Biology subjects

Ranjan, R.

Publications and source records attributed to Ranjan, R..

36 records · Page 2Linked to original sources

bioRxiv2023

Modeling and Simulation of Neocortical Micro- and Mesocircuitry. Part II: Physiology and Experimentation

Cortical dynamics underlie many cognitive processes and emerge from complex multi-scale interactions, which are challenging to study in vivo. Large-scale, biophysically detailed models offer a tool which can complement laboratory approaches. We present a model comprising eight somatosensory cortex subregions, 4.2 million morphological and electrically-detailed neurons, and 13.2 billion local and mid-range synapses. In silico tools enabled reproduction and extension of complex laboratory experiments under a single parameterization, providing strong validation. The model reproduced millisecond-precise stimulus-responses, stimulus-encoding under targeted optogenetic activation, and selective propagation of stimulus-evoked activity to downstream areas. The models direct correspondence with biology generated predictions about how multiscale organization shapes activity; for example, how cortical activity is shaped by high-dimensional connectivity motifs in local and mid-range connectivity, and spatial targeting rules by inhibitory subpopulations. The latter was facilitated using a rewired connectome which included specific targeting rules observed for different inhibitory neuron types in electron microscopy. The model also predicted the role of inhibitory interneuron types and different layers in stimulus encoding. Simulation tools and a large subvolume of the model are made available to enable further community-driven improvement, validation and investigation.

neuroscience↗

bioRxiv2023

Zinc nano-fertilization enhances wheat productivity and biofortification

Zinc (Zn) malnutrition has emerged as one of the major health challenges in developing nations across the globe. Development of Zn management protocols in staple food crops using modern scientific tools to enhance Zn concentration in grains along with augmented crop yields became utmost necessary. In this context a 2-year experiment was carried out to assess the effects of zinc oxide nanoparticles (ZnO-NPs) vis-a-vis bulk zinc sulfate (ZnSO4) on wheat growth, yield and Zn concentration in plant parts. Four levels of application of ZnO-NPs (0, 20, 25 and 30 mg kg-1) were compared with ZnSO4 (equal to Zinc concentration in ZnO-NPs). Results revealed that seed vigor was significantly (p <0.05) higher under 25 and 30 mg kg-1 soil ZnO-NPs treatments over ZnSO4. Among the crop yield parameters such as tillers (plant-1), grain weight (plant-1), biomass (plant-1) and grain yield were significantly (p <0.05) higher under ZnO-NPs 25 mg kg-1 treated soil as compared to any other treatment. Zinc concentration in grains increased with dose of ZnO-NPs and it was significantly more than ZnSO4 treated soil at each treatment level. ZnO-NPs and ZnSO4 treatments did not affect photosynthetic rate and chlorophyll (SPAD) content significantly. In conclusion, 25 mg kg-1 ZnO-NPs application could be recommended in wheat cultivation to improve growth, yield and grain Zn biofortification.

plant biology↗

bioRxiv2022

De novo identification of CD4+ T cell epitopes

CD4+ T cells recognize peptide antigens presented on class II Major Histocompatibility Complex (MHC-II) molecules to carry out their function. The remarkable diversity of T cell receptor (TCR) sequences and lack of antigen discovery approaches for MHC-II make profiling the specificities of CD4+ T cells challenging. We have expanded our platform of Signaling and Antigen-presenting Bifunctional Receptors to encode MHC-II molecules presenting covalently linked peptides (SABR-IIs) for CD4+ cell antigen discovery. SABR-IIs can present epitopes to CD4+ T cells and induce signaling upon their recognition, allowing a readable output. Here, we demonstrate that SABR-IIs libraries presenting endogenous and post-translationally modified epitopes can be used for antigen discovery. Using SABR-II libraries in conjunction with single cell RNA sequencing, we de-convoluted multiple highly expanded TCRs from pancreatic islets of Non-Obese Diabetic (NOD) mice. We compounded antigen discovery by incorporating computational TCR similarity prediction metrics followed by experimental validation. Finally, we showed SABR-IIs presenting epitopes in class II HLA alleles can be used for antigen discovery for human CD4+ T cells. Taken together, we have developed a rapid, flexible, scalable, and versatile approach for the de novo identification of CD4+ T cell ligands from single cell RNA sequencing data using experimental and computational approaches.

immunology↗

bioRxiv2022

Equalizing epigenetically imprinted centromeres in early mammalian embryos

The CENP-A histone variant epigenetically defines centromeres, where its levels and locations are precisely maintained through mitotic cell divisions. However, differences in centromere CENP-A propagation in soma versus female/male germline remains poorly understood. Here, we generated CenpamScarlet mice and followed CENP-A dynamics in gametes, zygotes, and embryos. We found that, unlike somatic cells, progenitor female and male germ cells carry high centromeric CENP-A levels that decrease upon terminal differentiation. The reduction in CENP-A is differentially regulated between sexes, resulting in a ten-fold higher level in oocytes compared to sperm. In the zygote, the parent-of-origin CENP-A asymmetry is equalized prior to initial S-phase by redistribution of nuclear CENP-A from maternal to paternal chromosomes. Redistribution of CENP-A requires both CDK1/2 and PLK1 centromeric machinery. These experiments provide direct evidence for resetting of epigenetically imprinted centromeres in early pronuclear stage embryos and imply a mechanism to sense the non-equivalency of parental chromosomes. HighlightsO_LIIncreased CENP-A density at centromeres is a conserved property of germline stem cells while CENP-A reduction is coincident with germ cell differentiation C_LIO_LIPaternal and maternal CENP-A containing nucleosomes are intergenerationally inherited C_LIO_LICENP-A density at centromeres differs between male and female mature gametes C_LIO_LIUpon fertilization, maternal nuclear CENP-A is redistributed to equalize with parental CENP-A C_LIO_LICENP-C and MIS18BP1 are asymmetrically enriched in the parental pronuclei in accordance with CENP-A asymmetry. C_LIO_LILicensing for centromere equalization begins before zygotic DNA replication C_LI

developmental biology↗

bioRxiv2022

A Canine Model of Chronic Ischemic Heart Failure

BackgroundThere is a paucity of novel therapeutics for chronic heart failure (HF). Preclinical large animal models of chronic HF are crucial to translating fundamental discoveries. Canine models of ischemic cardiomyopathy are frequently utilized but are severely limited by competing needs to both induce sufficient muscle injury which results in an appropriately severe degree of left ventricular dysfunction yet to also minimize early mortality. MethodsTwenty-eight healthy adult dogs (30{+/-}4 kg, 15/28 male) underwent thoracotomy procedures followed by one of three types of left anterior descending (LAD) coronary artery ligations: Simple LAD (proximal and distal LAD ligation), LAD and Lateral (proximal and distal LAD, distal first diagonal, and proximal first obtuse marginal), and Total LAD Devascularization (TLD including the proximal and distal LAD, proximal aspects of the first, second, and third diagonals, and proximal aspects of first and second epicardial right ventricular branches of the LAD). Invasive and non-invasive hemodynamics were followed until attainment of chronic severe HF, which was achieved when left ventricular ejection fraction (LVEF) was <40% and N-terminal prohormone B-type Natriuretic peptide (NT-proBNP) >900 pmol/L, both for two consecutive measurements. ResultsOverall post-ligation early mortality (within 48 h of coronary ligation) was 18% (n=5/28), and the overall survival rate at 5 weeks post-ligation was 68% (n=19/28). At post-ligation week 1, LVEF was acutely reduced in all animals, from 54{+/-}4 to 38{+/-}1%, p<0.001. Beyond week 1, systolic function recovered over 3-4 weeks and then slowly declined as the left ventricle dilated and both systolic and diastolic function worsened. The magnitude of the initial decline in LVEF predicted ultimate HF development. Dogs with Simple LAD and LAD plus Lateral had excellent and limited survival at 48 hours (100% and 47%, respectively), yet the second group more likely developed chronic HF at 9 - 14 weeks (50% versus 57%, respectively). Dogs with TLD had both improved survival at 48 hours (90%) and excellent development of chronic HF (100%). ConclusionsBy focusing on the LAD only and limiting collateral flow to it by ligating all side branches, optimal survival and attainment of chronic ischemic HF can be achieved in the canine heart.

developmental biology↗

bioRxiv2022

Roles of the distinct N-terminal amino acid between H3 and H3.3 in Drosophila male germline stem cell lineage

Adult stem cells undergo asymmetric cell divisions to produce two daughter cells with distinct cell fates: one capable of self-renewal and the other committed for differentiation. Mis-regulation of this delicate balance can lead to cancer and tissue degeneration. During asymmetric division of Drosophila male germline stem cells (GSCs), preexisting (old) and newly synthesized histone H3 are differentially segregated whereas old and new histone variant H3.3 are more equally inherited. However, what underlies these distinct inheritance patterns remains unknown. Here, we report that the N-terminal tails of H3 and H3.3 are critical for their inheritance patterns, as well as GSC maintenance and proper differentiation. H3 and H3.3 differ at the 31st position in their N-termini with Alanine for H3 and Serine for H3.3. By swapping these two amino acids, we generated two mutant histones (i.e., H3A31S and H3.3S31A). Upon expressing them in the early-stage germline, we identified opposing phenotypes: over-population of early-stage germ cells in the H3A31S-expressing testes and significant germ cell loss in testes expressing the H3.3S31A. Asymmetric H3 inheritance is disrupted in the H3A31S-expressing GSCs, due to mis-incorporation of old histones between sister chromatids during DNA replication. Furthermore, H3.3S31A mutation accelerates old histone turn-over in the GSCs. Finally, using a modified Chromatin Immunocleavage assay on early-stage germ cells, we found that H3A31S has an enhanced occupancy at the promoters and transcription starting sites than H3, while H3.3S31A is more enriched at transcriptionally silent intergenic regions compared to H3.3. Overall, these results suggest that the 31st amino acids for both H3 and H3.3 are critical for their proper genomic occupancy and function. Together, our findings indicate a critical role for the different amino acid composition of the N-terminal tails between H3 and H3.3 in an endogenous stem cell lineage, and provide insights into the importance of proper histone inheritance in specifying cell fates and regulating cellular differentiation.

developmental biology↗

bioRxiv2022

CryoEM structure of QacA, an antibacterial efflux transporter from Staphylococcus aureus

Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram-positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane (TM) helix containing major facilitator superfamily antiporter, mediates proton-coupled efflux of mono and divalent cationic antibacterial compounds. In this study, we report the cryoEM structure of QacA, with a single mutation D411N that improves homogeneity and retains efflux activity against divalent cationic compounds like dequalinium and chlorhexidine. The structure of substrate-free QacA, complexed to two single-domain camelid antibodies, was elucidated to a resolution of 3.6 [A]. The structure displays an outward-open conformation with an extracellular hairpin loop, which is conserved in a subset of DHA2 transporters and its deletion causes a loss of function in the transporter. Modeling and simulations of QacAs cytosol-facing and occluded conformations reveal asymmetry in the rocker-switch mode of QacAs conformational shifts, providing new insights into the organization and structural dynamics of DHA2 members.

biochemistry↗

bioRxiv2022

Illumina-based Whole De Novo Transcriptome Profiling and Diosgenin Biosynthetic pathway of Tribulus terrestris: A Medicinal herb

Tribulus terrestris L. (Chota gokhuru), an annual herb of the Zygophyllaceae family accumulates steroidal-type triterpenoids of diosgenin in root, leaf, fruit, which have been used as a traditional medicine to cure steroidal hormonal diseases such as estrogen, progesterone, and cardiovascular and urogenital. However, to date, the diosgenin biosynthetic pathway of T. terrestris is unknown. In this research paper, identification of candidate genes involved in bioactive compounds (diosgenin) was conducted via whole transcriptome profiling of T. terrestris using the Illumina HiSeq sequencing platform, which generated 482 million reads. RNA-Seq reads were assembled and clustered into 148871 unigenes. NCBI non-redundant (NR) and Swiss-Prot databases search anchored 50% unigenes with the functional annotations based on similarities of sequence. Further, in Gene Ontology and KEGG-KAAS pathways, a total of 126 unigenes were highly expressed in the terpenoids backbone biosynthesis, in which diosgenin pathways (two genes of glycolytic, three genes of mevalonate, two genes of MEP/DOX, one gene of mono-terpenoids, and seven genes of steroidal pathways) were identified. In addition, 9 genes were randomly selected for confirmation through real-time PCR. T. terrestris showed the highest homology with the Citrus sinensis and Juglans regia. 27478 unigenes of transcription factors were assembled, in which top ten transcription factors were highly expressed. A total of 17623 SSR was assembled, amongst them unigenes of di-nucleotide were highly repeated. This research work will improve our understanding of the diosgenin pathway and lay the groundwork for future molecular research on metabolic engineering, as well as raise diosgenin content in T. terrestris.

molecular biology↗

bioRxiv2022

Metagenomics Study of Bacterial Communities from Yamuna River water of city of Taj, Agra

Yamuna river water of Agra city is heavily contaminated with toxic pollutants including heavy metal that causes severe damage to ecological and social aspects. At present, the direct use of river water for the purpose of drinking causes severe hazards due to anthropogenic activities causing environmental pollution in rivers. In present study, Yamuna River water was collected from three different sites of Agra city. Various physico-chemical parameters were estimated by following the standard methods of APHA and the concentration of heavy metals were measured using Atomic Absorption Spectrophotometer (AAS). In case of physico-chemical parameters characterization, the obtained values were extremely above the permissible limits. On account of the research work; the Yamuna river water samples contains heavy metal concentrations (Cadmium, Chromium, Copper, Lead and Nickel) above the desirable and admissible levels except for Zinc. During analysis of non-culturable bacteria a substantial bacterial diversity was observed in the Yamuna river water samples collected from different sites. The water samples were subjected to metagenomic analysis using Illumina platform which revealed that Proteobacteria (phylum), Betaproteobacteria (class), Burkholderiales (order), Comamonadaceae (family), Hydrogenophaga (genus) and Chloroflexi bacterium OLB 14 (species) were found as the most dominant bacterial taxonomic abundance in the river water samples. The presence of such bacterial communities in water indicates the availability of pollutants and suggests the futuristic use in the field of bioremediation.

molecular biology↗

bioRxiv2022

Reconstruction and simulation of thalamoreticular microcircuitry

Thalamoreticular circuitry is known to play a key role in attention, cognition and the generation of sleep spindles, and is implicated in numerous brain disorders, but the cellular and synaptic mechanisms remain intractable. Therefore, we developed the first detailed computational model of mouse thalamus and thalamic reticular nucleus microcircuitry that captures morphological and biophysical properties of [~]14,000 neurons connected via [~]6M synapses, and recreates biological synaptic and gap junction connectivity. Simulations recapitulate multiple independent network-level experimental findings across different brain states, providing a novel unifying cellular and synaptic account of spontaneous and evoked activity in both wakefulness and sleep. Furthermore, we found that: 1.) inhibitory rebound produces frequency-selective enhancement of thalamic responses during wakefulness, in addition to its role in spindle generation; 2.) thalamic interactions generate the characteristic waxing and waning of spindle oscillations; and 3.) changes in thalamic excitability (e.g. due to neuromodulation) control spindle frequency and occurrence. The model is openly available and provides a new tool to interpret spindle oscillations and test hypotheses of thalamoreticular circuit function and dysfunction across different network states in health and disease.

neuroscience↗

bioRxiv2022

Complex effects of chytrid parasites on the growth of the cyanobacterium Planktothrix rubescens across interacting temperature and light gradients

Chytrids are important drivers of aquatic ecosystems as phytoplankton parasites. The interaction between these parasites and their hosts are shaped by abiotic factors such as temperature and light. Here, we performed a full-factorial experiment to study how temperature and light interact to affect the dynamics of the bloom-forming toxic cyanobacterium Planktothrix rubescens and its chytrid parasite. We used a dynamic host-parasite model to explore how temperature and light affect long term dynamics. At low temperatures, chytrids do not survive. Higher light and temperature levels stimulated both phytoplankton and chytrid growth, with complex effects on their dynamics. Model exploration indicates that increasing temperature and light shifts equilibrium outcomes from P. rubescens persisting alone to stable coexistence and then to limit cycles. This provides an alternative biological explanation for why P. rubescens is mainly found in the relatively cold and dark lake metalimnion - it may enable avoidance of its parasite. Our study emphasizes the importance of investigating how abiotic factors interact with biotic interactions to drive complex outcomes.

ecology↗

bioRxiv2021

Phyllosphere community assembly and response to drought stress on common tropical and temperate forage grasses

Grasslands represent a critical ecosystem important for global food production, soil carbon storage, and water regulation. Current intensification and expansion practices add to the degradation of grasslands and dramatically increase greenhouse gas emissions and pollution. Thus, new ways to sustain and improve their productivity are needed. Research efforts focus on the plant-leaf microbiome, or phyllosphere, because its microbial members impact ecosystem function by influencing pathogen resistance, plant hormone production, and nutrient availability through processes including nitrogen fixation. However, little is known about grassland phyllospheres and their response to environmental stress. In this study, globally dominant temperate and tropical forage grass species were grown in a greenhouse under current climate conditions and drought conditions that mimic future climate predictions to understand if (i) plant host taxa influence microbial community assembly, (ii) microbial communities respond to drought stress, and (iii) phyllosphere community changes correlate to changes in plant host traits and stress-response strategies. Community analysis using high resolution sequencing revealed Gammaproteobacteria as the dominant bacterial class, which increased under severe drought stress on both temperate and tropical grasses while overall bacterial community diversity declined. Bacterial community diversity, structure, and response to drought were significantly different between grass species. This community dependence on plant host species correlated with differences in grass species traits, which became more defined under drought stress conditions, suggesting symbiotic evolutionary relationships between plant hosts and their associated microbial community. Further understanding these strategies and the functions microbes provide to plants will help us utilize microbes to promote agricultural and ecosystem productivity in the future. IMPORTANCEGlobally important grassland ecosystems are at risk of degradation due to poor management practices compounded by predicted increases in severity and duration of drought over the next century. Finding new ways to support grassland productivity is critical to maintaining their ecological and agricultural benefits. Discerning how grassland microbial communities change in response to climate stress will help us understand how plant-microbe relationships may be useful to sustainably support grasslands in the future. In this study, phyllosphere community diversity and composition was significantly altered under drought conditions. The significance of our research is demonstrating how severe climate stress reduces bacterial community diversity, which previously was directly associated with decreased plant productivity. These findings guide future questions about functional plant-microbe interactions under stress conditions, greatly enhancing our understanding of how bacteria can increase food security by promoting grassland growth and resilience.

microbiology↗

bioRxiv2021

Differential condensation of sister chromatids coordinates with Cdc6 to ensure distinct cell cycle progression in Drosophila male germline stem cell lineage

Stem cells undergo asymmetric division to produce both a self-renewing stem cell and a differentiating daughter cell. During Drosophila male germline stem cell (GSC) asymmetric division, preexisting old histones H3 and H4 are enriched in the self-renewed stem daughter cell, whereas the newly synthesized H3 and H4 are enriched in the differentiating daughter cell. However, the biological consequences in the two daughter cells resulting from asymmetric histone inheritance remained to be elucidated. In this work, we track both old and new histones throughout GSC cell cycle using high spatial and temporal resolution microscopy. We find several unique features differentiating old versus new histone-enriched sister chromatids, including nucleosome density, chromosomal condensation, and H3 Ser10 phosphorylation. These distinct chromosomal features lead to their differential association with Cdc6, an essential component of the pre-replication complex, which subsequently contributes to asynchronous initiation of DNA replication in the two resulting daughter cells. Disruption of asymmetric histone inheritance abolishes both differential Cdc6 association and asynchronous S-phase entry, demonstrating that asymmetric histone acts upstream of these critical events during cell cycle progression. Furthermore, GSC defects are detected under these conditions, indicating a connection between histone inheritance, cell cycle progression and cell fate decision. Together, these studies reveal that cell cycle remodeling as a crucial biological readout of asymmetric histone inheritance, which precedes and could lead to other well-known readouts such as differential gene expression. This work also enhances our understanding of asymmetric histone inheritance and epigenetic regulation in other stem cells or asymmetrically dividing cells in multicellular organisms.

developmental biology↗

bioRxiv2021

Elucidating biosynthetic pathway of piperine using comparative transcriptome analysis of leaves, root, and spike in Piper longum L.

Piper longum (Pipli; Piperaceae) is an important spice valued for its pungent alkaloids, especially piperine. Albeit, its importance, the mechanism of piperine biosynthesis is still poorly understood. The Next Generation Sequencing (NGS) for P. longum leaves, root and spikes was performed using Illumina platform, which generated 16901456, 54993496 and 22900035, respectively of high quality reads. In de novo assembly P. longum 173381 numbers of transcripts were analyzed. Analysis of transcriptome data from leaf, root and spike showed gene families that were involved in the biosynthetic pathway of piperine and other secondary metabolites. To validate differential expression of the identified genes, 27 genes were randomly selected to confirm the expression level by quantitative real time PCR (qRT-PCR) based on the up regulation and down regulation of differentially expressed genes obtained through comparative transcriptome analysis of leaves and spike of P. longum. With the help of UniProt database the function of all characterized genes was generated.

plant biology↗

bioRxiv2019

Genetic and antigenic variation of foot-and-mouth disease virus during persistent infection in naturally infected cattle and Asian buffalo in India

The role of foot-and-mouth disease virus (FMDV) persistently infected ruminants in initiating new outbreaks remains controversial, and the perceived threat posed by such animals hinders international trade in FMD-endemic countries. In this study we report longitudinal analyses of genetic and antigenic variations of FMDV serotype O/ME-SA/Ind2001d sublineage during naturally occurring, persistent infection in cattle and buffalo at an organised dairy farm in India. The proportion of animals from which FMDV RNA was recovered was not significantly different between convalescent (post-clinical) and sub-clinically infected animals or between cattle and buffalo across the sampling period. However, infectious virus was isolated from a higher proportion of buffalo samples and for a longer duration compared to cattle. Analysis of the P1 sequences from recovered viruses indicated fixation of mutations at the rate of 1.816 x 10-2 substitution/site/year (s/s/y) (95% CI 1.362-2.31 x 10-2 s/s/y). However, the majority of point mutations were transitional substitutions. Within individual animals, the mean dN/dS ({omega}) value for the P1 region varied from 0.076 to 0.357, suggesting the selection pressure acting on viral genomes differed substantially across individual animals. Statistical parsimony analysis indicated that all of the virus isolates from carrier animals originated from the outbreak virus. The antigenic relationship value as determined by 2D-VNT assay revealed fluctuation of antigenic variants within and between carrier animals during the carrier state which suggested that some carrier viruses had diverged substantially from the protection provided by the vaccine strain. This study contributes to understanding the extent of within-host and within-herd evolution that occurs during the carrier state of FMDV.

evolutionary biology↗

bioRxiv2018

Stem cell mitotic drive ensures asymmetric epigenetic inheritance

Through the process of symmetric cell division, one mother cell gives rise to two identical daughter cells. Many stem cells utilize asymmetric cell division (ACD) to produce a self-renewed stem cell and a differentiating daughter cell. Since both daughter cells inherit the identical genetic information during ACD, a crucial question concerns how non-genic factors could be inherited differentially to establish distinct cell fates. It has been hypothesized that epigenetic differences at sister centromeres could contribute to biased sister chromatid attachment and segregation. However, direct in vivo evidence has never been shown. Here, we report that a stem cell-specific mitotic drive ensures biased sister chromatid attachment and segregation. We have found during stem cell ACD, sister centromeres become asymmetrically enriched with proteins involved in centromere specification and kinetochore function. Furthermore, we show that that temporally asymmetric microtubule activities direct polarized nuclear envelope breakdown, allowing for the preferential recognition and attachment of microtubules to asymmetric sister kinetochores and sister centromeres. This communication occurs in a spatiotemporally regulated manner. Abolishment of either the establishment of asymmetric sister centromeres or the asymmetric microtubule emanation results in randomized sister chromatid segregation, which leads to stem cell loss. Our results demonstrate that the cis-asymmetry at sister centromeres tightly coordinates with the trans-asymmetry from the mitotic machinery to allow for differential attachment and segregation of genetically identical yet epigenetically distinct sister chromatids. Together, these results provide the first direct in vivo mechanisms for partitioning epigenetically distinct sister chromatids in asymmetrically dividing stem cells, which opens a new direction to study how this mechanism could be used in other developmental contexts to achieve distinct cell fates through mitosis. One Sentence SummaryDuring Drosophila male germline stem cell asymmetric division, sister centromeres communicate with spindle microtubules for differential attachment and segregation of sister chromatids.

developmental biology↗

bioRxiv2018

Evaluating bacterial and functional diversity of human gut microbiota by complementary metagenomics and metatranscriptomics

It is well accepted that dysbiosis of microbiota is associated with disease; however, the biological mechanisms that promote susceptibility or resilience to disease remain elusive. One of the major limitations of previous microbiome studies has been the lack of complementary metatranscriptomic (functional) data to complement the interpretation of metagenomics (bacterial abundance). The purpose of the study was twofold, first to evaluate the bacterial diversity and differential gene expression of gut microbiota using complementary shotgun metagenomics (MG) and metatranscriptomics (MT) from same fecal sample. Second, to compare sequence data using different Illumina platforms and with different sequencing parameters as new sequencers are introduced and determine if the data are comparable on different platforms. In this study, we perform ultra-deep metatranscriptomic shotgun sequencing for a sample that we previously analyzed with metagenomics shotgun sequencing. We validated the sequencing and analysis methods using different Illumina platform, and with different sequencing and analysis parameters. Our results suggest that use of different Illumina platform did not lead to detectable bias in the sequencing data. The analysis of the sample using MG and MT approach shows that some species genes are more highly represented in the MT than in the MG, indicating that some species are highly metabolically active. Our analysis also shows that ~52% of the genes in the metagenome are in the metatranscriptome, and therefore are robustly expressed. The functions of the low and rare abundance bacterial species remain poorly understood. Our observations indicate that among the low abundant species analyzed in this study some were found to be more metabolically active compared to others and can contribute distinct profiles of biological functions that may modulate the host-microbiota and bacteria-bacteria interactions.

microbiology↗

bioRxiv2018

Asymmetric histone incorporation during DNA replication in Drosophila male germline stem cells

One Sentence SummaryDNA replication establishes asymmetric epigenomes SummaryOne of the most fundamental questions in developmental biology concerns how cells with identical genomes differentiate into distinct cell types. One important context for understanding cell fate specification is asymmetric cell division, where the two daughter cells establish different cell fates following a single division. Many stem cells undergo asymmetric division to produce both a self-renewing stem cell and a differentiating daughter cell1-5. Here we show that histone H4 is inherited asymmetrically in asymmetrically dividing Drosophila male germline stem cells, similar to H36. In contrast, both H2A and H2B are inherited symmetrically. By combining superresolution microscopy with the chromatin fiber method, we are able to study histone inheritance patterns on newly replicated chromatin fibers. Using this technique, we find asymmetric inheritance patterns for old and new H3, but symmetric inheritance patterns for old and new H2A on replicating sister chromatids. Furthermore, co-localization studies on isolated chromatin fibers and proximity ligation assays on intact nuclei reveal that old H3 are preferentially incorporated by the leading strand while newly synthesized H3 are enriched on the lagging strand. Finally, using a sequential nucleoside analog incorporation assay, we detect a high incidence of unidirectional DNA replication on germline-derived chromatin fibers and DNA fibers. The unidirectional fork movement coupled with the strand preference of histone incorporation could explain how old and new H3 are asymmetrically incorporated by replicating sister chromatids. In summary, our work demonstrates that the intrinsic asymmetries in DNA replication may help construct sister chromatids enriched with distinct populations of histones. Therefore, these results suggest unappreciated roles for DNA replication in asymmetrically dividing cells in multicellular organisms.

developmental biology↗