Biology subjects

Das, A.

Publications and source records attributed to Das, A..

11 recordsLinked to original sources

bioRxiv2018

A negative role for the interleukin-2-inducible T-cell kinase (ITK) in human Foxp3+ Treg differentiation

The Tec kinases ITK (interleukin-2-inducible T-cell kinase) and RLK (resting lymphocyte kinase) are critical components of the proximal TCR/CD3 signal transduction machinery, and data in mice suggest that ITK negatively regulates TREG differentiation. However, whether Tec kinases modulate TREG development and/or function in human T cells remains unknown. Using a novel self-delivery siRNA platform (sdRNA), we found that ITK knockdown in primary human naive peripheral blood CD4 T cells increased Foxp3+ TREG differentiation under both TREG and T effector (Teff) cell priming conditions. ITK knockdown also enhanced the expression of the co-inhibitory receptor PD-1 on FoxP3+ T cells. TREGS differentiated in vitro (iTREG) after ITK knockdown displayed suppressive capacity against effector CD4+ T cell proliferation. ITK knockdown decreased IL-17A production in T cells primed under Th17 conditions and increased Th1 differentiation. Finally, a dual ITK/RLK Tec kinase inhibitor blocked TREG differentiation and T cell activation in general. Our data suggest that targeting ITK in human T cells may be an effective approach to boost TREG in the context of autoimmune diseases, but non-specific inhibition of other Tec family kinases may broadly inhibit T cell activation.

immunology↗

bioRxiv2018

Migration rather than proliferation transcriptomic signatures are strongly associated with breast cancer patient survival

The efficacy of prospective cancer treatments is routinely estimated by in vitro cell-line proliferation screens. However, it is unclear whether tumor aggressiveness and patient survival are influenced more by the proliferative or the migratory properties of cancer cells. To address this question, we experimentally measured proliferation and migration phenotypes across more than 40 breast cancer cell-lines. Based on the latter, we built and validated individual predictors of breast cancer proliferation and migration levels from the cells transcriptomics. We then apply these predictors to estimate the proliferation and migration levels of more than 1000 TCGA breast cancer tumors. Reassuringly, both estimates increase with tumors aggressiveness, as qualified by its stage, grade, and subtype. However, predicted tumor migration levels are significantly more strongly associated with patient survival than the proliferation levels. We confirm these finding by conducting siRNA knock-down experiments on the highly migratory MDA-MB-231 cell lines and deriving gene knock-down based proliferation and migration signatures. We show that cytoskeletal drugs might be more beneficial in patients with high predicted migration levels. Taken together, these results testify to the importance of migration levels in determining patient survival.

bioinformatics↗

bioRxiv2018

Engineered cell-to-cell signalling within growing bacterial cellulose pellicles

Bacterial cellulose is a strong and flexible biomaterial produced at high yields by Acetobacter species and has applications in healthcare, biotechnology and electronics. Naturally, bacterial cellulose grows as a large unstructured polymer network around the bacteria that produce it, and tools to enable these bacteria to respond to different locations are required to grow more complex structured materials. Here, we introduce engineered cell-to-cell communication into a bacterial cellulose-producing strain of Komagataeibacter rhaeticus to enable different cells to detect their proximity within growing material and trigger differential gene expression in response. Using synthetic biology tools, we engineer Sender and Receiver strains of K. rhaeticus to produce and respond to the diffusible signalling molecule, acyl-homoserine lactone (AHL). We demonstrate that communication can occur both within and between growing pellicles and use this in a boundary detection experiment, where spliced and joined pellicles sense and reveal their original boundary. This work sets the basis for synthetic cell-to-cell communication within bacterial cellulose and is an important step forward for pattern formation within engineered living materials.

synthetic biology↗

bioRxiv2018

An Assessment of Skin Lesion Measurement Techniques for Use in Clinical Trials of Acute Bacterial Skin and Skin Structure Infections

BackgroundLimited data are available to support a reproducible measurement technique that could be used to assess the response of a skin lesion associated with a bacterial infection to antibacterial therapy.\n\nMethodsThis multicenter, observational study enrolled patients with a major cutaneous abscess, a traumatic wound or surgical site infection, or a cellulitis. The primary objective was to characterize the intra- and inter-observer variability inherent in measuring the size of the erythema associated with the presenting skin infection. At least two observers made ruler measurements of the infection site with the length of the infection measured as the longest dimension of the erythematous area and width measured as the largest dimension perpendicular to the longest length. Intra- and inter-observer variability was determined by the intraclass correlation coefficient (ICC). Photographs, tracings, and thermal imaging were also performed.\n\nResultsThe intra-observer ICC (95% CI) for lesion area as measured by ruler was 0.999 (0.998, 0.999), suggesting only a very small amount of the variability was due to measurement error. The difference in mean lesion area measurements by the same observer was <1%. The inter-observer ICC (95% CI) for lesion area as measured by ruler was 0.990 (0.981, 0.995) suggesting that the results between observers were also highly reliable.\n\nConclusionsMeasurement of infection area as defined by erythema and measured by ruler shows excellent intra- and inter-observer reliability and can be used in future clinical trials of acute bacterial skin infections.

pathology↗

bioRxiv2018

Vitexin alters Staphylococcus aureus surface hydrophobicity to interfere with biofilm formation.

Bacterial surface hydrophobicity is one of the determinant biophysical parameters of bacterial aggregation for being networked to form biofilm. Phytoconstituents like vitexin have long been in use for their antibacterial effect. The present work is aimed to characterise the effect of vitexin on S. aureus surface hydrophobicity and corresponding aggregation to form biofilm. We have found that vitexin shows minimum inhibitory concentration at 252 g/ml against S. aureus. Vitexin reduces cell surface hydrophobicity and membrane permeability at sub-MIC dose of 126 g/ml. The in silico binding analysis showed higher binding affinity of vitexin with surface proteins of S. aureus. Down regulation of dltA, icaAB and reduction in membrane potential under sub-MIC dose of vitexin, explains reduced S. aureus surface hydrophobicity. Vitexin has substantially reduced the intracellular adhesion of planktonic cells to form biofilm through interference of EPS formation, motility and subsequent execution of virulence. This was supported by the observation that vitexin down regulates the expression of icaAB and agrAC genes of S. aureus. In addition, vitexin also found to potentiate antibiofilm activity of sub-MIC dose of gentamicin and azithromycin. Furthermore, CFU count, histological examination of mouse tissue and immunomodulatory study justifies the in vivo protective effect of vitexin from S. aureus biofilm associated infection. Finally it can be inferred that, vitexin has the ability to modulate S. aureus cell surface hydrophobicity which can further interfere biofilm formation of the bacteria.\n\nImportanceThere has been substantial information known about role of bacterial surface hydrophobicity during attachment of single planktonic bacterial cells to any surface and the subsequent development of mature biofilm. This study presents the effect of flavone phytoconstituent vitexin on modulation of cell surface hydrophobicity in reducing formation of biofilm. Our findings also highlight the ability of vitexin in reducing in vivo S. aureus biofilm which will eventually outcompete the corresponding in vitro antibiofilm effect. Synergistic effect of vitexin on azithromycin and gentamicin point to a regime where development of drug tolerance may be addressed. Our findings explore one probable way of overcoming drug tolerance through application of vitexin in addressing the issue of S. aureus biofilm through modulation of cell surface hydrophobicity.

microbiology↗

bioRxiv2018

Inflammatory bowel disease microbiotas alter gut CD4 T-cell homeostasis and drive colitis in mice

To examine the functional contribution of Inflammatory Bowel Disease (IBD) microbes to immune homeostasis and colitis, we colonized unchallenged and colitis-susceptible germ-free mice with over twenty human intestinal microbiotas from healthy and IBD donors. Compared to healthy microbiotas, IBD microbiotas led to expanded ROR{gamma}t+Th17 cells and reduced ROR{gamma}t+Treg in the gut of unchallenged gnotobiotic mice and increased disease severity in colitis-susceptible mice. The proportions of ROR{gamma}t+Th17 and ROR{gamma}t+Treg induced by each microbiota were highly predictive of the human disease status and strongly correlated with disease severity in colitis-susceptible mice colonized with the same human microbiotas. The transmittable functional potential of IBD microbes suggests a mechanism for a microbial contribution to IBD pathogenesis and a potential route for its treatment and prevention.

immunology↗

bioRxiv2018

Beyond synthetic lethality: multiple gene interaction types play a key functional role in cancer

The phenotypic effect of perturbing a genes activity depends on the activity level of other genes, reflecting the notion that phenotypes are emergent properties of a network of functionally interacting genes. In the context of cancer, contemporary investigations have primarily focused on just one type of functional genetic interaction (GI) - synthetic lethality (SL). However, there may be additional types of GIs whose systematic identification would enrich the molecular and functional characterization of cancer. Here, we describe a novel data-driven approach called EnGIne, that applied to TCGA data identifies 71,946 GIs spanning 12 distinct types, only a small minority of which are SLs. The detected GIs explain cancer driver genes tissue-specificity and differences in patients response to drugs, and stratify breast cancer tumors into refined subtypes. These results expand the scope of cancer GIs and lay a conceptual and computational basis for future studies of additional types of GIs and their translational applications. The GI network is accessible online via a web portal [https://amagen.shinyapps.io/cancerapp/].

cancer biology↗

bioRxiv2017

Cross talk between photo-pigments and graphene electron cloud - Designing a biodiode

We report emergence of a new electrical material by growing photosynthetic biofilm on a Dirac material, graphene. The material showed new conducting as well as semiconducting properties. Frequency dependent capacitive spectra further indicated presence of electrical isosbestic points(at 0.8 and 9MHz), implying two state dieletric transitions at critical frequencies. A notable reult was a Schottky diode like behavior in the IV curve. Voltage dependent conductance with conductance peaks near the Schottky diode threshold was observed. We obtained facilitated growth of photosynthetic biofilm in presence of graphene. Lastly higher bacterial metabolism was seen in graphene incorporated biofilm. For this zero band gap Dirac material this can only be interpreted as coupling of the electron transport chain of the bacterial biofilm and the graphene electron cloud.

biophysics↗

bioRxiv2017

Emerging Electrical Properties of Graphene incorporated Photosynthetic Biofilms

We report emergence of a new electrical material by growing photosynthetic biofilm on a Dirac material, graphene. The material showed new conducting as well as semiconducting properties. Frequency dependent capacitive spectra further indicated presence of electrical isosbestic points(at 0.8 and 9MHz), implying two state dieletric transitions at critical frequencies. A notable reult was a Schottky diode like behavior in the IV curve. Voltage dependent conductance with conductance peaks near the Schottky diode threshold was observed. We obtained facilitated growth of photosynthetic biofilm in presence of graphene. Lastly higher bacterial metabolism i was seen in the biofilm in contact with graphene as compared to its normal growth condition. For this zero band gap Dirac material this can only be interpreted as coupling of the electron transport chain of the bacterial biofilm and the graphene electron cloud.

biophysics↗

bioRxiv2017

Ultrasound elicits behavioral responses through mechanical effects on neurons and ion channels in a simple nervous system

Focused ultrasound has been shown to stimulate excitable cells, but the biophysical mechanisms behind this phenomenon remain poorly understood. To provide additional insight, we devised a behavioral-genetic assay applied to the well-characterized nervous system of C. elegans nematodes. We found that pulsed ultrasound elicits robust reversal behavior in wild-type animals in a pressure-, duration-, and pulse protocol-dependent manner. Responses were preserved in mutants unable to sense thermal fluctuations and absent in mutants lacking neurons required for mechanosensation. Additionally, we found that the worm s response to ultrasound pulses rests on the expression of MEC-4, a DEG/ENaC/ASIC ion channel required for touch sensation. Consistent with prior studies of MEC-4-dependent currents in vivo, the worms response was optimal for pulses repeated 300 to 1000 times per second. Based on these findings, we conclude that mechanical, rather than thermal stimulation accounts for behavioral responses. Further, we propose that acoustic radiation force governs the response to ultrasound in a manner that depends on the touch receptor neurons and MEC-4-dependent ion channels. Our findings illuminate a complete pathway of ultrasound action, from the forces generated by propagating ultrasound to an activation of a specific ion channel. The findings further highlight the importance of optimizing ultrasound pulsing protocols when stimulating neurons via ion channels with mechanosensitive properties.\n\nSignificance StatementHow ultrasound influences neurons and other excitable cells has remained a mystery for decades. Although it is widely understood that ultrasound can heat tissues and induce mechanical strain, whether or not neuronal activation depends on heat, mechanical force, or both physical factors is not known. We harnessed C. elegans nematodes and their extraordinary sensitivity to thermal and mechanical stimuli to address this question. Whereas thermosensory mutants respond to ultrasound similar to wild-type animals, mechanosensory mutants were insensitive to ultrasound stimulation. Additionally, stimulus parameters that accentuate mechanical effects were more effective than those producing more heat. These findings highlight a mechanical nature of the effect of ultrasound on neurons and suggest specific ways to optimize stimulation protocols in specific tissues.

neuroscience↗

bioRxiv2017

Soft drug-resistant ovarian cancer cells invade via two distinct mechanisms utilizing myosin IIB

The failure of chemotherapeutic drugs in treatment of various cancers is attributed to the acquisition of drug resistance. However, the invasion mechanisms of drug-resistant cancer cells remains incompletely understood. Here we address this question from a biophysical perspective by mapping the phenotypic alterations in ovarian cancer cells (OCCs) resistant to cisplatin and paclitaxel. We show that cisplatin-resistant (CisR), paclitaxel-resistant (PacR) and dual drug-resistant (i.e., resistant to both drugs) OCCs are softer and more contractile than drug-sensitive cells. Protease inhibition suppresses invasion of CisR cells but not of PacR and dual cells, suggesting protease-dependent mode of invasion in CisR cells and protease-independent mode in PacR and dual cells. Despite these differences, actomyosin contractility, mediated by the RhoA-ROCK2-Myosin IIB signaling pathway regulates both modes of invasion. Myosin IIB modulates matrix metalloproteinase-9 (MMP-9) secretion in CisR cells and nuclear squeezing in PacR and dual cells, thereby highlighting its importance as a potential therapeutic target for treatment of drug-resistant ovarian cancer cells.\n\nFinancial SupportAuthors acknowledge financial support from IIT Bombay Healthcare Initiative, CSIR andDepartment of Biotechnology (Govt. of India) (Grant # BT/PR14658/MED/31/107/2010).AK and BTwere supported by fellowships from UGC and CSIR respectively (Govt. of India).\n\nAuthors declare no competing financial interests\n\nAuthor contributions: AK, PR and SS designed the experiments. AK performed most of the experiments and analyzed the data.BT and SG developed drug resistant cell lines and did RT-PCR. MM performed western blot. AD performed AFM experiments. SD performed gelatin zymography. ABB, PM, AM and AD helped with live cell imaging experiments. AK and SS wrote the manuscript. All authors read and approved the final manuscript.\n\nSummary statement: This study identifies drug-specific differences in the modes of invasion utilized by ovarian cancer cells, and demonstrates the role of myosin IIB in regulating both modes of invasion.

biophysics↗