechanisms remain unrevealed till date. In the present study, we have dealt with the mechanistic intervention of the disease by peripheral blood hemogram, bone marrow histopathology, cytopathology, hematopoietic kinetic study, scanning electron microscopy, DNA damage assessment and flowcytometric analysis of cellular proliferation and apoptosis in hematopoietic stem/progenitor cell (HSPC) rich marrow compartment using busulfan and cyclophosphamidemediated mouse model. To unveil the molecular mechanisms behind aplastic pathophysiology, we further investigated the role of some crucial mitotic and apoptotic regulators like Protein kinase-B (PKB), Gsk-3ß, Cyclin-D1, PP2A, Cdc25c, Plk-1, Aurora kinase-A, Chk-1 regarding the hematopoietic catastrophe. Our observations revealed that the alteration of PKB-GSK-3ß axis, Plk-1, and Aurora kinase-A expressions in HSPC compartment due to DNA damage response was associated with the proliferative impairment and apoptosis during aplastic anemia. The study established the correlation between the accumulation of DNA damage and alteration of the mentioned molecules in aplastic HSPCs that lead to the hematopoietic catastrophe. We anticipate that our findings will be beneficial for developing better therapeutic strategies for the dreadful disease concerned.

Effect of chronic pesticide exposure on murine cornea: a histopathological, cytological and flow cytometric approach to study ocular damage by xenobiotics.

Pesticide exposure can occur directly or indirectly in an occupational setting or otherwise. The health hazards of pesticides have long been studied; however, little is known about the ocular insult of these potent chemicals. In this study, we examined the consequences of long-term pesticide exposure on the ocular tissue in animal model with special focus on the cornea. Swiss Albino mice were sacrificed to obtain the eye globes and various cytological, cytotoxic and histological evaluations, in vitro growth kinetic studies and flow cytometric analyses of select cytokeratins were performed to determine the structural and functional damage due to pesticide exposure. Our study revealed the detrimental impact of this xenobiotic insult by cataloguing the damage to each layer of the cornea wherein it was discovered that all the functional layers as well as the membranes were compromised. We hope that our investigation will pave the way for future studies in this oft overlooked area of affront caused by pesticide exposure to the ocular surface.

Noncanonical Wnt5a-Ca(2+) -NFAT signaling axis in pesticide induced bone marrow aplasia mouse model: A study to explore the novel mechanism of pesticide toxicity.

According to case-control studies, long-term pesticide exposure can cause bone marrow aplasia like hematopoietic degenerative disease leading to impaired hematopoiesis and increased risk of aplastic anemia in human subjects. However, the exact mechanism of pesticide mediated hematotoxicity still remains elusive. In this study, we investigated the role of noncanonical Wnt signaling pathway, a crucial regulator of adult hematopoiesis, in pesticide induced bone marrow aplasia mouse model. Aplasia mouse model was developed following inhalation and dermal exposure of 5% aqueous mixture of common agriculturally used pesticides for 6 h/day for 5 days a week up to 90 days. After that, blood hemogram, marrow smear, cellularity, scanning electron microscopy, extramedullary hematopoiesis and flowcytometric expression analysis of noncanonical Wnt signaling components, such as Wnt 5a, fzd5, NFAT, IFN-γ, intracellular Ca(2+) level were evaluated in the bone marrow hematopoietic stem/progenitor compartment of the control and pesticide induced aplasia groups of animals. Results showed that pesticide exposed mice were anemic with peripheral blood pancytopenia, hypocellular degenerative marrow, and extramedullary hematopoiesis in the spleen. Upon pesticide exposure, Wnt 5a expression was severely downregulated with a decline in intracellular Ca(2+) level. Moreover, downstream of Wnt5a, we observed sharp downregulation of NFATc2 transcription factor expression, the major target of pesticide toxicity and its target molecule IFN-γ. Taken together, our result suggests that deregulation of Wnt5a-Ca(2+) -NFAT signaling axis in the hematopoietic stem/progenitor compartment plays a crucial role behind the pathogenesis of pesticide mediated bone marrow aplasia by limiting primitive hematopoietic stem cells' ability to maintain hematopoietic homeostasis and reconstitution mechanism in vivo during xenobiotic stress leading to ineffective hematopoiesis and evolution of bone marrow aplasia. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1163-1175, 2016.

Alteration of hedgehog signaling by chronic exposure to different pesticide formulations and unveiling the regenerative potential of recombinant sonic hedgehog in mouse model of bone marrow aplasia.

Chronic pesticide exposure-induced downregulation of hedgehog signaling and its subsequent degenerative effects on the mammalian hematopoietic system have not been investigated yet. However a number of concurrent studies have pointed out the positive correlation between chronic pesticide exposure induced bone marrow failure and immune suppression. Here, we have given an emphasis on the recapitulation of human marrow aplasia like condition in mice by chronic mixed pesticide exposures and simultaneously unravel the role of individual pesticides in the said event. Unlike the effect of mixed pesticide, individual pesticides differentially alter the hedgehog signaling in the bone marrow primitive hematopoietic compartment (Sca1 + compartment) and stromal compartment. Individually, hexaconazole disrupted hematopoietic as well as stromal hedgehog signaling activation through inhibiting SMO and facilitating PKC δ expression. On contrary, both chlorpyriphos and cypermethrin increased the sequestration and degradation of GLI1 by upregulating SU(FU) and ßTrCP, respectively. However, cypermethrin-mediated inhibition of hedgehog signaling has partly shown to be circumvented by non-canonical activation of GLI1. Finally, we have tested the regenerative response of sonic hedgehog and shown that in vitro supplemented recombinant SHH protein augmented clonogenic stromal progenitors (CFU-F) as well as primitive multipotent hematopoietic clones including CFU-GEMM and CFU-GM of mixed pesticide-induced aplastic marrow. It is an indication of the marrow regeneration. Finally, our findings provide a gripping evidence that downregulated hedgehog signaling contribute to pesticide-mediated bone marrow aplasia but it could be recovered by proper supplementation of recombinant SHH along with hematopoietic base cocktail. Furthermore, SU(FU) and GLI1 can be exploited as future theradiagnostic markers for early marrow aplasia diagnosis.

Limbal epithelial stem-microenvironmental alteration leads to pterygium development.

Maintenance of tissue homeostasis relies on the accurate regulation of tissue specific stem cell activity which is governed by the dynamic interaction between the positive and negative feedback modulating mechanism of stem cell microenvironmental niche. Alteration or deregulation of the "stem-microenvironmental networking" provokes disease development. Limbal epithelial stem cells (LESC) are the initiator hierarchy that maintains corneal integrity. Compartmentalization of LESC within the limbal vicinity provides an opportunity to understand the stem-microenvironmental relationship. The purpose of this study was to determine the microenvironmental alteration associated with LESCs fate in pterygium condition in comparison with healthy state. Clinical observations evaluated the ocular surface disorder with respect to corneal vascularization, tear film abnormality, and thickening of limbal area in pterygium patients. Structural alteration of limbal stem/progenitor cells and its neighboring niche components were observed using histology and scanning electron microscopy. Receptor overexpression of TGFß-R1, EGF-R1, and IL6-Rα and alteration of IL2-Rα expression pointed toward aberration of "stem-microenvironmental networking" in the limbal vicinity during disease development. Increased cell proliferation index along with TERT, Cyclin-D1, and PCNA over-expression in limbal part of pterygium epithelial cells indicated increased cellular proliferation and disturbed homeostatic equilibrium. We postulate that pterygium is associated with limbal microenvironmental anomaly where the resident epithelial cells became hyperproliferative.

Pesticide induced alterations in marrow physiology and depletion of stem and stromal progenitor population: an experimental model to study the toxic effects of pesticide.

Long-term exposure of agriculturally used organochloride and organophosphate pesticides have been shown to cause long-lasting hematotoxicity and increased incidence of aplastic anemia in humans. The mechanisms involved in pesticide induced hematotoxicity and the features of toxicity that may play a major role in bone marrow suppression are not known. The aim of the present study was to investigate the hematological consequences of pesticide exposure in swiss albino mice exposed to aqueous mixture of common agriculturally used pesticides for 6 h/day, 5 days/week for 13 weeks. After the end of last exposure, without a recovery period, the strong hematotoxic effect of pesticide was assessed in mice with long-term bone marrow explant culture (LTBMC-Ex) system and cell colony forming assays. Bone marrow explant culture from the pesticide exposed group of mice failed to generate a supportive stromal matrix and did not produce adequate number of hematopoietic cells and found to contain largely the adipogenic precursors. The decreased cell colony numbers in the pesticide exposed group indicated defective maturational and functional status of different marrow cell lineages. As a whole, exposure of mice to the mixture of pesticides reduced the total number of bone marrow cells (granulocytes are the major targets of pesticide toxicity), hematopoietic, and non-hematopoietic progenitor cells and most of the hematological parameters. Replication of primitive stem/progenitor cells in the marrow was decreased following pesticide exposure with G0/G1-phase arrest of most of the cells. The progenitor cells showed decreased percentage of cells in S/G2/M-phase. The increased apoptosis profile of the marrow progenitors (Increased CD95 expression) and primitive stem cells (High Annexin-V positivity on Sca1+ cells) with an elevated intracellular cleaved caspase-3 level on the Sca1+ bone marrow cells provided the base necessary for explaining the deranged bone marrow microenvironmental structure which was evident from scanning electron micrographs. These results clearly indicate a strong, long lasting toxic effect of pesticides on the bone marrow microenvironment and different microenvironmental components which ultimately leads to the formation of a degenerative disease like aplastic anemia.

Unveiling the paradoxical nature of myelodysplastic syndromes (MDS): why hypercellular marrow strongly favors accelerated apoptosis.

The pathogenesis of bone marrow failure in myelodysplastic syndromes (MDS) is an unresolved mystery. MDS causes peripheral blood cytopenias and increased bone marrow cellularity. This apparent paradox has been interpreted as a sign of intramedullary destruction of a substantial portion of the developing hematopoietic cells by apoptosis. The present study aimed to delineate the exact mechanistic relationship between the bone marrow hypercellularity and the accelerated apoptosis in an N-ethyl-N-nitrosourea (ENU)-induced experimental MDS mouse model. The observations made so far clarify the quantitative and qualitative changes that occur in the bone marrow microenvironment through cell cycle analysis, especially involving the telomerase reverse transcriptase (TERT) and p53 expression patterns. The survival fate of the bone marrow cells were observed by measuring the expression level of some intracellular protein molecules like apoptosis signal-regulating kinase 1 (ASK-1), c-Jun N-terminal kinase (JNK), and cleaved caspase-3 of the extrinsic pathway toward apoptosis. We found myelodysplasia damage occurs within one or more multipotent progenitor populations resulting in uncontrolled cellular proliferation within the MDS bone marrow. Then, due to homeostatic balance, this high cellular burden is minimized by activating the apoptosis pathway. As a result, the peripheral blood suffers cellular deprivation. This study can throw some light on the mechanism of disease progression and also help to reveal the paradoxical nature of the disease.

Phenotypic alteration of limbal niche-associated limbal epithelial stem cell deficiency by ultraviolet-B exposure-induced phototoxicity in mice.

Good vision requires a healthy cornea, and a healthy cornea needs healthy stem cells. Limbal epithelial stem cells (LESCs) are a traditional source of corneal epithelial cells and are recruited for the continuous production of epithelium without seizing throughout an animal's life, which maintains corneal transparency. Like the maintenance of other adult somatic stem cells, the maintenance of LESCs depends on the specific microenvironmental niche in which they reside. The purpose of this study was to determine the microenvironmental damage associated with LESCs fate due to ultraviolet (UV)-B exposure in a mouse model. Structural alteration and deregulation of the stem cell and its neighboring niche components were observed by using clinical, morphological, explant culture study, and flowcytometric analysis, which demonstrated that the limbal microenvironment plays an important role in cornea-related disease development. In UV-exposed mice, overexpression of vascular endothelial growth factor receptor 2 indicated neovascularization, decreased CD38 expression signified the alteration of limbal epithelial superficial cells, and the loss of limbal stem cell marker p63 indicated limbal stem cell deficiency in the limbal vicinity. We concluded that LESC deficiency diseases (LESCDDs) are associated with pathophysiological changes in the LESC niche, with some inhibitory interception such as UV-B irradiation, which results in corneal defects.

Differential remodeling of cadherins and intermediate cytoskeletal filaments influence microenvironment of solid and ascitic sarcoma.

Different forms of sarcoma (solid or ascitic) often pose a critical medical situation for pediatric or adolescent group of patients. To date, predisposed genetic anomalies and related changes in protein expression are thought to be responsible for sarcoma development. However, in spite of genetic abnormality, role of tumor microenvironment is also indispensable for the evolving neoplasm. In our present study, we characterized the deferentially remodeled microenvironment in solid and ascitic tumors by sequential immunohistochemistry and flowcytometric analysis of E-cdaherin, N-cadherin, vimentin, and cytokeratin along with angiogenesis and metastasis. In addition, we considered flowcytometric apoptosis and CD133 positive cancer stem cell analysis. Comparative hemogram was also considered as a part. Our investigation revealed that both types of tumor promoted neovascularization over time with sign of local inflammation. Invasion of neighboring skeletal muscle by solid sarcoma was more frequent than its ascitic counterpart. In contrary, rapid and earlier cadherin switching (E-cadherin to N-cadherin) in ascitic sarcoma made them more aggressive than that of solid sarcoma and helped to early metastasize distant tissue like liver through the hematogenous route. Differential cadherin switching and infidelity of cytokeratin expression in Vimentin positive sarcoma also influenced the behavior of ascitic CD133+ cancer initiating cell pool with respect to CD133+ cells housed in solid sarcoma. Therefore our study concludes that differential cadherin switching program and infidelity of intermediate filaments in part, sharply discriminate the severity and metastatic potentiality of either type of sarcoma accompanying with CD133+ cellular repertoire. Besides, tumor phenotype-based dichotomous cadherin switching program could be exploited as a future drug target to manage decompensated malignant ascitic and solid sarcoma.

Cord and peripheral blood erythrocyte analysis by scanning electron microscopy and flow cytometry.

INTRODUCTION: Human umbilical cord blood is rich in hematopoietic cells. We aimed to focus on the morphological, biochemical, membrane protein profile and surface protein expression differences of erythrocytes, isolated from cord and adult peripheral blood using techniques such as high-resolution scanning electron microscopy (SEM), gel electrophoresis (SDS-PAGE) and flow cytometry. METHODS: Adult peripheral blood was collected from consenting adults, and umbilical cord blood was procured from consenting mothers, post-delivery at Medical College, Kolkata. We emphasized on cord and adult peripheral blood erythrocytes' morphological variations using SEM images and protein expression by flow cytometric analysis. Some conventional biochemical analyses such as osmotic fragility of the cell membrane, haemoglobin co-oxidation study and lipid peroxidation assay were done for supporting evidence along with membrane protein content using gel electrophoresis. RESULTS: Our SEM images indicated clear morphological variations in cord erythrocyte with a higher degree of cellular deformities and difference in membrane texture. Flow cytometric analysis of cord erythrocyte showed a significant difference in CD235a expression than adults. We observed an overexpression of GLUT1 and decreased expression of Band 3 in cord erythrocyte membrane. Our results also showed cord erythrocytes have low osmotic fragility, a slower rate of co-oxidation of cord haemoglobin and a lesser lipid peroxidation level than that of adults. CONCLUSION: Cord blood erythrocytes have deeper indentations leading to higher flexibility, more oxygen-carrying capacity and less osmotic fragility in comparison with adult erythrocytes. The expression of CD235a and Band 4.5 (GLUT 1) was significantly higher in cord erythrocytes than peripheral adult erythrocytes.

Chronic pesticide exposure induced aberrant Notch signalling along the visual pathway in a murine model.

Pesticides aid in crop-protection against pests and increase yield. However, the xenobiotic stress exerted by pesticides leads to the deterioration of human and animal health. There is a lacuna in our knowledge about their impact on the ocular surface The present work sheds light on this gap by analysing the deterioration of visual acuity as a consequence of pesticide induced xenobiotic stress and Notch pathway dysregulation. Alteration in the expression of vital components of the notch signalling was analyzed along the visual pathway with special focus on its two terminals-the cornea and the visual cortex, by mimicking the on-field scenario regarding chronic pesticide exposure in experimental murine model (Swiss albino mice; Mus musculus). Various aspects were taken into consideration through visual acuity tests, histological evaluations, culture analyses, wound healing assays, flowcytometric evaluation, fluorescence microscopic studies etc. Complete dysregulation of key players of the Notch signalling pathway was observed in both: cells of the ocular surface as well as those in the murine visual cortex post pesticide exposure, indicating activities relating to cell proliferation, differentiation and wound healing in the pesticide exposed samples. Ultra-microscopic analyses corroborated our findings by revealing the loss of fine neural processes in the visual cortex of the pesticide exposed murine samples, thereby hinting at delayed perception to visual stimuli. In vivo evaluations of the functional capacity of the neuroanatomical structures along the visual pathway also confirmed that pesticide exposure leads to severe damage along the various parts of the visual pathway, right from the ocular surface to the visual cortex.