Biology subjects

Singh, D.

Publications and source records attributed to Singh, D..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

Comprehensive Structure and Functional Adaptations of the Yeast Nuclear Pore Complex

Nuclear Pore Complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the yeast NPC in which the inner ring is resolved by cryo-EM at - helical resolution to show how flexible connectors tie together different structural and functional layers in the spoke. These connectors are targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and karyopherins have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We also provide evidence for three major NPC variants that foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies to provide a comprehensive model of the in situ NPC with a radially-expanded inner ring. Our model reveals novel features of the central transporter and nuclear basket, suggests a role for the lumenal ring in restricting dilation and highlights the structural plasticity required for transport by the NPC.

biophysics↗

bioRxiv2021

Surface expressed Plasmodium circumsporozoite protein (CSP) modulates cellular flexibility and motility

Plasmodium falciparum circumsporozoite protein (CSP) is a critically required abundant surface protein of sporozoites and a major vaccine candidate. However, neither the structure nor the role of CSP in sporozoite motility is well understood. Our recent in vitro data, from single-molecule pulling experiments suggested a mechanically pliable structure for P. falciparum CSP. By engineering vegetative cells of the cellular slime-mold Dictyostelium discoideum with regulatable CSP surface expression, we report evidence for direct involvement of CSP towards conferring elastic properties and motility of the cells. With an increase in the surface-CSP levels by 5-8-fold, the Youngs moduli of the cells, observed through atomic force microscopy, decreased around 2-fold, with a concomitant increase in motility by about 2-fold. Interestingly, only full length CSP expression conferred maximal flexibility and motility, as opposed to repeat region alone or the flanking domains of CSP. The enhanced motility of the CSP-expressing cells was abrogated with anti-CSP antibodies as well as phospholipase cleavage of CSP, indicating specific contribution of CSP towards motility. Measurements of the Youngs moduli of Plasmodium berghei midgut (MG) and salivary gland (SG) sporozoites revealed an inverse correlation with CSP levels with a decrease from 1.1 kPa to 0.3 kPa as the CSP concentration doubled from MG to SG sporozoites. We hypothesize that high CSP level lowers the stiffness of sporozoites possibly through its pliable surface-coat, leading to cellular flexibility. These findings may explain a sporozoites developmental ability to enhance its CSP levels during transition from midgut to salivary glands to suit a migratory mode in the host, needed for successful hepatocyte invasion.

cell biology↗

bioRxiv2021

Distributed representations for human inference

Inferring relationships that go beyond our direct experience is essential for understanding our environment. This capacity requires either building representations that directly reflect structure across experiences as we encounter them, or computing the indirect relationships across experiences as the need arises. Building structure directly into overlapping representations allows for powerful learning and generalization in neural network models, but building these so-called distributed representations requires inputs to be encountered in interleaved order. We test whether interleaving similarly facilitates the formation of representations that directly integrate related experiences in humans, and what advantages such integration may confer for behavior. In a series of behavioral experiments, we present evidence that interleaved learning indeed promotes the formation of representations that directly link across related experiences. As in neural network models, interleaved learning gives rise to fast and automatic recognition of item relatedness, affords efficient generalization, and is especially critical for inference when learning requires statistical integration of noisy information over time. We use the data to adjudicate between several existing computational models of human memory and inference. The results demonstrate the power of interleaved learning and implicate the formation of integrated, distributed representations that support generalization in humans.

neuroscience↗

bioRxiv2021

Severe Acute Respiratory Syndrome Coronavirus-2 genome sequence variations relate to morbidity and mortality in Coronavirus Disease-19

Outcome of infection with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) may depend on the host, virus or the host-virus interaction-related factors. Complete SARS-CoV-2 genome was sequenced using Illumina and Nanopore platforms from naso-/oro-pharyngeal ribonucleic acid (RNA) specimens from COVID-19 patients of varying severity and outcomes, including patients with mild upper respiratory symptoms (n=35), severe disease ad-mitted to intensive care with respiratory and gastrointestinal symptoms (n=21), fatal COVID-19 outcome (n=17) and asymptomatic (n=42). Of a number of genome variants observed, p.16L>L (Nsp1), p.39C>C (Nsp3), p.57Q>H (ORF3a), p.71Y>Y (Membrane glycoprotein), p.194S>L (Nucleocapsid protein) were observed in similar frequencies in different patient subgroups. However, seventeen other variants were observed only in symptomatic patients with severe and fatal COVID-19. Out of the latter, one was in the 5UTR (g.241C>T), eight were synonymous (p.14V>V and p.92L>L in Nsp1 protein, p.226D>D, p.253V>V, and p.305N>N in Nsp3, p.34G>G and p.79C>C in Nsp10 protein, p.789Y>Y in Spike protein), and eight were non-synonymous (p.106P>S, p.157V>F and p.159A>V in Nsp2, p.1197S>R and p.1198T>K in Nsp3, p.97A>V in RdRp, p.614D>G in Spike protein, p.13P>L in nucleocapsid). These were completely absent in the asymptomatic group. SARS-CoV-2 genome variations have a significant impact on COVID-19 presentation, severity and outcome.

genomics↗

bioRxiv2021

A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, by direct counting using single-molecule fluorescence, we have determined that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise count of active/inactive subunit(s), we found, unexpectedly, that the packaging motor can tolerate an inactive sub-unit. However, motors containing an inactive subunit(s) exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a new packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, contrary to the prevailing notion of strict coordination amongst motor subunits of other packaging motors.

biophysics↗

bioRxiv2021

Traditional use of Cissampelos pareira L. for hormone disorder and fever provides molecular links of ESR1 modulation to viral inhibition

Bioactive fractions or compounds obtained from medicinal plants have been used for the treatment of multiple diseases. This effect could be due to common pathways underlying these conditions that are targeted by such medicines. In this study, we explored the molecular basis of action of one such herbal formulation Cissampelos pareira, used for the treatment of female hormone disorders and fever. Genome-wide expression studies on MCF7 cell lines treated with Cipa extract were carried out using Affymetrix arrays. Transcriptome analysis revealed a downregulation of signatures of estrogen response governed by estrogen receptor (ER). Molecular docking analysis identified 38 constituent molecules in Cipa that potentially bind ({Delta}G< -7.5) with ER at the same site as estrogen. Cipa transcriptome signatures show high positive connectivity (https://clue.io/) scores with protein translation inhibitors such as emetine (score: 99.61) and knockdown signatures of genes linked to the antiviral response such as ribosomal protein RPL7 (score: 99.92), which is also an ER coactivator. Cipa exhibits antiviral activity in dengue infected MCF7 cells that is decreased upon ESR1 (estrogen receptor 1) gene knockdown. This approach reveals a novel pathway involving ESR1-RPL7 axis that could be a potential target in dengue viral infection.

genomics↗

bioRxiv2021

PandoraGAN: Generating antiviral peptides using Generative Adversarial Network

The continuous increase in pathogenic viruses and the intensive laboratory research emphasizes the need for cost and time efficient drug development. This accelerates research for alternate drug candidates like antiviral peptides(AVP) that have therapeutic and prophylactic potential and gaining attention in recent times. However, diversity in their sequences, limited and non-uniform characterization often limit their applications. Isolating newer peptide backbones with required characteristics is a cumbersome process with many design-test-build cycles. Advanced deep learning approaches such as generative adversarial networks (GAN) can be helpful to expedite the initial stage of developing novel peptide drugs. In this study, we developed PandoraGAN that uses a manually curated training dataset of 130 highly active peptides that includes peptides from known databases (such as AVPdb) and literature to generate novel antiviral peptides. The underlying architecture in PandoraGAN is able to learn a good representation of the implicit properties of antiviral peptides. The generated sequences from PandoraGAN are validated based on physico-chemical properties. They are also compared with the training dataset statistically using Pearsons correlation and Mann-Whitney U-test. We therefore confirm that PandoraGAN is capable of generating a novel antiviral peptide backbone showing similar properties to that of the known highly active antiviral peptides. This approach exhibits a potential to discover novel patterns of AVP which may have not been seen earlier with traditional methods. To our knowledge this is the first ever use of GAN models for antiviral peptides across the viral spectrum.

bioinformatics↗

bioRxiv2021

Primary tumor associated macrophages activate programs of invasion and dormancy in disseminating tumor cells.

Metastases are initiated by disseminated tumor cells (DTCs) that depart from the primary tumor and colonize target organs. Growing evidence suggests that the microenvironment of the primary tumor lesion primes DTCs to display dormant or proliferative fates in target organs. However, the manner in which events taking place in the primary tumor influence DTC fate, sometimes long after dissemination, remains poorly understood. With the advent of a novel intravital imaging technique called the Window for High-Resolution Intravital Imaging of the Lung (WHRIL), we have, for the first time, been able to study the live lung longitudinally and follow the fate of individual DTCs that spontaneously disseminate from orthotopic breast tumors. We find, across several models, a high rate of success for tumor cells to complete the initial steps of the metastatic cascade in the secondary site, including retention of DTCs in the lung vasculature, speed of extravasation, and survival after extravasation. Importantly, initiation of metastatic growth was controlled primarily by a rate-limiting step that occurred post-extravasation and at the stage of the conversion of single DTCs from a dormant to a proliferative state. Detailed analysis of these events revealed that, even before dissemination, a subset of macrophages within the primary tumor induces, in tumor cells that are about to disseminate, the expression of proteins that regulate a pro- dissemination (MenaINV) and pro-dormancy (NR2F1) phenotype. Surprisingly, if cancer cells are intravenously injected, the rate limiting stages of MenaINV-associated extravasation, dormancy, and other parameters, are lost or altered in a way that impacts how DTCs progress through the metastatic cascade. Our work provides novel insight into how specific primary tumor microenvironments prime a subpopulation of cells for dissemination and dormancy. We also propose that dissecting mechanisms of metastasis, or testing anti-metastatic therapies, may yield results of limited application if derived from models that do not follow spontaneous dissemination. SIGNIFICANCEThis study provides important insight into the contribution of primary tumor microenvironmental niches to cancer metastasis by identifying the manner in which these niches spawn subpopulations of DTCs that are primed for dissemination and dormancy in the secondary site. This study may provide novel targets that could be inhibited to prevent successful colonization of the secondary site and, hence, metastasis.

cancer biology↗

bioRxiv2021

Assessment of immunogenicity and protective efficacy of ZyCoV-D DNA vaccine candidates in Rhesus macaques against SARS-CoV-2 infection

Vaccines remain the key protective measure to achieve herd immunity to control the disease burden and stop COVID-19 pandemic. We have developed and assessed the immunogenicity and protective efficacy of two formulations (1mg and 2mg) of ZyCoV-D (a plasmid DNA based vaccine candidates) administered through Needle Free Injection System (NFIS) and syringe-needle (intradermal) in rhesus macaques with three dose vaccine regimens. The vaccine candidate 2mg dose administered using Needle Free Injection System (NFIS) elicited a significant immune response with development of SARS-CoV-2 S1 spike region specific IgG and neutralizing antibody (NAb) titers during the immunization phase and significant enhancement in the levels after the virus challenge. In 2 mg NFIS group the IgG and NAb titers were maintained and showed gradual rise during the immunization period (15 weeks) and till 2 weeks after the virus challenge. It also conferred better protection to macaques evident by the viral clearance from nasal swab, throat swab and bronchoalveolar lavage fluid specimens in comparison with macaques from other immunized groups. In contrast, the animals from placebo group developed high levels of viremia and lung disease following the virus challenge. Besides this, the vaccine candidate also induced increase lymphocyte proliferation and cytokines response (IL-6, IL-5).The administration of the vaccine candidate with NFIS generated a better immunogenicity response in comparison to syringe-needle (intradermal route). The study demonstrated immunogenicity and protective efficacy of the vaccine candidate, ZyCoV-D in rhesus macaques.

microbiology↗

bioRxiv2021

Baseline cell proliferation rates and response to UV differ in Lymphoblastoid Cell Lines derived from healthy individuals of extreme constitution types

Differences in human phenotypes and susceptibility to complex diseases are an outcome of genetic and environmental interactions. This is evident in diseases that progress through a common set of intermediate patho-endophenotypes. Precision medicine aims to delineate the molecular players for individualized and early interventions. Functional studies in Lymphoblastoid Cell Line (LCL) model of phenotypically well characterized healthy individuals can help deconvolute and validate these molecular mechanisms. We developed LCLs from eight healthy individuals belonging to three extreme constitution types, deep phenotyped on the basis of Ayurveda. LCLs were characterized by karyotyping and immunophenotyping. Growth characteristics and response to UV was studied in these LCLs. We observed significant differences in cell proliferation rates between the contrasting groups such that one type (Kapha) proliferates significantly slower than the other two (Vata, Pitta). In response to UV, one fast growing group (Vata) shows higher cell death but recovers its numbers due to inherent higher rates of proliferation. The baseline differences in cell proliferation are key to understanding the survival of cells in UV stress. Variability in baseline cellular phenotypes not only explains the cellular basis of different constitutions types but can also help set priors during designing an individualized therapy with DNA damaging agents. This is the first study of its kind that shows variability of intermediate patho-phenotypes amongst healthy individuals that have implications in precision medicine.

cell biology↗

bioRxiv2020

Coupled feedback loops involving PAGE4, EMT and Notch signaling can give rise to non-genetic heterogeneity in prostate cancer cells

Non-genetic heterogeneity is emerging to be a crucial factor underlying therapy resistance in multiple cancers. However, the design principles of regulatory networks underlying non-genetic heterogeneity in cancer remain poorly understood. Here, we investigate the coupled dynamics of feedback loops involving a) oscillations in androgen receptor (AR) signaling mediated through an intrinsically disordered protein PAGE4, b) multistability in epithelial-mesenchymal transition (EMT), and c) Notch-Delta-Jagged signaling mediated cell-cell communication, each of which can generate non-genetic heterogeneity through multistability and/or oscillations. Our results show how different coupling strengths between AR and EMT signaling can lead to possible bistability in the levels of AR. These results reveal the emergent dynamics of coupled oscillatory and multi-stable systems and unravel mechanisms by which non-genetic heterogeneity in AR levels can be generated, which can act as a barrier to most existing therapies for prostate cancer patients.

systems biology↗

bioRxiv2020

Mitragyna alkaloids derived from the medicinal plant Mitragyna speciosa Korth. and cisplatin synergistically inhibit cell proliferation of Nasopharyngeal Carcinoma (NPC) cell lines

Ethnopharmacological relevanceMitragyna speciosa (Korth.) or kratom is a medicinal plant indigenous to Southeast Asia. The leaves of M. speciosa is used as a medication in pain management including cancer related pain, in a similar way as opioids and cannabis. Despite its well-known analgesic effect, there is a scarce of information on the cancer-suppressing potential of M. speciosa and its active constituents. Aim of the studyTo assess the potential applicability of M. speciosa alkaloids (mitragynine, speciociliatine or paynantheine) as chemosensitizers for cisplatin in Nasopharyngeal carcinoma (NPC) cell lines. Materials and MethodsThe cytotoxic effects of the extracts, fractions and compounds were determined by conducting in vitro cytotoxicity assays. Based on the cytotoxic screening, the alkaloid extract of M. speciosa exhibited potent inhibitory effect on the NPC cell line HK-1, and therefore, was chosen for further fractionation and purification. NPC cell lines HK-1 and C666-1 were treated with combinations of cisplatin and M. speciosa alkaloids in 2D monolayer culture. The effect of the drug combination on cell migration was tested using in vitro wound healing and spheroid invasion assays. ResultsIn our bioassay guided isolation, both methanolic and alkaloid extracts showed mild to moderate cytotoxic effect against the HK-1 cell line. Both NPC cell lines were insensitive to single agent and combination treatments of the M. speciosa alkaloids. However, mitragynine and speciociliatine sensitised the HK-1 and C666-1 to cisplatin ~4- and >5-fold, respectively in 2D monolayer culture. The combination of mitragynine and cisplatin also significantly inhibited cell migration of the NPC cell lines. Similarly, combination of mitragynine and cisplatin inhibited growth and invasion of HK-1 spheroids in a dose-dependent manner. Moreover, the spheroids did not rapidly develop resistance to the drug combinations at high concentrations over 10 days. ConclusionCollectively, data shows that both mitragynine and speciociliatine could be potential chemosensitizers for cisplatin. Further extensive drug mechanistic studies and investigations in animal models are necessary to delineate the applicability of M. speciosa alkaloids for NPC therapy.

cancer biology↗

bioRxiv2020

Python Inferfaces for the Smoldyn Simulator

MotivationSmoldyn is a particle-based biochemical simulator that is frequently used for systems biology and biophysics research. Previously, users could only define models using text-based input or a C/C++ applicaton programming interface (API), which were convenient, but limited extensibility. ResultsWe added a Python API to Smoldyn to improve integration with other software tools such as Jupyter notebooks, other Python code libraries, and other simulators. It includes low-level functions that closely mimic the existing C/C++ API and higher-level functions that are more convenient to use. These latter functions follow modern object-oriented Python conventions. AvailabilitySmoldyn is open source and free, available at http://www.smoldyn.org, and can be installed with the Python package manager pip. It runs on Mac, Windows, and Linux. Contactsteven.s.andrews@gmail.com Supplementary informationDocumentation is available at http://www.smoldyn.org/SmoldynManual.pdf and https://smoldyn.readthedocs.io/en/latest/python/api.html.

bioinformatics↗

bioRxiv2020

Influence of spatial arrangement, biofertilizers and bioirrigation on the performance of legume-millet intercropping system in rainfed areas of southern India

Biofertilization via the inoculation with arbuscular mycorrhizal fungi (AMF), combined with rhizobia and plant growth promoting rhizobacteria (PGPR), are beginning to become established as an effective and sustainable measure to improve yields. Biofertilization might have a particular potential to boost the yield of intercropping systems in rainfed areas because AMF can form a common mycorrhizal network (CMN) that can transfer nutrients and water between two plants and balance as such belowground competition. In this study, we tested if biofertilizers can enhance the yield of intercropping systems using a pigeon pea (PP) - finger millet (FM) intercropping system grown for two consecutive growing seasons (2016/17 and 2017/18) at two contrasting sites in Bengaluru and Kolli Hills, India. To validate the process of bioirrigation (transfer of water between rhizosphere of two plants), we tested, for the first time, if the spatial arrangement of intercropped plants using either a row-wise or a mosaic design affected yield and water relations with and without biofertilizers. Our results demonstrate that intercropping can improve the straw and grain yield in PP-FM intercropping compared to the respective monocultures but that intercropping effects vary depending on the site characteristic such as climate and soil type. Spatial arrangement of component plants affected the total, straw and grain biomass in intercropping treatments, but this effect also varied across sites. Most importantly, the results from the 2017-18 growing season clearly demonstrated a positive effect of biofertilizer on biomass yield, and this effect was irrespective of site, spatial arrangement, mixed or monoculture. Despite a yield increase in intercropping, we did not see a positive effect of biofertilization on water relations of FM possibly due to interspecific competition for soil moisture where PP dominated. In summary, our study shows the potential of biofertilizers to increase the yield of intercropping systems in rainfed dryland agriculture.

ecology↗

bioRxiv2020

Structural insights on the substrate-binding proteins of the Mycobacterium tuberculosis mammalian-cell-entry (Mce) 1 and 4 complexes

Tuberculosis (Tb), caused by Mycobacterium tuberculosis (Mtb), is responsible for more than a million deaths annually. In the latent phase of infection, Mtb uses lipids as the source of carbon and energy for its survival. The lipid molecules are transported across the cell wall via multiple transport systems. One such set of widely present and less-studied transporters is the Mammalian-cell-entry (Mce) complexes. Here, we report the properties of the substrate-binding proteins (SBPs; MceA-F) of the Mce1 and Mce4 complexes from Mtb which are responsible for the import of mycolic acid/fatty acids, and cholesterol respectively. MceA-F are composed of four domains namely, transmembrane, MCE, helical and tail domains. Our studies show that MceA-F are predominantly monomeric when purified individually and do not form homohexamers unlike the reported homologs (MlaD, PqiB and LetB) from other prokaryotes. The crystal structure of MCE domain of Mtb Mce4A (MtMce4A39-140) determined at 2.9 [A] shows the formation of an unexpected domain-swapped dimer in the crystals. Further, the purification and small-angle X-ray scattering (SAXS) analysis on MtMce1A, MtMce4A and their domains suggest that the helical domain requires hydrophobic interactions with the detergent molecules for its stability. Combining all the experimental data, we propose a heterohexameric arrangement of MtMceA-F SBPs, where the soluble MCE domain of the SBPs would remain in the periplasm with the helical domain extending to the lipid layer forming a hollow channel for the transport of lipids across the membranes. The tail domain would reach the cell surface assisting in lipid recognition and binding.

biochemistry↗

bioRxiv2020

Cocculus hirsutus -derived phytopharmaceutical drug has potent anti-dengue activity

BackgroundDengue is a serious public health concern worldwide, with ~3 billion people at risk of contracting dengue virus (DENV) infections. Currently, no effective vaccine or drug is available for the prevention or treatment of dengue, which leaves only anti-mosquito strategies to combat this disease. The present study was initiated to determine the in-vitro and in vivo protective effects of a plant-derived phytopharmaceutical drug for the treatment of dengue. Methodology/Principal FindingsIn our previous report, we had identified methanolic extract of the aerial parts of Cissampelos pareira to exhibit in vitro and in vivo anti-dengue activity against all the four DENV serotypes. In the current study, we have identified another Indian medicinal plant, Cocculus hirsutus, which has a more potent anti-dengue activity than C. pareira. The activity has been evaluated through flow-cytometry-based virus inhibition assay. Interestingly, the stem of C. hirsutus was found to be more potent than the aerial part irrespective of the extraction solvent used viz., denatured spirit, hydro-alcohol (50:50) and water. Hence, the aqueous extract of stem of C. hirsutus (AQCH) was further advanced for investigations because of greater regulatory acceptance. The AQCH exhibited dose-dependent inhibition of release of DENV and its secretory antigen, NS1. Five chemical markers viz. Sinococuline, 20-Hydroxyecdysone, Makisterone-A, Magnoflorine and Coniferyl alcohol were identified as the major chemical ingredients of the AQCH extract. These chemicals were subsequently used for extract standardisation. Importantly, AQCH completely protected AG129 mice at 25 mg/kg/dose body weight when fed 4 times a day post-infection with a lethal dose of DENV-2 S221 strain. Because of its potential as an effective phytopharmaceutical drug against dengue, AQCH, has been formulated into tablets for further pre-clinical and clinical developments. Conclusions/SignificanceWe provide evidence of the pan anti-dengue potential of C. hirsutus-based phytopharmaceutical drug as determined through in vitro and in vivo experiments. We have also characterized five chemical entities in the drug substance, which provides means for standardization of drug substance and drug product. Based on these findings, a program to develop a safe and effective C. hirsutus-derived phytopharmaceutical drug for the treatment of dengue has been initiated. Author summaryThere is an urgent need to develop a safe and effective drug against dengue, which is a rapidly expanding mosquito-borne viral disease. Half of the worlds population has been estimated to be at risk of contracting this disease and the situation remains grim due to lack of an approved drug. We aimed to develop an ethnopharmacological drug against dengue by exploring traditional Indian medicinal science, Ayurveda. This led us to identify a creeper, Cocculus hirsutus, as a more potent anti-dengue plant than Cissampelos pareira, reported in our earlier published study. The stem part of C. hirsutus was found to be more efficacious in inhibiting the propagation of dengue viruses (DENVs) in cell culture than its aerial part. Hence, we chose to advance aqueous extract of stem of C. hirsutus (AQCH) for further studies. Importantly, AQCH also protected immune-compromised mice from lethal DENV infection, which is suggestive of its potential clinical relevance. We have identified five chemical marker compounds in AQCH to gauge the quality and consistency of extract preparation and its formulation into stable tablets. Based on the findings of this study, we have undertaken the development of a safe and effective C. hirsutus-derived phytopharmaceutical drug for the treatment of dengue.

microbiology↗

bioRxiv2020

Divergent DNA methylation signatures underlying X chromosome regulation in marsupials and eutherians

X chromosome inactivation (XCI) mediated by differential DNA methylation between sexes is well characterized in eutherian mammals. Although XCI is shared between eutherians and marsupials, the role of DNA methylation in marsupial XCI remains contested. Here we examine genome-wide signatures of DNA methylation from methylation maps across fives tissues from a male and female koala (Phascolarctos cinereus) and present the first whole genome, multi-tissue marsupial "methylome atlas." Using these novel data, we elucidate divergent versus common features of marsupial and eutherian DNA methylation. First, tissue-specific differential DNA methylation in marsupials primarily occurs in gene bodies. Second, females show significant global reduction (hypomethylation) of X chromosome DNA methylation compared to males. We show that this pattern is also observed in eutherians. Third, on average, promoter DNA methylation shows little difference between male and female koala X chromosomes, a pattern distinct from that of eutherians. Fourth, the sex-specific DNA methylation landscape upstream of Rsx, the primary lncRNA associated with marsupial XCI, is consistent with the epigenetic regulation of female-(and presumably inactive X chromosome-) specific expression. Finally, we utilize the prominent female X chromosome hypomethylation and classify 98 previously unplaced scaffolds as X-linked, contributing an additional 14.6 Mb (21.5 %) to genomic data annotated as the koala X chromosome. Our work demonstrates evolutionarily divergent pathways leading to functionally conserved patterns of XCI in two deep branches of mammals.

evolutionary biology↗

bioRxiv2020

The SARS-CoV-2 Nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that a centrally located 40 amino acid intrinsically disordered domain drives phase separation of N protein when bound to RNA, with the morphology of the resulting condensates affected by inclusion in the RNA of the putative SARS-CoV-2 packaging signal. The SARS-CoV-2 M protein, normally embedded in the virion membrane with its C-terminus extending into the virion core, independently induces N protein phase separation that is dependent on the N proteins C-terminal dimerization domain and disordered region. Three-component mixtures of N+M+RNA form condensates with mutually exclusive compartments containing N+M or N+RNA, including spherical annular structures in which the M protein coats the outside of an N+RNA condensate. These findings support a model in which phase separation of the N protein with both the viral genomic RNA and the SARS-CoV-2 M protein facilitates RNA packaging and virion assembly.

biochemistry↗