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.

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.

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.

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.

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.

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.

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.

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.

Myelodysplastic Syndrome related alterations of MAPK signaling in the bone marrow of experimental mice including stem/progenitor compartment.

Myelodysplastic syndrome is considered globally as heterogenous group of neoplasm which often proclaims leukemic progression. The heterogeneity is reflected not only in clinical manifestations of the disease but also in salient causes of disease development. In spite of multiple therapeutic modalities, shortfall towards treatment of this disorder still persists. The focal point of tussle suggested toward defects, which are not confined to any unifying cellular signalling. The pathobiology of the disease often experiences an intriguing paradox involving 'hyperproliferative bone marrow with pancytopenic peripheral blood'. In our present study we have reported about MAPK signaling in the hematopoietic stem progenitor compartmental (HSPC) dysregulation during the course of alkylator(ENU) induced myelodysplasia. The phospho-protein status of RTK's(FLT3, PDGFR, EGFR) were markedly increased that activated MAPK signaling proteins which finally executed their tasks by transcription of c-Myc and Rb leading to uncontrolled cellular proliferation, simultaneously the activated c-Jun revealed stress related apoptosis. Altogether, the role of activated MAPK signaling in the HSPC's may have led to hyperproliferation and concurrent enhanced apoptosis of abnormal cells which gradually headed towards premalignant transformations during the course of disease. The phenotypic expression of the HSPC markers CD 150 and CD 90 also established a mechanistic correlation with MAPK signalling alterations and overall scenario.

Genomic insult oriented mitochondrial instability and proliferative hindrance in the bone marrow of aplastic mice including stem/progenitor population.

Aplastic anemia is the bone marrow failure condition characterized by the development of hypocellularity in both marrow and peripheral blood compartments. Anti-tumor chemotherapeutic agents often exert secondary effect on hematopoietic system leading to aplastic anemia by marrow failure. The precise mechanisms behind the marrow ablative effects of the drugs remain yet to be established. The present study holds a mechanistic approach to unveil the mystery. Aplastic anemia was generated in mice with the administration of busulfan and cyclophosphamide followed by the characterization of the disease with peripheral blood hemogram, histopathological and cytochemical examinations of bone marrow. To gain deep knowledge about the molecular mechanisms of the hematopoietic disruption, cytotoxicity assay, DNA damage measurement, apoptosis study, replicative senescence analysis, redox balance study, mitochondrial membrane potential change assessment, flowcytometric expressional analysis of p21, p53, ATM, Chk-2, Necdin, Gfi-1, c-myc, KU-80 and Sod-2 were done with marrow hematopoietic stem/ progenitor cells (HSPCs). Severe blood pancytopenia and marrow hypocellularity was found in aplastic mice. Proliferative hindrance and apoptosis of marrow cells were identified as the cause behind the hematopoietic catastrophe. The genotoxic effects of the drugs triggered chromatin damage and induced replicative senescence in aplastic HSPCs by upregulating p21 in a p53 independent manner. Moreover, accumulation of genomic insults also caused apoptotic elimination of marrow cells due to disruption of mitochondrial membrane potential by generating redox imbalance. The study established the underlying mechanisms behind hematopoietic disruption during drug induced marrow aplasia. Outcome of the study may be helpful in successful designing of therapeutic strategies for the disease concerned.

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'.

Aberrant Wnt Signaling Pathway in the Hematopoietic Stem/Progenitor Compartment in Experimental Leukemic Animal.

The evolutionarily conserved Wnt signaling pathway regulates physiological hematopoiesis, a process of formation of blood cells and has been shown to play crucial role in the development of both myeloid and lymphoid malignancies. The Wnt signaling pathway can be broadly divided into canonical and non-canonical pathways. In the present study, we investigated the pathobiology of leukemia by studying the expression profile of Wnt proteins, receptors, key signaling intermediates and endogenous Wnt antagonist involved in canonical and non-canonical pathways in the bone marrow (BM) hematopoietic stem/progenitor cell (HSPC) compartment of experimental leukemic mice. Cell adhesion molecule N-Cadherin and leukemic BM microenvironment with reference to Wnt were also studied. We used ENU, a potent carcinogen, to induce leukemia in wild type Swiss albino mice and malignant transformation was cofirmed by peripheral blood and BM studies. Flow cytometric expression analysis revealed profound up-regulation of canonical Wnt3a/ß-catenin/CyclinD1 signaling axis along with N-Cadherin whereas down-regulation of non-canonical Wnt5a/Ca2+/CaMKII signaling axis in the leukemic HSPC compartment. Subsequent use of anti-Wnt3a antibody in the in vitro clonogenicity assay uncovered that anti-Wnt3a antibody preferentially inhibited the growth and number of the primitive leukemic hematopoietic CFU-GEMM and BFU-E colonies. Stromal cells derived from the leukemic BM also exhibited aberrant Wnt3a and Wnt5a protein expression. Taken together, alteration of canonical and non-canonical Wnt signaling pathways in the HSPC compartment along with classical Wnt protein expression pattern in the leukemic stromal microenvironment resulted in progression of leukemia.

Role of tumor microenvironment in cancer stem cell chemoresistance and recurrence.

Chemoresistance to the anticancer therapy is the main challenge for the recurrence of cancer and it is responsible for treatment failure and unfavorable clinical outcome. Understanding the mechanisms of chemoresistance in cancer would help to predict disease progression, develop new therapies and personalize systemic therapy. In the last few decades, cancer stem cells (CSC) have proven to play a key role in tumor initiation and may also act as a key factor for chemoresistance and recurrence of the disease following chemotherapy. Resistance to anticancer therapy in patients has been attributed towards a number of factors controlling the stemness character of the cancer stem cells which leads to therapeutic resistance. Accumulating evidences show that the bone marrow (BM) niche is critical to the maintenance and retention of cancer stem cells. Undeniably, stromal cells within the BM niche provide a sanctuary where different signaling pathways and cell-cell interaction gives a protective nature to the diseased condition, and thereby evade chemotherapy-induced death. In this review we have tried to highlight the factors which have important role in chemoresistance and are associated with bone marrow microenvironment. We have also discussed about the recent progresses regarding CSC-targeted therapies to throw light toward developing more effective therapeutic strategies to eradicate cancer in the patients concerned.

Therapeutic management of peritoneal ascitic sarcomatosis by Ruta graveolens: A study in experimental mice.

RELEVANCE: Malignant peritoneal sarcomatosis related ascitic formation often leads to grave consequences but the therapeutic management of the fatal pathophysiological condition remains a rarely discussed issue. The present study investigates the anti-neoplastic activity of the plant alkaloid from Ruta graveolens on ascitic Sarcoma-180 bearing mice as a model of human malignant peritoneal ascites. MATERIALS AND METHODS: The efficacy of the loco-regional administration of Ruta graveolens on tumour cells was explored with cytopathological and cytotoxicological studies, along with the expressional modulation vital regulatory molecules viz. Chk2, c-Myc, CD95 and Aurora kinase. RESULTS: The study revealed a series of anti-neoplastic events exerted by Ruta graveolens that included the boosting of anti-tumour immunity, generation of tumour cell cytotoxicity and disruption of cellular energetics which lead to the induction of apoptosis and simultaneous impairment of cell division in tumour cells. Expressional decline of c-Myc oncoproteins and mitosis promoter Aurora kinase A together with up regulation of vital tumour suppressor Chk-2 and apoptosis inducer CD 95 in ascitic tumour cells was also found to be associated with Ruta administration. CONCLUSION: Our observations revealed that loco-regional Ruta administration resulted in the anti-neoplastic effect on peritoneal sarcoma related ascites and the alteration of vital regulatory molecules which depicted the therapeutic utility of Ruta in the management of peritoneal malignant ascites.

Epigenetic and microenvironmental alterations in bone marrow associated with ROS in experimental aplastic anemia.

Aplastic anemia or bone marrow failure often develops as an effect of chemotherapeutic drug application for the treatment of various pathophysiological conditions including cancer. The long-term bone marrow injury affects the basic hematopoietic population including hematopoietic stem/progenitor cells (HSPCs). The present study aimed in unearthing the underlying mechanisms of chemotherapeutics mediated bone marrow aplasia with special focus on altered redox status and associated effects on hematopoietic microenvironment and epigenetic status of hematopoietic cells. The study involves the development of busulfan and cyclophosphamide mediated mouse model for aplastic anemia, characterization of the disease with blood and marrow analysis, cytochemical examinations of bone marrow, flowcytometric analysis of hematopoietic population and microenvironmental components, determination of ROS generation, apoptosis profiling, expressional studies of Notch-1 signaling cascade molecules, investigation of epigenetic modifications including global CpG methylation of DNA, phosphorylation of histone-3 with their effects on bone marrow kinetics and expressional analysis of the anti-oxidative molecules viz; SOD-2 and Sdf-1. Severe hematopoietic catastrophic condition was observed during aplastic anemia which involved peripheral blood pancytopenia, marrow hypocellularity and decreased hematopoietic stem/progenitor population. Generation of ROS was found to play a central role in the cellular devastation in aplastic marrow which on one hand can be correlated with the destruction of hematopoiesis supportive niche components and alteration of vital Notch-1 signaling and on other hand was found to be associated with the epigenetic chromatin modifications viz; global DNA CpG hypo-methylation, histone-3 phosphorylation promoting cellular apoptosis. Decline of anti-oxidant components viz; Sdf-1 and SOD-2 hinted towards the irreversible nature of the oxidative damage during marrow aplasia. Collectively, the findings hinted towards the mechanistic correlation among ROS generation, microenvironmental impairment and epigenetic alterations that led to hematopoietic catastrophe under aplastic stress. The findings may potentiate successful therapeutic strategy development for the dreadful condition concerned.

Microenvironmental Scenario of the Bone Marrow of Inorganic Arsenic-Exposed Experimental Mice.

Exposure to arsenic on a regular basis, mainly through drinking water, agricultural pesticide, and sometimes therapeutic dose, results in various diseases of different tissues including the bone marrow hematopoietic system. Hematopoiesis is a dynamic process by which bone marrow (BM) hematopoietic stem/progenitor cells (HSPCs) generate a relatively constant pool of functionally mature blood cells by the support of microenvironmental components. The present study has been aimed to understand stem cell microenvironmental status during arsenic toxicity and the consequent reflection of dysregulation involving the hematopoietic machinery in experimental mice. Swiss albino mice were experimentally exposed to 10 µg arsenic trioxide/g body weight through oral gavage and 5 µg arsenic trioxide/g body weight intraperitoneally for a period of 30 days. Altered hemogram values in peripheral blood reflected the impaired hematopoiesis which was further validated by the reduced BM cellularity along with the deviated BM cell morphology as observed by scanning electron microscopy post arsenic exposure. The stromal cells were unable to establish a healthy matrix and the sustainability of hematopoietic progenitors was drastically affected in arsenic-exposed mouse groups, as observed in in vitro explant culture. The inability of stromal cells to establish supportive matrix was also explained by the decreased adherent colony formation in treated animals. Furthermore, the flow cytometric characterization of CXCR4+ and Sca-1+ CD44+ receptor expressions confirmed the dysregulation in the hematopoietic microenvironment. Thus, considering the importance of microenvironment in the maintenance of HSPC, it can be concluded that arsenic toxicity causes microenvironmental damage, leading to niche derangement and impaired hematopoiesis.

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.

Hematopathological alterations of major tumor suppressor cascade, vital cell cycle inhibitors and hematopoietic niche components in experimental myelodysplasia.

Myelodysplastic syndrome (MDS) is a poorly understood dreadful hematopoietic disorder that involves maturational defect and abnormalities in blood cell production leading to dysplastic changes and peripheral blood pancytopenia. The present work aims in establishing the mechanistic relationship of the expressional alterations of major tumor suppressor cascade, vital cell cycle inhibitors and hematopoietic microenvironmental components with the disease pathophysiologies. The study involves the development of N-N' Ethylnitrosourea (ENU) induced mouse model of MDS, characterization of the disease with blood film and bone marrow smear studies, scanning electron microscopic observation, mitochondrial membrane potential determination, flowcytometric analysis of osteoblastic and vascular niche components along with the expressional study of cleaved caspase-3, PCNA, Chk-2, p53, Ndn, Gfi-1, Tie-2, Sdf-1, Gsk-3ß, p18 and Myt-1 in the bone marrow compartment. Dysplastic features were found in peripheral blood of MDS mice which seemed to be the consequence of three marrow pathophysiological conditions viz; aberrant rise of cellular proliferation, increased apoptosis and crowding of abnormal blast population. Expressional decline of the p53 cascade involving Chk-2, p53, Ndn, Gfi-1 along with the downregulation of major cell cycle inhibitors seemed to be associated with the hyper-proliferative nature of bone marrow cells during MDS. Moreover the disruption of osteoblastic niche components added to the decreased hematopoietic quiescency. Increased marrow vascular niche components signified the pre-malignant state of MDS. Elevated cellular apoptosis and rise in the blast burden were also found to be associated with the p53 expression dependent collapsing of mitochondrial membrane potential and upregulation of Tie-2 respectively. The study established the mechanistic correlation between the alterations of the mentioned signaling components and hematopoietic anomalies during MDS which may be beneficial for the development of therapeutic strategies for the disease.

Analysis of hematopathology and alteration of JAK1/STAT3/STAT5 signaling axis in experimental myelodysplastic syndrome.

Hematological disorders like myelodysplastic syndrome (MDS) may arise due to cumulative dysregulation of various signalling pathways controlling proliferation, differentiation, maturation and apoptosis of bone marrow cells. This devastating bone marrow condition can be due to consequential abnormalities in haematopoiesis as well as its supportive microenvironment. Although mutations related to JAK/STAT pathway are common in myeloproliferative neoplasms, further studies are required to fully explore the myelodysplastic scenario regarding the concerned pathway. In this study, we have investigated the JAK-STAT signalling pathway which inevitably plays a crucial role in haematopoiesis. MDS was mimicked in a mouse model with an induction of ENU in adult mice. The bone marrow of the control and MDS groups of animals were subjected to a variety of tests, including cell morphology study in peripheral blood and bone marrow, cytochemistry and histochemistry of bone marrow smears, karyotyping and flowcytometric expression analysis of the phosphorylated forms of proteins like JAK1, STAT3 and STAT5 (denoted as pJAK1, pSTAT3 and pSTAT5) and the phenotypic expression of proteins like CD45 and CD71. The results revealed that the morphology of the blood and bone marrow cells were dysplastic compared to the affected blast populations of different lineages. The expression of common leucocyte antigen CD45 was less in comparison to the expression of transferrin receptor CD71 which was increased in the ENU induced MDS mouse model. Moreover, we have observed an upregulated expression of JAK1 followed by STAT5. Therefore, we can conclude that downregulation of CD45 may have helped in the upregulation of JAK-STAT signaling and CD71 expression. This aberrant signaling may be among one of the activated signaling axes that lead to affected hematopoietic lineages in Myelodysplastic syndrome.

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.