Biology subjects

Das, A.

Publications and source records attributed to Das, A..

At least 199 records · Page 11Linked to original sources

bioRxiv2021

Differential Regulation of Rice Transcriptome to Rhizoctonia solani infection

Sheath Blight (SB) disease in rice crop caused by the infection of the fungal pathogen Rhizoctonia solani (R. solani) is one of the severe rice diseases that can cause up to 50% yield losses. Naturally occurring rice varieties resistant to SB have not been reported yet. We have performed a Time-Series RNA-Seq analysis on a widely cultivated rice variety BPT-5204 for identifying its transcriptomic response signatures to R. solani infection at 1st, 2nd and 5th day post inoculation (dpi). In total, 428, 3225 and 1225 genes were differentially expressed in the treated rice plants post 1, 2 and 5 dpi, respectively. GO and KEGG enrichment analysis identified significant processes and pathways differentially altered in the rice plant after the fungal infection. Machine learning and network based integrative approach was used to construct Transcriptional Regulatory Networks (TRNs) of the rice plant at the three Time Points. Regulatory network analysis identified SUB1B, MYB30 and CCA1 as important regulatory hub Transcription Factors in rice during R. solani infection. Jasmonic acid signaling pathway was activated and in contrast, photosynthesis and carbon fixation processes were significantly compromised. Involvement of MAPK, CYPs, Peroxidases and PAL genes was observed in response to the fungal infection. Circadian clock was also strongly influenced by R. solani infection. Our integrative analysis identified 7 putative SB resistant genes altered in rice after R. solani infection and provided a better understanding of rice plant response to R. solani infection. One sentence summaryTime series expression analysis of rice variety BPT-5204 identifies key molecular signatures involved in rice plant response to R. solani infection.

systems biology↗

bioRxiv2021

Why does Δ9-Tetrahydrocannabinol act as a partial agonist of cannabinoid receptors?

Cannabinoid receptor 1 (CB1) is a therapeutically relevant drug target for controlling pain, obesity, and other central nervous system disorders. However, full agonists and antagonists of CB1 have been reported to cause serious side effects in patients. Therefore, partial agonists have emerged as a viable alternative to full agonists and antagonists as they avoid overstimulation and side effects. One of the key bottlenecks in the design of partial agonists is the lack of understanding of the molecular mechanism of partial agonism. In this study, we examine two mechanistic hypotheses for the origin of partial agonism in cannabinoid receptors and explain the mechanistic basis of partial agonism exhibited by {Delta}9-Tetrahydrocannabinol (THC). In particular, we inspect whether partial agonism emerges from the ability of THC to bind in both agonist and antagonist binding pose or from its ability to only partially activate the receptor. Extensive molecular dynamics simulations and the Markov state model capture the THC binding in both antagonist, and agonist binding poses in CB1 receptor. Furthermore, we observe that binding of THC in the agonist binding pose leads to rotation of toggle switch residues and causes partial outward movement of intracellular transmembrane helix 6 (TM6). Our simulations also suggest that the alkyl side chain of THC plays a crucial role in determining partial agonism by stabilizing the ligand in the agonist and antagonist-like poses within the pocket. This study provides us fundamental insights into the mechanistic origin of the partial agonism of THC.

biophysics↗

bioRxiv2021

Identification of oxalyl-CoA synthetase gene (LsAAE3) and its regulatory role in β-ODAP biosynthesis in grasspea (Lathyrus sativus L.)

Grasspea is a popular pulse crop due to its hardiness and low cost of production. Presence of anti-nutritive factor {beta}-ODAP in its seeds and other plant parts hinder its widespread cultivation and usage. Oxalyl-CoA synthetase is one of the key enzyme of {beta}-ODAP biosynthesis pathway, catalyses the conversion of oxalate to oxalyl-CoA. ACYL ACTIVATING ENZYME 3 (AAE3) gene has been characterised to encode an oxalyl-CoA synthetase enzyme in many plant species. We report here the isolation of full length AAE3 homolog in grasspea with a combination of PCR based strategy and in silico analysis. We first identified AAE3 homolog by PCR using degenerate primers. The partial LsAAE3 sequence showed 88% amino acids sequence identity with the characterised AAE3 gene of M. truncatula. We then predicted the full length AAE3 sequence using the publically available transcriptome datasets of grasspea. Determination of LsAAE3 gene and protein structure and phylogenetic relationship analysis strongly suggested that LsAAE3 is a true homolog of AAE3 gene. Expression profiling of LsAAE3 in grasspea varieties with contrast in {beta}-ODAP content revealed its inverse relationship with the {beta}-ODAP content, LsAAE3 thus negatively regulates the synthesis of {beta}-ODAP. Involvement of AAE3 encoded oxalyl-CoA synthetase in a CoA-dependent pathway of oxalate degradation is well proven in many plant species. We also identified the CoA-dependent pathway of oxalate degradation in grasspea. Based on these observations, we hypothesized that LsAAE3 may regulate {beta}-ODAP content, possibly, by CoA-dependent pathway of oxalate degradation in grasspea. If this hypothesis is substantiated, genetic manipulation of LsAAE3 presents viable option for reducing {beta}-ODAP content in grass pea.

molecular biology↗

bioRxiv2021

The RNA-binding protein RBP42 regulates cellular energy metabolism in mammalian-infective Trypanosoma brucei

RNA-binding proteins are key players in coordinated post-transcriptional regulation of functionally related genes, defined as RNA regulons. RNA regulons play particularly critical roles in parasitic trypanosomes, which exhibit unregulated co-transcription of long arrays of unrelated genes. In this report, we present a systematic analysis of an essential RNA-binding protein, RBP42, in the mammalian-infective slender bloodstream form of African trypanosome, and we show that RBP42 is a key regulator of parasites central carbon and energy metabolism. Using individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify genome-wide RBP42-RNA interactions, we show that RBP42 preferentially binds within the coding region of mRNAs encoding core metabolic enzymes. Using global quantitative transcriptomic and proteomic analyses, we also show that loss of RBP42 reduces the abundance of target mRNA-encoded proteins, but not target mRNA, suggesting a plausible role of RBP42 as a positive regulator of target mRNA translation. Analysis reveals significant changes in central carbon metabolic intermediates following loss of RBP42, further supporting its critical role in cellular energy metabolism.

molecular biology↗

bioRxiv2021

Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Methods that dynamically monitor these intracellular effects are currently lacking. Here, we leveraged the environmental sensitivity and structural plasticity of intrinsically disordered regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use in environmentally-responsive molecular tools.

cell biology↗

bioRxiv2021

Nef dimerization defect abrogates HIV viremia and associated immune dysregulation in the Bone Marrow-Liver-Thymus-Spleen (BLTS) humanized mouse model

Loss of function mutations in the human immunodeficiency virus (HIV) negative factor (Nef) gene are associated with reduced viremia, robust T cell immune responses, and delayed acquired immunodeficiency syndrome (AIDS) progression in humans. In vitro studies have shown that mutations in the Nef dimerization interface significantly attenuate viral replication and impair host defense. However, in vivo, mechanistic studies on the role of Nef dimerization in HIV infection are lacking. Humanized rodents with human immune cells are robust platforms for investigating the interactions between HIV and the human immune system. The bone marrow-liver-thymus-spleen (BLTS) humanized mouse model carries human immune cells and lymphoid tissues that facilitate anti-viral immune responses. Here, we employed the BLTS-humanized mouse model to demonstrate that preventing Nef dimerization abrogates HIV viremia and the associated immune dysregulation. This suggests that Nef dimerization may be a therapeutic target for future HIV cure strategies.

immunology↗

bioRxiv2021

-actinin-4 drives invasiveness by regulating myosin IIB expression and myosin IIA localization

The mechanisms by which the mechanoresponsive actin crosslinking protein -actinin-4 (ACTN4) regulates cell motility and invasiveness remains incompletely understood. Here we show that in addition to regulating protrusion dynamics and focal adhesion formation, ACTN4 transcriptionally regulates expression of non-muscle myosin IIB (NMM IIB), which is essential for mediating nuclear translocation during 3D invasion. We further demonstrate association between NMM IIA and ACTN4 at the cell front ensures retention of NMM IIA at the cell periphery. A protrusion-dependent model of confined migration recapitulating experimental observations predicts a dependence of protrusion forces on the degree of confinement and on the ratio of nucleus to matrix stiffness. Together, our results suggest that ACTN4 is a master regulator of cancer invasion that regulates invasiveness by controlling NMM IIB expression and NMM IIA localization.

cell biology↗

bioRxiv2021

Inhibition of Extracellular Vesicle-Associated MMP2 Abrogates Intercellular Transfer of Hepatic miR-122 to Tissue Macrophages and Curtails Liver Inflammation

microRNA-122 (miR-122), a liver specific regulatory RNA, plays an important role in controlling metabolic homeostasis in mammalian liver cells. Interestingly, miR-122 is also a proinflammatory microRNA and when exported to tissue resident macrophage induces expression of inflammatory cytokines there. We found intercellular transfer of miR-122 in lipid exposed liver plays a role in liver inflammation. Exploring the mechanism of intercellular miR-122 transfer from hepatic cells, we detected MMP2 on the membrane of extracellular vesicles derived from hepatic cells which proved to be essential for transfer of extracellular vesicles and their miRNA content from hepatic to non-hepatic cells. Matrix metalloprotease 2 or MMP2 is a metalloproteinase that plays a key role in shaping and remodelling the extracellular matrix of human tissue by targeting degradation of matrix proteins. MMP2 was found to increase the movement of the EVs along the extracellular matrix to enhance their uptake in recipient cells. Inhibition of MMP2 restricts functional transfer of hepatic miRNAs across the hepatic and non-hepatic cell boundaries. By targeting MMP2, we could reduce the innate immune response in mammalian liver by preventing intra-tissue miR-122 transfer. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/425217v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@aec3b0org.highwire.dtl.DTLVardef@1b18e87org.highwire.dtl.DTLVardef@47e75org.highwire.dtl.DTLVardef@10a48e0_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIHuman hepatocytes on exposure to high lipid export out miRNAs including proinflammatory miR-122. C_LIO_LIExtracellular miR-122 is taken up by tissue macrophages to get them activated to produce inflammatory cytokines. C_LIO_LIMMP2 present on the surface of the EVs released by hepatocyte is essential for miRNA transfer to macrophage cells C_LIO_LIInhibition of MMP2 prevents miR-122 transfer to macrophage and stops activation of recipient macrophage. C_LI

cancer biology↗

bioRxiv2020

A quick and cost-effective method for DNA-free total RNA isolation using magnetic silica beads

Current RNA purification methods widely use silica-based columns that allow quick isolation of high-quality and good quantities of RNA. However, the major limitations include high cost, the requirement of different kits for small RNA isolation, genomic DNA contamination, and not being flexible. Here, we used the in-house RNA isolation reagent (RIR) for cell lysis, followed by RNA precipitation using isopropanol. RNA isolated using the in-house RIR resulted in a similar quantity and quality compared to the commercial TRIzol. Furthermore, the commercial RNA isolation kits with silica-based columns recommend genomic DNA digestion during or after RNA purification, adding time and cost to RNA purification. Here, we developed an optimized in-house protocol for isolating high-quality RNA free of genomic DNA contamination using magnetic silica beads without needing DNase digestion. Additionally, our method purifies total RNA along with the small RNA fraction, including miRNAs, which usually require a separate kit for extraction. Additionally, the RNA prepared with our method was equally suitable for mRNA and miRNA expression analysis using RT-qPCR. Together, the in-house method of RNA isolation using the magnetic silica beads has exhibited comparable or better total RNA extraction compared to commercial kits at a fraction of the cost and across various cells and tissues.

molecular biology↗

bioRxiv2020

The molecular and metabolic program for adaptation of white adipocytesto cool physiologic temperatures

Although visceral adipocytes located within the bodys central core are maintained at ~37{degrees}C, adipocytes within bone marrow, subcutaneous, and dermal depots are found primarily within the peripheral shell, and generally exist at cooler temperatures. Responses of brown and beige/brite adipocytes to cold stress are well-studied; however, comparatively little is known about mechanisms by white adipocytes adapt to temperatures below 37{degrees}C. Here we report that adaptation of cultured adipocytes to 31{degrees}C, the temperature at which distal marrow adipose tissues and subcutaneous adipose tissues often reside, induces extensive changes in gene expression, increased anabolic and catabolic lipid metabolism, and elevated oxygen consumption with reduced reliance on glucose and preferential use of pyruvate, glutamine and fatty acids as energy sources. Cool temperatures up-regulate stearoyl-CoA desaturase-1 expression and monounsaturated lipid levels in cultured adipocytes and distal bone marrow adipose tissues, and stearoyl-CoA desaturase-1 activity is required for acquisition of maximal oxygen consumption at 31{degrees}C.

physiology↗

bioRxiv2020

Semiconducting bacterial biofilm based on graphene-MoS2 template and component dependent gating behavior

In this paper, we report for the first time, the synthesis of a semiconducting biofilm. Photosynthetic bacterial biofilm has been used to weave together MoS2 nanosheets into an adherent film grown on interdigitated electrodes. Liquid-phase exfoliation of bulk MoS2 powder was used to obtain MoS2 nanosheets. A synchronous-fluorescence scan revealed the presence of two emission maxima at 682nm and 715nm for the MoS2 suspension. Such maxima with bandgap energy 1.82 and 1.73 eV corresponded to the single and double layer of MoS2. The presence of such single and multi-layered structures was confirmed by Raman spectroscopy, FTIR studies, and electron microscopy. The current-voltage (I-V) studies of such a bio-nano hybrid revealed the emergence of the gated nature of the current flow. This Schottky diode like behavior, reported earlier for Graphene-biofilm junctions, is also observed in this case. Gating voltage depended on the composition of the biofilm. The semiconductor biofilms, when studied using electrochemical impedance spectroscopy, revealed characteristic Nyquist and Bode plots, suggesting special circuit-equivalence for each film. While Mos2 was marked with stability with respect to variations in RMS voltage and bias voltage, the graphene biofilm was unique by the absence of any Warburg element.

synthetic biology↗

bioRxiv2020

Nicotinamide mononucleotide (NMN) de-amidation by the gut microbiome and evidence for indirect upregulation of the NAD+ metabolome

Oral administration of nicotinamide mononucleotide (NMN) is a prominent strategy to elevate nicotinamide adenine dinucleotide (NAD+) levels to treat age-related pathologies, where it is assumed to be directly incorporated into the NAD+ metabolome through the canonical recycling pathway. During oral delivery, NMN is exposed to the gut microbiome, which can modify the NAD+ metabolome through enzyme activities that are not present in mammals. Here, we show that orally delivered NMN can undergo direct deamidation and incorporation in mammalian tissue via the de novo pathway, and that this deamidation is reduced in animals treated with antibiotics to ablate the gut microbiome. Further, we show that antibiotics treatment increases the overall availability of NAD+ metabolites in the gut epithelium, with one possibility that the gut microbiome could be in competition with the host for dietary NAD+ precursors. Together, these data highlight previously undescribed interactions between orally delivered NMN and the gut microbiome.

biochemistry↗

bioRxiv2020

Decoding of attentional state using high-frequency local field potential is as accurate as using spikes

Local field potentials (LFPs) in visual cortex are reliably modulated when the subjects focus of attention is cued into versus out of the receptive field of the recorded sites, similar to modulation of spiking activity. However, human psychophysics studies have used an additional attention condition, neutral cueing, for decades. The effect of neutral cueing on spiking responses was examined recently and found to be intermediate between cued and uncued conditions. However, whether LFPs are also precise enough to represent graded states of attention is unknown. We found that LFPs during neutral cueing were intermediate between cued and uncued conditions, and, for a single electrode, attention was more discriminable using high frequency (>30 Hz) LFP power than spikes. Surprisingly, spikes did not outperform LFPs even when discriminability was computed using multiple electrodes. These results constrain the spatial scale attention operates over and highlight the usefulness of LFPs in studying attention.

neuroscience↗

bioRxiv2020

Protein Phosphatase 2A-B56 maintains the meiotic spindle, kinetochore attachments and cohesion by antagonizing Aurora B Drosophila Oocytes

Meiosis in female oocytes lack centrosomes, the major microtubule-organizing center, which makes them especially vulnerable to aneuploidy. In the acentrosomal oocytes of Drosophila, meiotic spindle assembly depends on the chromosomal passenger complex (CPC). Aurora B is the catalytic component of the CPC while the remaining subunits regulate its localization. Using an inhibitor of Aurora B activity, Binucleine 2, we found that continuous Aurora B activity is required to maintain the oocyte spindle during meiosis I, and this activity is antagonized by phosphatases acting on spindle associated proteins such as kinesins. Protein Phosphatase 2A (PP2A) exists in two varieties, B55 and B56. While both antagonize Aurora B, B55 has only minor roles in meiosis I spindle function. The B56 subunit is encoded by two partially redundant paralogs in the Drosophila genome, wdb and wrd. Knocking down both paralogs showed that the B56 subunit is critical for maintaining sister chromatid cohesion, establishing end-on microtubule attachments, and the metaphase I arrest in oocytes. We found that WDB recruitment to the centromeres depends on BUBR1, MEI-S332, and kinetochore protein SPC105R. While BUBR1 has been shown previously to stabilize microtubule attachments in Drosophila oocytes, only SPC105R is required for cohesion maintenance during meiosis I. We propose that SPC105R promotes cohesion maintenance by recruiting two proteins that recruit PP2A, MEI-S332, and the Soronin homolog Dalmatian.

genetics↗

bioRxiv2020

Electrophysiological foundations of the human default-mode network revealed by brain-wide intracranial-EEG recordings during resting-state and cognition

Investigations using noninvasive functional magnetic resonance imaging (fMRI) have provided significant insights into the unique functional organization and profound importance of the human default mode network (DMN), yet these methods are limited in their ability to resolve network dynamics across multiple timescales. Electrophysiological techniques are critical to address these challenges, yet few studies have explored the neurophysiological underpinnings of the DMN. Here we investigate the brain-wide electrophysiological organization of the DMN in a common large-scale network framework consistent with prior fMRI studies. We used brain-wide intracranial EEG (iEEG) recordings, and evaluated intra- and cross-network interactions during the resting-state and cognition. Our analysis revealed significantly greater intra-DMN phase iEEG synchronization in the slow-wave (< 4 Hz) while DMN interactions with other brain networks was higher in all higher frequencies. Crucially, slow-wave intra-DMN synchronization was observed in the task-free resting-state and during verbal memory encoding and recall. Compared to resting-state, intra-DMN phase synchronization was significantly higher during both memory encoding and recall. Slow-wave intra-DMN phase synchronization increased during successful memory retrieval, highlighting its behavioral relevance. Finally, analysis of nonlinear dynamic causal interactions revealed that the DMN is a causal outflow network during both memory encoding and recall. Our findings identify dynamic spectro-temporal network features that allow the DMN to maintain a balance between stability and flexibility, intrinsically and during task-based cognition, provide novel insights into the neurophysiological foundations of the human DMN, and elucidate network mechanisms by which it supports cognition.

neuroscience↗

bioRxiv2020

Genome-wide binding analysis of 195 DNA Binding Proteins reveals "reservoir" promoters and human specific SVA-repeat family regulation

A key aspect in defining cell state is the complex choreography of DNA binding events in a given cell type, which in turn establishes a cell-specific gene-expression program. In the past two decades since the sequencing of the human genome there has been a deluge of genome-wide experiments which have measured gene-expression and DNA binding events across numerous cell-types and tissues. Here we re-analyze ENCODE data in a highly reproducible manner by utilizing standardized analysis pipelines, containerization, and literate programming with Rmarkdown. Our approach validated many findings from previous independent studies, underscoring the importance of ENCODEs goals in providing these reproducible data resources. This approach also revealed several new findings: (i) 1,362 promoters, termed reservoirs, have up to 111 different DNA binding-proteins localized on one promoter yet do not have any expression of steady-state RNA (ii) The human specific SVA repeat element may have been co-opted for enhancer regulation. Collectively, this study performed by the students of a CU Boulder computational biology class (BCHM 5631 - Spring 2020) demonstrates the value of reproducible findings and how resources like ENCODE that prioritize data standards can foster new findings with existing data in a didactic environment.

genomics↗

bioRxiv2020

Receptor-independent membrane mediated pathways of serotonin action

Serotonin is a neurotransmitter as well as a somatic signaling molecule, and the serotonergic system is a major target for psychotropic drugs. Serotonin, together with a few related neurotransmitters, has recently been found to exhibit an unexpectedly high lipid membrane affinity1–3. It has been conjectured that extrasynaptic serotonin can diffuse in the lipid membrane to efficiently reach remote receptors (and receptors with buried ligand-binding sites)4, providing a mechanism for the diffuse ‘volume’ neurotransmission that serotonin is capable of5–10. Here we show that membrane binding by serotonin can directly modulate membrane properties and cellular function, independent of its receptor-mediated actions. Atomic force microscopy shows that serotonin binding makes artificial lipid bilayers softer. It induces nucleation of liquid disordered domains inside the raft-like liquid-ordered domains in a ternary bilayer displaying phase separation. Solid-state NMR spectroscopy corroborates this data, revealing a rather homogeneous decrease in the order parameter of the lipid chains in the presence of serotonin. In the RN46A immortalized serotonergic neuronal cell line, extracellular serotonin enhances transferrin receptor endocytosis, an action exerted even in the presence of both broad-spectrum serotonin receptor and transporter inhibitors. Similarly, it increases the binding and internalization of Islet Amyloid Polypeptide (IAPP) oligomers, suggesting a connection between serotonin, which is co-secreted with IAPP by pancreatic beta cells, and the cellular effects of IAPP. Our results uncover a hitherto unknown serotonin-bilayer interaction that can potentiate key cellular processes in a receptor-independent fashion. Therefore, some pathways of serotonergic action may escape potent pharmaceutical agents designed for serotonin transporters or receptors. Conversely, bio-orthogonal serotonin-mimetics may provide a new class of cell-membrane modulators.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Monocyte Subsets with High Osteoclastogenic Potential and Their Epigenetic Regulation Orchestrated by IRF8

Osteoclasts (OCs) are bone resorbing cells formed by the serial fusion of monocytes. In mice and humans, three distinct subsets of monocytes exist; however, it is unclear if all of them exhibit osteoclastogenic potential. Here we show that in wild-type mice, Ly6Chi and Ly6Cint monocytes are the primary source of OC formation when compared to Ly6C- monocytes. Their osteoclastogenic potential is dictated by increased expression of signaling receptors and activation of pre-established transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of IRF8, a transcription factor important for myelopoiesis and osteoclastogenesis, all three monocyte subsets are programmed to display higher osteoclastogenic potential. Enhanced NFATc1 nuclear translocation and amplified transcriptomic and epigenetic changes initiated at early developmental stages direct the increased osteoclastogenesis in Irf8 deficient mice. Collectively, our study provides novel insights into the transcription factors and active cis-regulatory elements that regulate OC differentiation.

immunology↗