Biology subjects

Ghosh, S. K.

Publications and source records attributed to Ghosh, S. K..

27 records · Page 2Linked to original sources

bioRxiv2020

Novel insights into Entamoeba cyst wall formation and the importance of actin cytoskeleton

The cyst wall of Entamoeba histolytica, the causative agent of the amoebiasis, is a potential target for new drugs. The "Wattle and Daub" model of cyst wall formation of Entamoeba invadens had already been reported. In this study, we demonstrate in more detail the morphological stages of chitin wall formation in E. invadens using fluorescent chitin-binding dyes and immunolocalization of the cyst wall proteins. Here, the expression and localization of chitin synthase and the importance of actin cytoskeleton dynamics at cellular level, during encystations have been demonstrated for the first time. Chitin deposition was found to be initiated on the cell surface mostly from one distinct point, though multipoint initiation was also observed sometimes. From these points, the wall grew outwards and gradually covered the entire cyst surface with time. The initiation of chitin deposition was guided by the localization of chitin synthase 1 on the plasma membrane. The gradual formation of the cyst wall follows the Wattle and daub model. The chitin deposition occurred on the foundation of Jacob lectin at the cell membrane, and the other cyst wall components, like chitinase, and Jessie were also found to be present in the growing incomplete walls. In contrary to the Wattle and daub model, Jessie was found to be expressed and localized in the growing wall at the early hours of encystations. During encystation, F-actin was reorganized into the cortical region within an hour encystation initiation and remained intact until the completion of the chitin wall. Disruption of cortical actin polymerization with 2, 3-Butanedione monoxime inhibited proper wall formation but produced wall-less cysts or cysts with defective chitin wall. Malformations of cyst-walls were mainly due to improper localization and activity of chitin synthases, which indicates the indispensability of cortical actin cytoskeleton for the proper cyst wall formation. Author SummaryEntamoeba parasites reach new hosts using the resistant cyst form, so preventing its formation can stop the spread of amoebiasis. The resistant nature of the cyst is due to the chitin wall, and thus identifying the critical steps of wall formation could provide targets for designing new drugs. Here we studied the morphological stages of the cyst wall formation by observing how the chitin and other cell wall components were deposited on the cell surface using fluorescent chitin-binding dyes and antibodies against cyst wall proteins. In most cases, the chitin wall was found to start from one distinct point from which it spread all over the cell surface, guided by chitin synthase. The composition of these incomplete walls was the same as a mature cyst wall indicating that the wall may be a result of extracellular self-assembly of its constituents from one starting point. We have also observed that F-actin polymerized in the cortex of encysting cells and its disruption resulted in wall-less cysts or cysts with aberrant walls showing the importance of actin cytoskeleton in proper chitin deposition.

cell biology↗

bioRxiv2019

Polarized Entamoeba: A model for stable bleb driven motility

Protozoan parasites Entamoeba histolytica and Entamoeba invadens formed a polarized phenotype, an elongated shape with a single leading edge and a trailing edge when treated with pentoxifylline. The leading edge of the polarized morphology was a spherical protrusion devoid of F-actin but with occasional F-actin scars, indicating the presence of bleb. The polarized form was stable bleb driven since the blebbing was limited to the leading edge. Pentoxifylline induced chemokinesis in Entamoeba as it switched the motility pattern from slow and random to fast and directionally persistent. Pentoxifylline speeded up the cell aggregation in E. invadens during growth and encystation due to enhanced chemotaxis of the polarized form. The transformation of non-polarized adherent trophozoites to nonadherent stable bleb driven form occurred via lamellipodial and bleb driven adherent intermediate phenotypes. The nonadherent polarized phenotype was highly motile under confinement and moved by rearward plasma membrane flow. In contrast to pentoxifylline, adenosine, the adenosine receptor agonist, stimulated the formation of multiple protrusions leading to random motility. Thus pentoxifylline might prevent lateral protrusions by inhibiting adenosine receptor, producing the monopodial polarized morphology.\n\nSummary statementPentoxifylline, the adenosine receptor antagonist induced a stable bleb driven polarized morphology in Entamoeba characterized by fast, directionally persistent and highly chemotactic motility.

cell biology↗

bioRxiv2019

Down-regulated GDF9 and BMP15 mRNAs are the important determining factors for the unexplained female infertility cases advised for assisted reproductive technology

BackgroundIn India, the potential risk factors influencing female infertility are not well-characterized. Hence, this study was initially aimed to determine the multi-factor risk-predictive models for the infertile females from different Indian ethnics. Afterwards, when treatment was performed for correcting the abnormal demographic and clinical parameters obtained from our initial findings, some of the patients were observed to be treatment-non-responsive. This further recommended assessing the expression pattern of GDF9 and BMP15 mRNAs in those unexplained infertile females. MethodologyThis study included 470 Primary, 209 Secondary infertile and 419 age-matched Healthy females, where baseline demographic and clinicopathological factors were assessed. Additionally, from the treatment-non-responsive patients, laparoscopy and trans-vaginal ultrasound-guided collection of ovarian follicular fluid were done for studying the expression pattern of GDF9 and BMP15 mRNAs using q-PCR. Principal FindingsMid-education level, no contraception usage and duration of infertility for >2 years were the critical risk factors for female infertility (OR>>1; p<0.05), whereas elevated platelet count showed ~10-fold more risk of having secondary infertility and high ESR showed 3.33-fold increased threat of primary infertility. Besides, abnormal MCV, LH/FSH ratio, mild-anaemia and hypothyroidism were the best risk-predictive multi-factor model. After correcting these abnormal clinical factors, 6.8% of the patients were treatment-non-responsive, where elevated ESR, hypothyroidism and anaemia remained uncorrected. Interestingly, in those unexplained cases, GDF9 and BMP15 mRNAs were significantly down-regulated (2-4 fold) irrespective of age groups. Additionally, for predicting female infertility, the cut-off for the expression level of GDF9 and BMP15 mRNAs were estimated -2.09 and -2.75, respectively, with a relatively high sensitivity ([≥]62.5%) and specificity (75%). ConclusionsGDF9 and BMP15 mRNAs may be considered as molecular markers for predicting female infertility. Their down-regulation along with hypothyroidism and anaemia probably led to the treatment-non-responsiveness, thus, those patients may be advised to assisted reproductive technology with supplementation of exogenous GDF9 and/or BMP15.

epidemiology↗

bioRxiv2019

Stress-responsive Entamoeba topoisomerase II: a potential anti-amoebic target

Topoisomerases are ubiquitous enzymes, involved in all DNA processes across the biological world. These enzymes are also targets for various anticancer and antimicrobial agents. The causative organism of amoebiasis, Entamoeba histolytica (Eh), has seven unexplored genes annotated as putative topoisomerases. One of the seven topoisomerases in this parasite was found to be highly up-regulated during heat shock and oxidative stress. The bioinformatic analysis shows that it is a eukaryotic type IIA topoisomerase. Its ortholog was also highly up-regulated during the late hours of encystation in E. invadens (Ei), the encystation model of Eh. Immunoprecipitated endogenous EhTopoII showed topoisomerase II activity in vitro. Immunolocalization studies show that this enzyme colocalized with newly forming nuclei during encystation, which is a significant event in maturing cysts. Double-stranded RNA mediated down-regulation of the TopoII both in Eh and Ei reduced the viability of actively growing trophozoites and also reduced the encystation efficiency in Ei. Drugs, targeting eukaryotic topoisomerase II, e.g., etoposide, ICRF193, and amsacrine, show 3-5 times higher EC50 in Eh than that of mammalian cells. Sequence comparison with human TopoII showed that key amino acid residues involved in the interactions with etoposide and ICRF193 are different in Entamoeba TopoII. Interestingly, ciprofloxacin an inhibitor of prokaryotic DNA gyrase showed about six times less EC50 value in Eh than that of human cells. The parasites notable susceptibility to prokaryotic topoisomerase drugs in comparison to human cells opens up the scope to study this invaluable enzyme in the light of an antiamoebic target.

biochemistry↗

bioRxiv2019

Unique PKD like protein kinase in Entamoeba histolytica regulates vital actin mediated processes

Entamoeba histolytica causes widespread amoebiasis in humans. Multiple lines of emerging evidence have identified a repertoire of proteins involved in the process of erythrophagocytosis. However, the early initiation of the erythrophagosome at the site of erythrocytes attachment is not well understood. Here in our study we have identified and characterized a small Protein kinase D like protein (EhPKDL) in Eh, which nucleates actin polymerization and thus mediates many vital processes in Eh including erythrophagocytosis. Following multiple biochemical and biophysical approaches, we have characterized EhPKDL and have shown that EhPKDL can indeed interact and prime the nucleation of monomeric actin for polymerization. Furthermore, we went on to demonstrate the vitality of the EhPKDL in major actin-mediated processes like capping, motility, and erythrophagocytosis following knockdown of EhPKDL in the cellular context. Our study thus provides novel insights into the early actin nucleation in Eh and thus bridges the gap with our previous understanding of the assembly of erythrophagosomes.

cell biology↗

bioRxiv2019

Transcriptomic Analysis of the Neurogenesis Signature suggests Continued but Minimal Neurogenesis in the Adult Human Hippocampus

PurposeSince immunohistological investigations have given rise to divergent perspectives about continued hippocampal neurogenesis in adult humans, a comprehensive transcriptomic analysis of the neurogenesis signature markers supplemented with insights from gliogenesis and apoptotic markers (in context to the developmental stages across age) may discern important aspects and may well be the appropriate methodology for resolving this conflict.\n\nMaterials and MethodsRNA expression data for the salient neurogenesis, gliogenesis, and apoptotic marker genes in post-mortem human hippocampal tissue of the Prenatal (n=15), Infant/Early childhood (n= 5), Adolescence (n=4), and Adulthood (n=6) ages were downloaded from Allen Brain Atlas database (http://www.brainspan.org/rnaseq). Gene expression data was categorized, median values were computed, and age group specific differential expression was statistically analyzed (the confidence level of 95%, p value [≤] 0.05 is used).\n\nResultsA sharp fall in prenatal to infant/early childhood expression was observed for all studied neurogenesis markers, except that for the post-mitotic late maturation (CALB1, CALB2, MAP2, NEUN, STMN2) which showed no significant differences in expression profiles. A continued post childhood decrease across advancing age was observed in the neural stem cells and progenitor markers with insignificant differences across close age groups. Uniquely, the postnatal sharp fall of KI67 and TBR2 continued across advancing age groups, reached near baseline until adolescent age. The immature granule cell, post mitotic early maturation, and late maturation markers showed a maintained or slightly increased (albeit insignificant) post childhood expression. The gliogenesis markers mostly showed a significant downregulation between prenatal and infant /early childhood age groups; this decline was persistent thereafter with insignificant differences between close age groups. A continued postnatal decrease occurred in apoptotic markers with observable, but insignificant, differences between adolescent and adult age.\n\nConclusionsOur findings indicate a possibility of continued but minimal neurogenesis in the adult human hippocampus. A significant part of the newborn cells in the neurogenic niche may be glial cells.\n\n(Findings of this study were first presented at the Annual Meeting of Society for Neuroscience (SFN), 3rd-7th November, 2018, San Diego, USA. https://abstractsonline.com/pp8/#!/4649/presentation/37213)\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC=\"FIGDIR/small/664995v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (43K):\norg.highwire.dtl.DTLVardef@3268f3org.highwire.dtl.DTLVardef@18855bborg.highwire.dtl.DTLVardef@1b46bdaorg.highwire.dtl.DTLVardef@14b929_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract\n\nC_FIG HighlightsO_LIA varying but continued fall in expression was observed for all studied neurogenesis markers across advancing age groups, except that for the immature granule cells, early and post-mitotic late neuronal maturation markers.\nC_LIO_LIA continued postnatal sharp fall of progenitor markers KI67 and TBR2 reached near baseline until adolescent age.\nC_LIO_LIGliogenesis markers showed continued but insignificant fall in expression post infant/early childhood.\nC_LIO_LIApoptotic markers showed continued post infant/early childhood downfall but changes were negligible between adolescent and adult age.\nC_LI

neuroscience↗

bioRxiv2019

Anti-cancer property of Lenzites betulina (L) Fr. on cervical cancer cell lines and its anti-tumor effect on HeLa-implanted mice

In global scenario cervical cancer is increasing. New drugs from natural compounds are in search. Mushrooms are now recognized as miniature pharmaceutical factories producing hundreds of novel constituents. We have taken ethanolic extract Lenzities betulina (LBE) wild mushroom for evaluation of its as anti-cancer property against cervical cancer cell lines e.g. HeLa, CaSki and SiHa and anti tumor activity against HeLa implanted tumor on mice. The extraction was done by dip and stirring method in 90% ethanol for 72 h. For evaluation of anti-cervical cancer, several assays were performed such as MTT assay, cell morphology by phase contrast microscope and F-action polymerization by Laser scanning confocal microscope and nuclear morphology DAPI staining under inverted fluorescence microscope, MMP, ROS, cell cycle, autophagy and stem cell population by flow cytometry and DNA laddering were done. Western blotting was done for protein expression. To evaluate anti-metastatic activity, anti-cologenic assay and wound healing assay were adopted. For chemo-analysis of the LBE, GC-MS was done. The results from Cytotoxicity assay showed that at highest dose of LBE (1000 {micro}g/ml) after 24 h, percentage of cell inhibitions were 85.13 %, 77.13 % and 47.70 % against HeLa, CaSki and SiHa respectively and the calculated IC50 values were 492.52 {+/-} 2.6 {micro}g/ml, 612.22 {+/-} 4.2 {micro}g/ml, and 1210.30 {+/-} 6.4 {micro}g/ml respectively. Depending upon the cytotoxicity screening, HeLa cell line was considered for the further studies. Cell morphology study exhibited that LBE treated HeLa cells became round from normal spindle shape. DAPI staining showed that LBE treated nucleus became condensed and fragmented. DNA fragmentation at 230 and 300 base pair zone from agarose gel assay was observed. LBE induced ROS generation and reduced MMP. It up regulated the expression of apoptotic genes and p53 while down regulated Bcl2, pro-caspase 3 and pro caspase-9 gene. Cell cycle was arrested at G2/M checkpoint. Autophagic induction was exhibited by vacuole formation in treated cells. CSC population of treated cells was reduced and F-actin polymerization was observed in treated cells. In addition, LBE suppressed metastatic nature by inhibition of cell migration and colonization. The inhibition of growth of the tumors in HeLa cell-implanted mice showed that treatment with 50 mg LBE/kg of body weight of mice led to a marked reduction in the volume (93.22 {+/-} 9.2 %) and weight (90.42 {+/-}9.55 %) of the tumors. The GC-MS profile of LBE shows that out of 69 compounds, 9, 12-Octadecadienoic acid (Z, Z) and Ergosta-5, 8, 22-trien-3-ol, (3.beta22E) are in a significantly higher proportion with the percentage peak area 22.13 and 19.72 respectively. Library search for bioactivity showed that these compounds are anti-cancerous and interestingly 4-Hydroxy-6-methoxyaurone binding with P-glycoprotein inhibits the cancer cells to become drug resistant. In conclusion, LBE is very prominent anti-cervical cancer having a lot of anti-cancerous compounds which are probably acting synergistically. This report of anti-cervical cancer property of L. betulina is probably first time in oncology. Its therapeutic use in human model is urgent for new drug development.

cancer biology↗

bioRxiv2018

Structural plasticity of CENP-A regulated by H4 influences cellular levels and kinetochore assembly

The Histone variant CENP-ACse4 is a core component of the specialized nucleosome at the centromere in budding yeast. The level of Cse4 in cells is tightly regulated, primarily by ubiquitin-mediated proteolysis. However, the structural transitions in Cse4 that regulate centromere localization and interaction with regulatory components are poorly understood. Using time resolved fluorescence, NMR and molecular dynamics we show for the first time that soluble Cse4 can exist in a closed conformation, inaccessible to various regulatory components. We further determined that binding of its obligate partner H4, alters the inter-domain interaction within Cse4, ensuring an open state that will lend itself to proteolysis. This dynamic model allows kinetochore formation only in presence of H4, as the N-terminus, which is required for interaction with centromeric components will be unavailable in absence of H4. The specific requirement of H4 binding for the conformational regulation of Cse4 suggests a unique structure-based regulatory mechanism for Cse4 localization and prevention of premature kinetochore assembly.

biophysics↗

bioRxiv2017

How epigenome drives chromatin folding and dynamics, insights from efficient coarse-grained models of chromosomes

The 3D organization of chromosome is crucial for regulating gene expression and cell function. Many experimental and polymer modeling efforts are dedicated to deciphering the mechanistic principles behind chromosome folding. Chromosomes are long and densely packed - topologically constrained - polymers. The main challenge is therefore to develop adequate models and simulation methods to investigate properly the multi spatio-temporal scales of such macromolecules. Here, we discussed a generic strategy to develop efficient coarse-grained models for self-avoiding polymers on a lattice. Accounting accurately for the polymer entanglement length and the volumic density, we show that our simulation scheme not only captures the steady-state structural and dynamical properties of the system but also tracks the same dynamics at different coarse-graining. This strategy allows a strong power-law gain in numerical efficiency and offers a systematic way to define reliable coarse-grained null models for chromosomes and to go beyond the current limitations by studying long chromosomes during an extended time period with good statistics. We use our formalism to investigate in details the time evolution of the 3D organization of chromosome 3R (20 Mbp) in drosophila during one cell cycle (20 hours). We show that a combination of our coarse-graining strategy with a one-parameter block copolymer model integrating epigenomic-driven interactions quantitatively reproduce experimental data at the chromosome-scale and predict that chromatin motion is very dynamic during the cell cycle.

biophysics↗