bioRxiv Science⌕ Search

Biology subjects

Thakur, A.

Publications and source records attributed to Thakur, A..

29 records · Page 2Linked to original sources
bioRxiv2020

Regeneration of lung epithelial cells by Fullerene C60 nanoformulation: A possible treatment strategy for acute respiratory distress syndrome (ARDS)

Acute respiratory distress syndrome (ARDS) involves death of lung epithelial cells. ARDS is a leading reason behind mortality in respiratory infections. Here we show a proof-of-concept that a Fullerene nanoformulation can be used for the regeneration of cells treated with apoptosis-inducing molecules, suggeting its potential for ARDS therapy.

pharmacology and toxicology↗
bioRxiv2020

UHRF1 suppresses viral mimicry through both DNA methylation-dependent and -independent mechanisms

Some chemotherapeutic agents which cause loss of DNA methylation have been recently shown to induce a state of "viral mimicry" involving upregulation of endogenous retroviruses (ERV) and a subsequent innate immune response. This approach may be useful in combination with immune checkpoint cancer therapies, but relatively little is known about normal cellular control of ERV suppression. The UHRF1 protein can interact with the maintenance methylation protein DNMT1 and is known to play an important role in epigenetic control in the cell. To examine potential roles of this protein in differentiated cells, we first established stable knockdowns in normal human lung fibroblasts. While these knockdown cells showed the expected loss of DNA methylation genome-wide, transcriptional changes were instead dominated by a single response, namely activation of innate immune signalling, consistent with viral mimicry. We confirmed using mechanistic approaches that activation of interferons and interferon-stimulated genes involved in double-stranded RNA detection was crucial to the response. ERVs were demethylated and transcriptionally activated in UHRF1 knockdown cells. As in these normal cell lines, ERV activation and interferon response also occurred following the transient loss of UHRF1 in both melanoma and colon cancer cell lines. Restoring UHRF1 in either transient- or stable knockdown systems abrogated ERV reactivation and interferon response, but without substantial restoration of DNA methylation. Rescued cell lines were hypersensitive to depletion of SETDB1, implicating H3K9me3 as crucial to UHRF1-mediated repression in the absence of DNA methylation. Confirming this, cells rescued with UHRF1 containing point mutations affecting H3K9me3 binding could not mediate silencing of ERV transcription or the innate immune response. Finally, by introducing similar point mutations in the mouse homologue, we could show that this pathway is conserved in mice. Our results therefore implicate UHRF1 as a key regulator of ERV suppression and strengthen the basis for cancer cell hypomethylation therapy.

genomics↗
bioRxiv2020

Disambiguating stress-buffering and relationship-renewal in reunions between female degus

Many animals become more motivated to interact after a period of isolation. This phenomenon may involve general drives, e.g. for social touch or companionship, as well as drives that are specific to particular peers, and which ultimately serve to reestablish relationships between the individuals. Female degus are known to be affiliative with multiple other individuals, including unrelated and unfamiliar conspecifics, offering an opportunity to study social motivation independent from exclusive pair-bonds or overt, same-sex competition. We attempted to disentangle factors driving peer interaction by examining reunion behavior across several social isolation and separation manipulations. High levels of interaction were observed between adult females who had been separated even without isolation, revealing a drive to re-establish relationships with specific peers. The content of separation-only reunions differed from isolation, with the latter involving more early-session interaction, higher levels of allogrooming before rear-sniffing, and a higher ratio of chitter vocalizations. To assess whether post-isolation behavior was related to stress, we examined reunions following a non-social (footshock) stressor. Like isolation, footshock increased early-session interactions, but did not increase allogrooming before rear-sniffing or chittering, as compared with controls. To test whether separation-only reunion behavior shared qualities with relationship formation, we also examined reunions of new (stranger) dyads. Strangers exhibited higher levels of interaction than cagemates, with particularly high levels of late-session rear-sniffing. Like separation-only reunions, strangers showed more non-chitter vocalizations and lower levels of allogrooming before rear-sniffing. Across experiments, an exploratory clustering method was used to identify vocalizations that differed between conditions. This yielded promising leads for future investigation, including a chaff-type syllable that may have been more common during relationship renewal. Overall, results are consistent with the hypothesis that female degu reunions are supported by both general and peer-stimulus specific drives, expressed through the structure of physical and vocal interactions over time.

animal behavior and cognition↗
bioRxiv2020

Novel PE and APC Tandems: Additional Bright Near-Infrared Tandem Fluorochromes for Use in Spectral Flow Cytometry

Recent advances in flow cytometry instrumentation and fluorochrome chemistries have greatly increased fluorescent conjugated antibody combinations that can be used reliably and easily in routine experiments. The Cytek Aurora flow cytometer has three excitation lasers (405nm, 488nm, and 640nm) and incorporates the latest Avalanche Photodiode (APD) technology, demonstrating significant improvement in sensitivity for fluorescent emission signals longer than 800nm. However, there are limited commercially available fluorochromes capable of excitation with peak emission signals beyond 800nm. To address this gap, we engineered 6 new fluorochromes: PE-750, PE-800, PE-830 for the 488nm laser and APC-750, APC-800, APC-830 for the 640nm laser. Utilizing the principal of fluorescence resonance energy transfer (FRET), these novel structures were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Additionally, each of these fluorochrome conjugates were shown to be compatible with Fixation/Permeabilization buffer reagents, and demonstrated acceptable brightness and stability when conjugated to antigen-specific monoclonal antibodies. These six novel fluorochrome reagents can increase the numbers of fluorochromes that can be used on a 3-laser spectral flow cytometer.

immunology↗
bioRxiv2020

Rub1/NEDD8, a ubiquitin-like modifier, is also a ubiquitin modifier

Of all ubiquitin-like small protein modifiers, Rub1/NEDD8 is the closest kin of ubiquitin in sequence and in structure. Despite their profound similarities, prevalence of ubiquitin and of Rub1 is starkly different: targets of ubiquitin modification reach into the thousands, whereas unique targets of Rub1/NEDD8 appear limited to one family of proteins, Cullins. This distinction is likely due to dedicated E1 activating enzymes that select either one or the other and relay the modifier until it is covalently attached to a target. To convert typical neddylation targets for modification by ubiquitin, and vice versa, we designed reciprocal substitutions at position 72 of Rub1 and of ubiquitin to render them substrates for activation by their non-cognate E1 activating enzymes. We found that this single amino acid is sufficient to distinguish between Ub and Rub1 in living cells, and determine their targets. Thus, modification of Cullins by UbR72T could compensate for loss of Rub1, even as it maintained its ability to polymerize and direct conjugates for degradation. Conversely, Rub1T72R activated by ubiquitin-activating enzyme entered into the ubiquitination cascade, however was not efficiently polymerized, essentially capping polyubiquitin chains. Upon shortage of free ubiquitin under stress, even native Rub1 spilled-over into the ubiquitinome suppressing polyubiquitination. By contrast, the need to maintain monomeric modifications on unique targets is a likely explanation for why the Rub1-activating enzyme strictly discriminates against ubiquitin. Swapping Rub1 and ubiquitin signals uncovered a reason for maintaining two separate pathways across eukaryotic kingdom.

biochemistry↗
bioRxiv2020

CYCLODEXTRIN DERIVATIVE ENHANCES THE OPHTHALMIC DELIVERY OF POORLY SOLUBLE AZITHROMYCIN.

Azithromycin (AZM), a macrolide antibiotic used for the treatment of Chlamydial conjunctivitis, is less effective for the treatment of this disease due to its poor bioavailability (38%). Various alternatives have been developed for improving the physico-chemical properties (i.e., solubility) of the AZM without much success. To overcome the problems associated with AZM, an inclusion complex employing a modified cyclodextrin i.e., sulfobutylether-{beta}-cyclodextrin (SBE-{beta}-CD) was prepared and characterized by phase solubility studies, pXRD and FTIR techniques. The results portrayed the formation of the inclusion complex of AZM with sulfobutylether {beta}-cyclodextrin (SBE-{beta}-CD) in 1:2 molar stoichiometric ratios. This inclusion complex was later incorporated into a polymer matrix to prepare an in situ gel. Various combinations of carbopol 934P and hydroxypropyl methylcellulose (HPMC K4M) polymers were used and evaluated by rheological and in vitro drug release studies. The optimized formulation (F4), containing carbopol 934P 0.2% (w/v) and HPMC K4M 0.2% (w/v), was evaluated for clarity, pH, gelling capacity, drug content, rheological properties, in vitro drug release pattern, ocular irritation test and antimicrobial efficacy. Finally, owing to the improved antimicrobial efficacy and increased residence time, AZM:SBE-{beta}-CD in situ gel was found to be a promising formulation for the efficient treatment of bacterial ocular disease.

microbiology↗
bioRxiv2020

Investigation of a night soil compost psychrotrophic bacterium Glutamicibacter arilaitensis LJH19 for its safety, efficient hydrolytic and plant growth-promoting potential

Night soil compost (NSC) has traditionally been a source of organic manure in north-western Himalaya. Lately, this traditional method is declining due to modernization, its unhygienic conditions and social apprehensions. Reduction in the age-old traditional practice has led to excessive usage of chemical fertilizers and shortage of water in the eco-sensitive region. Microbiological intervention was attempted to obtain bacterial consortia for accelerated degradation of human faeces in cold climate to improvise this traditional knowledge. Glutamicibacter arilaitensis LJH19, a psychrotrophic bacteria was identified as one such potential candidate for the proposed consortia. The bacterium was isolated from NSC of Lahaul valley and exhibited potential hydrolytic activities, the specific activities of amylase, cellulase and xylanase was observed as 186.76 U/mg, 21.85 U/mg and 11.31 U/mg respectively. Additionally, the strain possessed multiple plant growth-promoting (PGP) traits. The bacterium produced 166.11 {micro}g/ml indole acetic acid and 85.72 % siderophore units, and solubilized 44.76 {micro}g/ml phosphate. Whole genome sequence (3,602,821 bps) endorsed the cold adaptation, polysaccharide metabolism, PGP potential of the bacterium. Genome mining revealed biosynthetic gene clusters for type III polyketide synthase (PKS), terpene, and siderophore in agreement with its potential PGP traits. Comparative genomics within the genus revealed 217 unique genes specific to hydrolytic and PGP activity. Negative haemolysis and biofilm production and susceptibility towards all 12 tested antibiotics indicated the bacterium to be a safe bioinoculant. Genomic investigation supported the bacterium safety with absence of any virulence and antibiotic resistance genes. We propose the strain LJH19 to be a potentially safe bioinoculant candidate for efficient degradation of night soil owing to its survivability in cold and its efficient hydrolytic and PGP potential.

microbiology↗
bioRxiv2020

A Drosophila model of oral peptide therapeutics for adult intestinal stem cell tumors

The proto-oncogene YAP /Yki, a transcription co-factor of the Hippo pathway, has been linked to many cancers. YAP interacts with DNA-binding TEAD/Sd proteins to regulate expression of its transcriptional targets. Disruption of YAP-TEAD therefore offers a potential therapeutic strategy. The mammalian Vestigial Like (VGLL) protein, specifically its TONDU domain, has been shown to competitively inhibit YAP-TEAD interaction and a TONDU peptide can suppress YAP-induced cancer. As TONDU could potentially be developed into a therapeutic peptide for multiple cancers, we evaluated its efficacy in Yki-driven adult Intestinal Stem Cell (ISC) tumors in Drosophila. We show that oral uptake of the TONDU peptide is highly effective at inhibiting Yki-driven gut tumors by suppressing YAP-TEAD interaction. Comparative proteomics of early and late stage Yki-driven ISC tumors revealed enrichment of a number of proteins, including members of the integrin signaling pathway, such as Talin, Vinculin and Paxillin. These, in turn displayed a decrease in their levels in TONDU-peptide treated tumors. Further, we show that Sd binds to the regulatory region of integrin-coding gene, mew, which codes for PS1, a key integrin of the ISCs. In support to a possible role of integrins in Yki-driven ISC tumors, we show that genetic downregulation of mew arrests Yki-driven ISC proliferation, reminiscent of the effects of TONDU peptide. Altogether, our findings present a novel platform for screening therapeutic peptides and provide insights into tumor suppression mechanisms. SIGNIFICANCE STATEMENTDiscovering novel strategies to inhibit oncogene activity is a priority in cancer biology. As signaling pathways are widely conserved between mammals and Drosophila, these questions can be effectively addressed in this model organism. Here, we show that progression of Drosophila Intestinal Stem Cell (ISC) tumors induced by gain of an oncogenic form of the transcription co-factor Yki can be suppressed by feeding a peptide corresponding to the conserved TONDU domain of Vestigial (Vg), which blocks binding of Yki to the Sd transcription factor. Further, we show that down regulation of the integrin signaling pathway is causally linked to TONDU-peptide-mediated ISC tumor suppression. Our findings reveal that Drosophila can be successfully used to screen peptides for their therapeutic applications.

cancer biology↗
bioRxiv2019

Predicting glaucoma prior to its onset using deep learning

PurposeTo assess the accuracy of deep learning models to predict glaucoma development from fundus photographs several years prior to disease onset.\n\nDesignA deep learning model for prediction of glaucomatous optic neuropathy or visual field abnormality from color fundus photographs.\n\nParticipantsWe retrospectively included 66,721 fundus photographs from 3,272 eyes of 1,636 subjects to develop deep leaning models.\n\nMethodFundus photographs and visual fields were carefully examined by two independent readers from the optic disc and visual field reading centers of the ocular hypertension treatment study (OHTS). When an abnormality was detected by the readers, subject was recalled for re-testing to confirm the abnormality and further confirmation by an endpoint committee. Using OHTS data, deep learning models were trained and tested using 85% of the fundus photographs and further validated (re-tested) on the remaining (held-out) 15% of the fundus photographs.\n\nMain Outcome MeasuresAccuracy and area under the receiver-operating characteristic curve (AUC).\n\nResultsThe AUC of the deep learning model in predicting glaucoma development 4-7 years prior to disease onset was 0.77 (95% confidence interval 0.75, 0.79). The accuracy of the model in predicting glaucoma development about 1-3 years prior to disease onset was 0.88 (0.86, 0.91). The accuracy of the model in detecting glaucoma after onset was 0.95 (0.94, 0.96).\n\nConclusionsDeep learning models can predict glaucoma development prior to disease onset with reasonable accuracy. Eyes with visual field abnormality but not glaucomatous optic neuropathy had a higher tendency to be missed by deep learning algorithms.

bioengineering↗
bioRxiv2017

Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons

The eukaryotic 43S pre-initiation complex (PIC) bearing Met-tRNA[Formula] in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable \"Kozak\" context. AUG recognition provokes rearrangement from an open PIC conformation with TC bound in a state not fully engaged with the P site (\"POUT\") to a closed, arrested conformation with TC tightly bound in the \"PIN\" state. Yeast ribosomal protein Rps3/uS3 resides in the mRNA entry channel of the 40S subunit and contacts mRNA via conserved residues whose functional importance was unknown. We show that substitutions of these residues reduce bulk translation initiation and diminish initiation at near-cognate UUG start codons in yeast mutants in which UUG selection is abnormally high (Sui-), conferring the Ssu- phenotype. Two such Ssu- substitutions--R116D and R117D--also increase discrimination against an AUG codon in suboptimal Kozak context. Consistently, the Arg116 and Arg117 substitutions destabilize TC binding to 48S PICs reconstituted in vitro with mRNA harboring a UUG start codon, indicating destabilization of the closed PIN state with a UUG:anticodon mismatch. Using model mRNAs lacking contacts with either the mRNA entry or exit channels of the 40S subunit, we demonstrate that Arg116/Arg117 are crucial for stabilizing PIC:mRNA contacts at the entry channel, complementing the function of eIF3 at both entry and exit channels. The corresponding residues in bacterial uS3 promote the helicase activity of the elongating ribosome, suggesting that uS3 contacts with mRNA enhance multiple phases of translation across different domains of life.

genetics↗