Biology subjects

Chandra, A.

Publications and source records attributed to Chandra, A..

44 records · Page 3Linked to original sources

bioRxiv2020

Genetically encoded live cell sensor for tyrosinated microtubules

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin post-translational modification (PTMs). These PTMs are known to affect microtubule stability, dynamics and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present there exist no tool that can specifically mark tubulin PTMs in live cells, thus severely limiting our understanding of tubulin PTMs. Using yeast display library, we identified a binder against terminal tyrosine of alpha tubulin, a unique PTM site. Extensive characterization validates the robustness and non-perturbing nature of our binder as tyrosination sensor, a live cell tubulin nanobody specific towards tyrosinated or unmodified microtubules. Using which, in real time we followed nocodazole, colchicine and vincristine induced depolymerization events of unmodified microtubules, and found each distinctly perturb microtubule polymer. Together, our work describes the tyrosination sensor and potential applications to study microtubule and PTM processes in living cells.

cell biology↗

bioRxiv2020

Cholesterol metabolism drives regulatory B cell function

Regulatory B cells restrict immune and inflammatory responses across a number of contexts. This capacity is mediated primarily through the production of IL-10. Here we demonstrate that the induction of a regulatory program in human B cells is dependent on a metabolic priming event driven by cholesterol metabolism. Synthesis of the metabolic intermediate geranylgeranyl pyrophosphate (GGPP) was required to specifically drive IL-10 production, and to attenuate Th1 responses. Furthermore, GGPP-dependent protein modifications controlled signaling through PI3K{delta}-AKT-GSK3, which in turn promoted BLIMP1-dependent IL-10 production. Inherited gene mutations in cholesterol metabolism result in a severe autoinflammatory syndrome, termed mevalonate kinase deficiency (MKD). Consistent with our findings, B cells from MKD patients induced poor IL-10 responses and were functionally impaired. Moreover, metabolic supplementation with GGPP was able to reverse this defect. Collectively, our data define cholesterol metabolism as an integral metabolic pathway for the optimal functioning of human IL-10 producing regulatory B cells. Graphical abstractMKD = Mevalonate kinase deficiency O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/893982v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@e23fd0org.highwire.dtl.DTLVardef@1cb9611org.highwire.dtl.DTLVardef@e9769corg.highwire.dtl.DTLVardef@16a1d0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2019

Insight into the genetic aetiology of retinal detachment by combining small clinical and large population-based datasets

Idiopathic retinal detachment is a serious common condition, but genetic studies to date have been hampered by the small size of the assembled cohorts. Genetic correlations between retinal detachment and high myopia or cataract operation were high, respectively 0.46 (SE=0.08) and 0.44 (SE=0.07), in the UK Biobank dataset and in line with known epidemiological associations. Meta-analysis of genome-wide association studies using UK Biobank retinal detachment cases (N=3977) and two cohorts, each comprising [~]1000 rhegmatogenous retinal detachment patients, uncovered 11 genome-wide significant association signals, near or within ZC3H11B, BMP3, COL22A1, DLG5, PLCE1, EFEMP2, TYR, FAT3, TRIM29, COL2A1 and LOXL1. Replication in the 23andMe dataset, where retinal detachment is self-reported by participants, firmly establishes association at six loci FAT3, COL22A1, TYR, BMP3, ZC3H11B and PLCE1. The former two seem to particularly impact on retinal detachment, the latter three shed light on shared aetiologies with cataract, myopia and glaucoma.\n\nAuthor SummaryRetinal detachments are common conditions that may lead to permanent severe sight reduction or blindness; they are a major cause of emergency eye surgery. The most common type of retinal detachment follows a break in the retina and is thought to be in part genetically determined but little is known about the contributing individual genetic risk variants. The condition prevalence increases with age and with common eye conditions such as myopia, cataract or glaucoma. We showed that the retinal detachment cases derived from self-report or hospitalisation records in the large UK Biobank dataset show very similar characteristics to samples of carefully clinically evaluated retinal detachment with break cases and therefore could be used to perform genetic analysis of the condition. Association studies require large sample of cases and by pooling Biobank and clinical cases, this study identifies 11 novel significant associations, six of which were further replicated in an independent population-based dataset (23andMe). Two of the replicated findings seem to specifically underline retinal detachment risk while three others highlight shared genetic risk with myopia, cataract and/or glaucoma, paving the way to better understanding of these conditions and of their overlap.

genetics↗

bioRxiv2018

O-GlcNAc modification of oncogenic transcription factor Sox2 promotes protein stability and regulates self-renewal in pancreatic cancer

Pancreatic cancer is among the 3rd leading cause of cancer related deaths in the United States along with a 5-year survival rate of 7%. The aggressive biology of the disease is responsible for such dismal outcome and is manifested by an increase in self-renewal capacity of the cancer cells, which leads to an increased rate of tumor-recurrence, contributing to poor prognosis. Transcription factor SOX2 maintains a critical balance between differentiation and \"stemness\" and is thus tightly regulated within a cell. In cancer, SOX2 is aberrantly \"turned-on\" leading to activation of self-renewal pathways in cancer. Regulation of Sox2 in cancer is poorly understood. In the current study, we show for the first time that in pancreatic cancer, Sox2 is modified by addition of O-GlcNAc moiety, catalyzed by OGT (O-GlcNAc Transferase) at S246. This activates Sox2 transcriptional activity by stabilizing the protein in the nucleus. A CRISPR-OGT knockout in pancreatic cancer cell line S2VP10 resulted in a delayed tumor initiation. We further showed that mutation of this site (S246A) prevents the modification of Sox2 and its downstream activity. Our study also demonstrated that targeting OGT in vivo with a small molecule inhibitor OSMI, results in decreased tumor burden, delayed tumor progression and a decreased expression of SOX2 in pancreatic cancer cells. Our study highlights for the first time that that the O-GlcNAc transferase dependent SOX2 glycosylation has a profound effect on the transcriptional activity of SOX2 and is instrumental in determining self-renewal in pancreatic cancer.\n\nSignificanceOur study highlights for the first time that that the O-GlcNAc transferase dependent SOX2 glycosylation determines self-renewal in pancreatic cancer which is responsible for tumor initiation.

cancer biology↗

bioRxiv2018

PI3Kδ hyper-activation promotes the development of B cells that exacerbate Streptococcus pneumoniae infection in an antibody-independent manner

Streptococcus pneumoniae is a major cause of pneumonia and a leading cause of death world-wide. Antibody-mediated immune responses can offer protection against repeated exposure to S. pneumoniae, yet vaccines only offer partial protection. Patients with Activated PI3K{delta} Syndrome (APDS) are highly susceptible to S. pneumoniae. We generated a conditional knockin mouse model of this disease and identified a CD19+B220- B cell subset that is induced by PI3K{delta} signaling, is resident in the lungs, and which promotes increased susceptibility to S. pneumoniae during the early phase of infection via an antibody-independent mechanism. We show that an inhaled PI3K{delta} inhibitor improves survival rates following S. pneumoniae infection in wild-type mice and in mice with activated PI3K{delta}. These results suggest that a subset of B cells in the lung can promote the severity of S. pneumoniae infection, representing a novel therapeutic target.

immunology↗

bioRxiv2018

A novel xenograft model reveals invasive mesenchymal transition and ineffective angiogenic response during the evolution of resistance to anti-angiogenic therapy

Bevacizumab treatment of glioblastoma is limited by transient responses and acquired resistance. Because of the lengthy duration of treatment that can precede resistance in patients, in order to study changes underlying the evolution of bevacizumab resistance, we created a novel multigenerational xenograft model of acquired bevacizumab resistance. Glioblastoma xenografts were treated with bevacizumab or IgG, and the fastest growing tumor re-implanted into new mice, generating paired isogeneic responsive or resistant multigenerational xenografts. Microarray analysis revealed significant overexpression across generations of the mesenchymal subtype gene signature, paralleling results from patient bevacizumab-resistant glioblastomas (BRGs) that exhibited increasing mesenchymal gene expression correlating with increased bevacizumab treatment duration. Key mesenchymal markers, including YKL-40, CD44, SERPINE1, and TIMP1 were upregulated across generations, with altered morphology, increased invasiveness, and increased neurosphere formation confirmed in later xenograft generations. Interestingly, YKL-40 levels were elevated in serum of patients with bevacizumab-resistant vs. bevacizumab-naive glioblastomas. Finally, despite upregulation of VEGF-independent pro-angiogenic genes across xenograft generations, immunostaining revealed increased hypoxia and decreased vessel density with increasing generation of treatment, mirroring our findings in patient BRGs and suggesting tumor growth despite effective devascularization caused by VEGF blockade. Besides identifying novel targets for preventing the evolution of resistance and offering a xenograft model for testing resistance inhibitors, our work suggests YKL-40 as a blood biomarker of bevacizumab resistance worthy of further evaluation.

cancer biology↗

bioRxiv2017

Cells With Treg-Specific FOXP3 Demethylation But Low CD25 Are Prevalent In Autoimmunity

Identification of alterations in the cellular composition of the human immune system is key to understanding the autoimmune process. Recently, a subset of FOXP3+ cells with low CD25 expression was found to be increased in peripheral blood from systemic lupus erythematosus (SLE) patients, although its functional significance remains controversial. Here we find in comparisons with healthy donors that the frequency of FOXP3+ cells within CD127lowCD25low CD4+ T cells (here defined as CD25lowFOXP3+ T cells) is increased in patients affected by autoimmune disease of varying severity, from combined immunodeficiency with active autoimmunity, SLE to type 1 diabetes. We show that CD25lowFOXP3+ T cells share phenotypic features resembling conventional CD127lowCD25highFOXP3+ Tregs, including demethylation of the Treg-specific epigenetic control region in FOXP3 that is highly enriched in HELIOS+ cells, and lack of IL-2 production. As compared to conventional Tregs, more CD25lowFOXP3+HELIOS+ T cells are in cell cycle (33.0% vs 20.7% Ki-67+; P = 1.3 x 10-9) and express the late-stage inhibitory receptor PD-1 (67.2% vs 35.5%; P = 4.0 x 10-18), while having reduced expression of the early-stage inhibitory receptor CTLA-4, as well as other Treg markers, such as FOXP3 and CD15s. The number of CD25lowFOXP3+ T cells are highly correlated (P = 1.2 x 10-19) with the proportion of CD25highFOXP3+ T cells in cell cycle (Ki-67+). These findings suggest that CD25lowFOXP3+ T cells represent a subset of Tregs that are derived from CD25highFOXP3+ T cells, and are a peripheral marker of recent Treg expansion in response to an autoimmune reaction in tissues.\n\nHighlights- FOXP3+ compartment within CD127lowCD25low T cells is expanded in autoimmune patients.\n\n- Increased numbers of CD25lowFOXP3+ T cells are a circulating marker of autoimmunity.\n\n- CD25lowFOXP3+ HELIOS+ T cells are fully demethylated at the FOXP3 TSDR.\n\n- CD25lowFOXP3+ T cells could represent a terminal stage of regulatory T cells.

immunology↗

bioRxiv2017

Modulation Of Type IV Pili Phenotypic Plasticity Through A Novel Chaperone-Usher System In Synechocystis sp 6803

Controlling the transition from a multicellular motile state to a sessile biofilm is an important eco-physiological decision for most prokaryotes, including cyanobacteria. Photosynthetic and bio geochemically significant cyanobacterium Synechocystis sp. PCC6803 (Syn6803) uses Type IV pili (TFP) for surface-associated motility and light-directed phototaxis. We report the identification of a novel Chaperone-Usher (CU) system in Syn6803 that regulate secretion of minor pilins as a means of stabilizing TFP morphology. These secreted minor-pilins aid in modifying TFP morphology to suit the adhesion state by forming cell to surface contacts when motility is not required. This morphotype is structurally distinct from TFP assembled during motile phase. We further demonstrate by examining mutants lacking either the CU system or the minor-pilins, which produce aberrant TFP, that are morphologically and functionally distinct from wild-type (WT). Thus, here we report that in Syn6803, CU system work independent of TFP biogenesis machinery unlike reported for other pathogenic bacterial systems and contributes to provide multifunctional plasticity to TFP. cAMP levels play an important role in controlling this switch. This phenotypic plasticity exhibited by the TFP, in response to cAMP levels would allow cells and cellular communities to adapt to rapidly fluctuating environments by dynamically transitioning between motile and sessile states.\n\nSignificance of this workHow cyanobacterial communities cope with fluctuating or extreme environments is crucial in understanding their role in global carbon and nitrogen cycles. This work addresses the key question: how do cyanobacteria modulate external appendages, called Type IV pili, to effectively switch between motile and sessile biofilm states? We demonstrate that cells transition between forming strong cell-surface interactions indispensable for biofilm formation to forming cell-cell interactions that allow for coordinated movement crucial for social motility by functional/ structural modification of same TFP appendage. The second messenger, cAMP and a Chaperone-Usher secretion are indispensible to achieve these structural modifications of TFP and control the complex phenotypic transition. We have uncovered a strategy that Syn6803 has evolved to deal with molecular decision-making under uncertainty, which we call phenotypic plasticity. Here we demonstrate how a single motility appendage can be structurally modified to attain two antagonistic functions in order to meet the fluctuating environmental demands.

microbiology↗