Debashish Das - Academia.edu (original) (raw)

Papers by Debashish Das

Current Drug Targets, Apr 12, 2016

MicroRNAs (miRNAs) are small (19-25 nucleotides) non-coding single-stranded RNAs that control pos... more MicroRNAs (miRNAs) are small (19-25 nucleotides) non-coding single-stranded RNAs that control post-transcriptional gene expression. miRNAs are abundantly expressed in the brain, where they play key roles during neuronal differentiation, synaptogenesis, and plasticity. It is also becoming increasingly evident that miRNAs are involved in the etiology of several neurological disorders. Mounting evidence indicates that miRNAs have the ability to regulate the expression profiles of genes in signaling pathways associated with cerebrovascular diseases such as ischemic stroke, subarachnoid hemorrhage, and vascular dementia. For instance, miR-21 is involved in ischemic stroke pathology through atherosclerosis and provides neuroprotection by its anti-apoptotic features. miR-497 induces neuronal death and miR-210 is upregulated in hypoxic cells. Deregulated expression of miRNAs in response to ischemic stroke has enabled the use of miRNA as an efficient non-invasive biomarker. Antagomirs are often effective against neuronal apoptosis and can induce neuroregeneration following ischemia. Moreover, the advent of systems biology has introduced novel computational tools to identify the link between miRNAs, target genes and transcription factors involved in the stroke pathology and its treatment. This review describes the emerging role of miRNAs in neuroprotection and focuses on a subset of miRNAs that act as central players in ischemic stroke.

Investigative Ophthalmology & Visual Science, Jul 13, 2018

3.1 Down regulation of cell surface CALEB is an outcome of ectodomain shedding...................... more 3.1 Down regulation of cell surface CALEB is an outcome of ectodomain shedding........................................ 59 3.2 Presence of the shed ectodomain fragment of CALEB protein........................................................................ 61 3.4 Presence of the membrane ...

PubMed, 2015

Purpose: Limbal epithelial stem cell deficiency is caused by exposure of the cornea to thermal, c... more Purpose: Limbal epithelial stem cell deficiency is caused by exposure of the cornea to thermal, chemical, or radiation burns or by diseases (aniridia and Stevens-Johnson syndrome). Autologous cell transplantation is a widely used therapeutic modality for restoring the corneal surface in such pathological conditions. Ex vivo cultured limbal, conjunctival, and oral biopsies have been widely used to reconstruct the corneal surface with variable outcomes. Culture characterization of the ex vivo cultured cells would provide insight and clues into the underlying signaling mechanisms that would aid in determining the probable transplantation outcome. Comparison of the vital proteins and genes among the three ex vivo cultured tissues has implications in clinical practice. To address this issue, we characterized and compared the proliferative and differentiated properties of ex vivo cultured limbal, conjunctival, and oral biopsies used for cell-based therapy for corneal surface restoration. Methods: Limbal, conjunctival, and oral biopsies were collected with informed patient consent. Explant cultures were established on the denuded human amniotic membrane with corneal lineage differentiation medium. The day 14 cultures were characterized for epithelial and corneal lineage-specific markers using reverse transcription (RT)-PCR for cytokeratin 3, 4, 12, 13, 15, connexin 43, vimentin, p63α, and ABCG2 markers. mRNA expression was estimated in day 14 cultures with real-time quantitative real time (qRT)-PCR for pluripotency markers (OCT4, SOX2, NANOG), putative corneal stem cell markers (ABCG2 and p63α), proliferation markers (cyclin d1, Ki-67, PCNA, and CDC20), apoptotic markers (BCL2, BAX, caspase 3, and caspase 9), Notch signaling pathway markers (Notch1, Jagged1, Hes1, Hes3, Hes5, and Hey1), and autophagic markers (LC3A, LC3B, ATG7, RAB7, LAMP1, and LAMP2). Fluorescence-activated cell sorter profiling was performed for pluripotent markers and putative corneal stem cell markers ABCG2 and p63α. Results: The protein and mRNA expression levels of the pluripotent markers were lower, whereas those of the putative stem/progenitor markers ABCG2, ΔNp63α, and Notch signaling molecules (Notch1 and Jagged1) were elevated in limbal cultures. The gene expression levels of the autophagy markers (LC3A, LC3B, and LAMP1) were significantly increased in the limbal cultures compared to the oral and conjunctival cultures. Conclusions: In conclusion, the limbal epithelial cultures showed higher expression of proliferative, limbal stem cell marker, Notch signaling, and autophagy markers suggesting a role in stem cell maintenance and differentiation. This implicates the probable factors that might drive a successful transplantation. Our findings provide the initial steps toward understanding transplantation medicine in an ex vivo model.

Investigative Ophthalmology & Visual Science, Jun 1, 2019

The purpose of this study was to study the status and association among tear-soluble factors, cor... more The purpose of this study was to study the status and association among tear-soluble factors, corneal dendritic cell density, vitamin D, and signs and symptoms in dry eye disease (DED). METHODS. A total of 33 control subjects and 47 evaporative dry eye patients were included in the study. DED diagnosis and classification was based on the 2017 Report of the Tear Film & Ocular Surface Society International Dry Eye Workshop (TFOS DEWS II). DED workup, including tear film break-up time (TBUT), Schirmer's test I (STI), corneal and conjunctival staining, ocular surface disease index (OSDI) scoring, and in vivo confocal microscopy (to assess corneal dendritic cell density [cDCD] and subbasal nerve plexus [SBNP] features) was performed in the study subjects. Tear fluid using Schirmer's strip and serum were collected from the subjects. Multiplex ELISA or single analyte ELISA was performed to measure 34 tearsoluble factors levels including vitamin D. RESULTS. Significantly higher OSDI discomfort score, lower TBUT, and lower STI were observed in DED patients. cDCD was significantly higher in DED patients. No significant difference was observed in SBNP features. Tear fluid IL-1b, IL-17A, MMP9, MMP10, MMP9/ TIMP ratio, and VEGF-B were significantly higher in DED patients. Significantly lower tear fluid IL-2, IP-10, NPY, VEGF-A, and vitamin D was observed in DED patients. These dysregulated tear factors showed significant associations with DED signs and symptoms. CONCLUSIONS. Altered tear fluid soluble factors with potential to modulate nociception exhibited a distinct association with ocular surface discomfort status, TBUT, STI, and cDCD. This implies a functional relationship between the various tear-soluble factors and dry eye pathogenesis, indicating new molecular targets for designing targeted therapies.

Cornea, Sep 15, 2020

Purpose: Dry eye disease (DED) is a common ocular surface condition across age groups. Recently, ... more Purpose: Dry eye disease (DED) is a common ocular surface condition across age groups. Recently, vitamin D deficiency has gained importance as a causative factor, and its supplementation alleviates symptoms of DED. Resveratrol (RES) regulates vitamin D receptors (VDRs) and Notch signaling. We investigated the role of RES on vitamin D levels and Notch signaling under hyperosmolar conditions. Methods: Human corneal epithelial (HCE-T) cells were treated with RES in hyperosmolar and normal conditions. Quantitative real-time polymerase chain reaction (PCR), immunofluorescence, enzymelinked immunosorbent assay, and western blot analysis were performed for estimating reactive oxygen species, VDR, secreted 25-hydroxyvitamin D3, and Notch signaling pathway molecules in treated and control cells. Results: HCE-T cells in hyperosmolar conditions had increased reactive oxygen species levels and decreased vitamin D levels that got restored in the presence of RES. Hyperosmolarity also reduced VDR expression and Notch activity that normalized to original levels with RES. In the presence of Notch blocker LY-411575, RES could not restore VDR expression or secreted vitamin D levels in HCE-T cells exposed to hyperosmolar conditions, whereas recombinant Jagged1 restored vitamin D and VDR levels. Conclusions: RES restores vitamin D levels in hyperosmolar conditions most likely through activation of Notch signaling. Hence, RES can be a potential adjuvant in DED for patients considered for vitamin D treatment.

Cytotherapy, Apr 1, 2016

Background aims. Autologous transplantation of ex vivo cultured cells the treatment of choice for... more Background aims. Autologous transplantation of ex vivo cultured cells the treatment of choice for patients with limbal stem cell deficiency. The most commonly used cell sources for transplantation limbal, conjunctival or oral mucosal tissue. Protocols vary for culturing each tissue type, and there are no comparative studies on transplantation outcomes using these different culture techniques. To overcome this limitation, we devised a simple protocol that can uniformly promote growth and differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. Methods. Biopsies were cultured as explants on de-epithelialized human amniotic membrane in the presence of recombinant epidermal growth factor and insulin. Cultured cells were characterized using immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction for stem/progenitor markers (ABCG2 and P63α) and differentiation markers (CK3, CK12, CK4, CK13, CK15 and CONNEXIN 43). Fluorescence-activated cell sorter analysis was performed for ABCG2. Results. The results revealed that cells of all three biopsies differentiated into the corneal lineage. Positivity of CK3/12, CK4, CK12 and CONNEXIN 43 immunostaining and the relative mRNA expression of CK3, CK4, CK12, CK13, CK15 and CONNEXIN 43 could be detected in the cultured biopsies. Conclusions. Unlike tissue-specific protocols, our protocol can unequivocally promote differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. This simple standardized protocol can be adapted for ocular surface reconstruction using stem cell transplantation.

Investigative Ophthalmology & Visual Science, Mar 26, 2012

Indian Journal of Ophthalmology, 2023

Dry eye disease (DED) is a multifactorial and common ophthalmic disease that has a tremendous eff... more Dry eye disease (DED) is a multifactorial and common ophthalmic disease that has a tremendous effect on the quality of life. It is now becoming a public health concern because of our changing lifestyle and environment. The current treatment modalities, artificial tear substitutes, and anti-inflammatory therapy are directed at dry eye symptoms. One of the major drivers for DED is oxidative stress, and the polyphenol group of natural compounds has the potential to reduce the same. Resveratrol, widely found in the skin of grapes and nuts, has antioxidative and anti-inflammatory properties. It has been shown to have beneficial effects in glaucoma, age-related macular degeneration, retinopathy of prematurity, uveitis, and diabetic retinopathy. Studies have also explored the beneficial effects of resveratrol in DED, making it as a promising therapeutic molecule. Resveratrol has not yet reached clinical application because of difficulty in deliverability and low bioavailability. In this review, we explore the potential of resveratrol in DED treatment based on various in vitro and in vivo studies.

Investigative Ophthalmology & Visual Science, Jun 23, 2017

Oral Oncology, May 1, 2013

Purpose The primary objective of this study is to evaluate the effect chemotherapy on tumor initi... more Purpose The primary objective of this study is to evaluate the effect chemotherapy on tumor initiating cells/cancer stem cell populations (TICs) resistant to drugs and the latter’s’ association with recurrence in head and neck cancer. Materials and methods TICs were identified from cell lines and primary cultures by markers (CD133, CD44 and ALDHA1) and enriched by spheroid cultures. The cell lines were treated with the drugs cisplatin, docetaxel and 5FU individually; the stem cell assays and expression profiles were carried out on both the parental and treated cell lines. These isolated populations are being characterized for the presence of a panel of markers associated with stem cells, drug resistance and epithelial mesenchymal transition. Further characterization by in vivo tumorigenicity assays is in progress. Results Analysis of the TIC population in HNSCC cell lines (HEp-2, FaDu, CAL27) shows that the percentage of tumor initiating cells was up to 2–3%. Spheroids generated from cell lines showed a higher expression of markers such as CD44, CD133, ABCG2, Notch 1 as compared to monolayer cells. Drug treatment of these cell lines with docetaxel, cisplatin and 5-FU also resulted in a similar expression profile indicating an enrichment of the stem cell population. Validation in the patients with HNSCC by immunohistochemical analysis (IHC) and QRT PCR showed a definite association of these markers with the treatment outcome. Spheroids isolated from fresh tumor specimens from responder/non-responder group of patients were also assessed for their tumorigenicity and marker expression profile. Further characterization and marker profiling of the stem cell content of these spheroids/treated cells is currently ongoing. Discussion Preliminary results suggest that the resistant TICs get enriched upon drug exposure and might be responsible for treatment failure in a subset of the HNSCC patients; gene signatures specific to non-responders/responders may have a potential role in stratifying them.

Scientific Reports

Deregulation of vascular endothelial growth factor (VEGF) levels leads to retinopathy of prematur... more Deregulation of vascular endothelial growth factor (VEGF) levels leads to retinopathy of prematurity (ROP). Vitamin D (VIT-D) is known to regulate VEGF in an oxygen dependent manner. The purpose of this study was to correlate tear levels of VEGF and VIT-D with different ROP stages in preterm infants. In this prospective cross-sectional study, we enrolled 104 pre-term infants. They were grouped into: Group-1 (Classical ROP) and Group-2 (Aggressive ROP), which were further subdivided into Group-1A (progressing), Group-1B (regressing), Group-2A (pre-treatment), and Group-2B (post-treatment). Tear VEGF and VIT-D levels and their association with different ROP stages were assessed. Stage 1 and stage 2 had higher whereas stage 3 had lower VEGF levels in Group-1B compared to Group-1A. Stage 1 and stage 3 showed higher levels of VIT-D with no difference in stage 2 in Group-1B compared to Group-1A., Group-2B showed higher VEGF and lower VIT-D levels compared to Group-2A. Presence of a positi...

Cancer genetics and cytogenetics, Jun 1, 2001

A method has been developed to eliminate melanin pigments from chromosomal plates of uveal melano... more A method has been developed to eliminate melanin pigments from chromosomal plates of uveal melanoma tumors. On preparing the chromosomal plates of tumor masses of posterior uveal melanoma for cytogenetic analysis in intermediate pigmented population, it was found that the melanin pigments obscured the cells and thereby the chromosomes. Thus, cytogenetic analysis could not be carried out. Hence, a method was developed in our laboratory to eliminate these obscuring pigments. Several different attempts were made to eliminate the melanin pigments. Finally, washing the cells with phosphate buffered saline before the harvesting stage served the purpose. After harvesting and preparing the chromosomal plates, the cells were found to be devoid of melanin pigments. The protocol would help researchers trying to carry out cytogenetic analysis on melanoma tumor masses in populations with intermediate to dark pigmentation.

Graefe's Archive for Clinical and Experimental Ophthalmology

Indian Journal of Ophthalmology

Synaptogenesis is a collaborative effort of activity-independent processes specifying neuronal co... more Synaptogenesis is a collaborative effort of activity-independent processes specifying neuronal connections, and activity-dependent processes refining these initial synaptic connections. CALEB, an activity-dependent modulated protein, might be implicated in this refinement. The aim of my work was to investigate the mechanism of CALEB (Chicken Acidic Leucine rich EGF-like domain containing brain protein) down regulation, which was identified in an initial screen. I showed that cell surface CALEB down regulation was enhanced in retinal cultures incubated with KCl or agonist of glutamate receptors. Modulation of extracellular calcium concentration and experiments using blockers of calmodulin revealed the involvement of calcium in the mechanism of down regulation. Since CALEB belongs to the EGF family of growth and differentiation factors that undergo cell surface cleavage, I hypothesized a similar phenomenon responsible for the cell surface down regulation of CALEB. Isolation of a solub...

Journal of Glaucoma, 2022

PRCIS NOTCH signaling is significantly upregulated in the lens capsules of eyes with pseudoexfoli... more PRCIS NOTCH signaling is significantly upregulated in the lens capsules of eyes with pseudoexfoliation syndrome (PXF) but not in those with pseudoexfoliation glaucoma (PXG) when compared with healthy controls. PURPOSE NOTCH signaling has neuroprotective functions and altered NOTCH signaling is associated with neurodegenerative diseases with protein aggregation such as Alzheimer disease. As PXG is also a protein aggregate disease associated with neural degeneration, NOTCH molecular expression was explored in the lens capsules of patients with PXF, PXG, primary open-angle glaucoma (POAG), and healthy controls. METHODS Anterior lens capsules were collected from 106 patients (27 PXF, 24 PXG, 22 POAG, and 33 controls) undergoing cataract surgery. Gene expression profiling for NOTCH pathway molecules (ligands, receptors, and downstream target genes) was performed on the tissue using a quantitative reverse transcription-polymerase chain reaction. The results were confirmed by protein analysis using dot-blot or immunostaining techniques. RESULTS There was no difference in the demographic characteristics between the groups. There was an increase in NOTCH4 receptor expression (>14-fold) in the PXF group as compared with the controls. Similarly, the Delta-like 3 and Delta-like 4 ligands were significantly elevated in the PXF group compared with controls (P<0.05). Downstream targets HES3, HES5, and HEY1 expression were significantly elevated (P<0.005) in PXF lens capsules, confirming a higher activity of NOTCH signaling in this cohort. Immunostaining also corroborated the gene expression profile. CONCLUSION The finding that NOTCH signaling is significantly upregulated in the lens capsule of eyes with PXF and not in PXG or POAG patients suggests a possible protective role in the development of glaucoma.

Investigative Ophthalmology & Visual Science, 2013

Scientific Reports, 2021

Corneal haze post refractive surgery is prevented by mitomycin c (MMC) treatment though it can le... more Corneal haze post refractive surgery is prevented by mitomycin c (MMC) treatment though it can lead to corneal endothelial damage, persistent epithelial defects and necrosis of cells. Suberanilohydroxamic acid (SAHA) however has been proposed to prevent corneal haze without any adverse effects. For clinical application we have investigated the short and long term outcome of cells exposed to SAHA. Human donor cornea, cultured limbal epithelial cells, corneal rims and lenticules were incubated with SAHA and MMC. The cells/tissue was then analyzed by RT-qPCR, immunofluorescence and western blot for markers of apoptosis and fibrosis. The results reveal that short term exposure of SAHA and SAHA + MMC reduced apoptosis levels and increased αSMA expression compared to those treated with MMC. Epithelial cells derived from cultured corneal rim that were incubated with the MMC, SAHA or MMC + SAHA revealed enhanced apoptosis, reduced levels of CK3/CK12, ∆NP63 and COL4A compared to other treatm...