Biology subjects

Sahu, S.

Publications and source records attributed to Sahu, S..

32 records · Page 2Linked to original sources

bioRxiv2022

Circadian rhythm orthologs drive pulses of heterochronic miRNA transcription in C. elegans

Developmental robustness relies on precise control of the timing and order of cellular events. In C. elegans, the invariant sequence of post-embryonic cell fate specification is controlled by oscillatory patterns of heterochronic microRNA transcription that are phase-locked with the larval molting cycle1-4. How these transcriptional patterns are generated and how microRNA dosage is controlled is unknown. Here we show that transcriptional pulses of the lin-4 heterochronic microRNA are produced by two nuclear hormone receptors, NHR-85 and NHR-23, whose mammalian orthologs, Rev-Erb and ROR, function in the circadian clock. While Rev-Erb and ROR play antagonistic roles in regulating once-daily transcription5-7, we find that NHR-85 and NHR-23 bind cooperatively as heterodimers to lin-4 regulatory elements to induce a single brief pulse of expression during each larval stage. We demonstrate that the timing and duration of lin-4 transcriptional pulses are programmed by the phased overlap of NHR-85 and NHR-23 protein expression and that these regulatory interactions are post-transcriptionally controlled by LIN-42, the circadian Period ortholog in C. elegans. These findings suggest that an evolutionary rewiring of the circadian clock machinery is co-opted in nematodes to generate periodic transcriptional patterns that define cell fate progression.

developmental biology↗

bioRxiv2021

Ecdysone exerts biphasic control of regenerative signaling, coordinating the completion of regeneration with developmental progression

In Drosophila melanogaster, loss of regenerative capacity in wing imaginal discs coincides with an increase in systemic levels of the steroid hormone ecdysone, a key coordinator of their developmental progression. Regenerating discs release the relaxin hormone Dilp8, which limits ecdysone synthesis and extends the regenerative period. Here, we describe how regenerating tissues produce a biphasic response to ecdysone levels: lower concentrations of ecdysone promote local and systemic regenerative signaling, whereas higher concentrations suppress regeneration through the expression of broad splice isoforms. Ecdysone also promotes the expression of wingless during both regeneration and normal development through a distinct regulatory pathway. This dual role for ecdysone explains how regeneration can still be completed successfully in dilp8- mutant larvae: higher ecdysone levels increase the regenerative activity of tissues, allowing regeneration to reach completion in a shorter time. From these observations, we propose that ecdysone hormone signaling functions to coordinate regeneration with developmental progression. Summary StatementEcdysone coordinates regenerative activity with developmental progression through the biphasic, concentration-dependent activation, and suppression of regenerative signaling.

developmental biology↗

bioRxiv2021

Berberine as siderophore from Talaromyces trachyspermus: augmentation and characterization".

In the present study, a siderophore compound produced by an endophytic fungus, Talaromyces trachyspermus was optimized for maximum production, 88.9 % SU by applying Plackett-Burman design and Response Surface Methodology through Central Composite Design that showed the succinic acid (1.141 g/L), sucrose (31.028 g/L) and temperature (27.475 {degrees}C) as significant factors. On scale up, a further increase in siderophore yield was obtained (by 3%) The compound was extracted, purified and detected chemically as catecholate siderophore showing max. {lambda} absorbance at 279nm. Contained of hydroxy benzene as shown by GC-MS analysis and further identified as berberine by HRLC-MS studies. The compound berberine is clinically a very important drug with several ethnobotanical properties. This is rare to report fungal catecholate and first to report the production of berberine from Talaromyces species.

microbiology↗

bioRxiv2021

A pro-endocrine pancreatic transcriptional program established during development is retained in human gallbladder epithelial cells

ObjectivePancreatic islet {beta}-cells are factories for insulin production; however ectopic expression of insulin is also well recognized. The gallbladder is a next-door neighbour to the developing pancreas. Here, we wanted to understand if gallbladders contain functional insulin-producing cells. DesignWe compared developing and adult mouse as well as human gallbladder epithelial cells and islets using immunohistochemistry, flow cytometry, ELISAs, RNA-sequencing, real-time PCR, chromatin immunoprecipitation and functional studies. ResultsWe demonstrate that the epithelial lining of developing, as well as adult mouse and human gallbladders naturally contain interspersed cells that retain the capacity to actively transcribe, translate, package, and release insulin. We show for the first time that human gallbladders also contain functional insulin-secreting cells with the potential to naturally respond to glucose in vitro and in situ. Notably, in a NOD mouse model of type 1 diabetes, we observed that insulin-producing cells in the gallbladder are not targeted by autoimmune cells. Conclusion: In summary, our biochemical, transcriptomic, and functional data in human gallbladder epithelial cells collectively demonstrate their potential for insulin-production under pathophysiological conditions, and open newer areas for type 1 diabetes research and therapy. Significance of the study What is already known about this subject?O_LIDeveloping pancreas and gallbladder are next-door neighbours and share similar developmental pathways. C_LIO_LIHuman Gallbladder-derived progenitor cells were shown to differentiate into insulin-producing cells. C_LI What are the new findings?O_LIGallbladder epithelium contains interspersed cells that can transcribe, translate, package and secrete insulin. C_LIO_LIInsulin-producing cells in the gallbladder are not destroyed by immune cells in an animal model of type 1 diabetes (T1D). C_LIO_LIOur studies demonstrating the absence of insulin splice variants in human gallbladder cells, and higher splice forms in human islets, suggest a potential mechanism (via defective ribosomal products) in escaping islet autoimmunity. C_LI How might it impact clinical practice?O_LIDeciphering mechanisms of protection of insulin-producing cells from immune cells in the gallbladder could help in developing strategies to prevent islet autoimmunity in T1D. C_LI

developmental biology↗

bioRxiv2020

Troxerutin acts on complement mediated inflammation to ameliorate arthritic symptoms in rats.

Troxerutin (TXR), is a phytochemical reported to possess anti-inflammatory and hepatoprotective effects. In this study, we aimed to exploit anti-arthritic properties of TXR using an adjuvant induced arthritic (AIA) rat model. AIA induced rats showed highest arthritis score at disease onset and by oral administration of TXR (50, 100, 200 mg/kg body weight), reduced to basal level in a dose dependent manner. Isobaric tag for relative and absolute quantitative (iTRAQ) proteomics tool was employed to identify deregulated joint homogenate proteins in AIA and TXR treated rats to decipher probable mechanism of the TXR action in arthritis. iTRAQ analysis identified a set of 434 joint homogenate proteins with 65 deregulated proteins (log2 case/control [≥]1.5) in AIA. Expressions of a set of important proteins (AAT, T-kininogen, vimentin, desmin, and nucleophosmin) that could classify AIA from healthy were validated using Western blot analysis. Western blot data corroborated proteomics findings. In silico protein-protein interaction study of joint homogenate proteome revealed that complement component 9, the major building blocks of the membrane attack complex (MAC) responsible for sterile inflammation, gets perturbed in AIA. Our dosimetry study suggests that a TXR dose of 200 mg/kg body weight for 15 days is sufficient to bring the arthritis score to basal levels in AIA rats. We have shown the importance of TXR as an anti-arthritis agent in AIA model and after additional investigation its arthritis ameliorating properties could be exploited for clinical usability.

biochemistry↗

bioRxiv2020

Single cell mutational profiling delineates clonal trajectories in myeloid malignancies

Myeloid malignancies, including acute myeloid leukemia (AML), arise from the proliferation and expansion of hematopoietic stem and progenitor cells which acquire somatic mutations. Bulk molecular profiling studies on patient samples have suggested that somatic mutations are obtained in a step-wise fashion, where mutant genes with high variant allele frequencies (VAFs) are proposed to occur early in disease development and mutations with lower VAFs are thought to be acquired later in disease progression1-3. Although bulk sequencing informs leukemia biology and prognostication, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity and clone size, or offer definitive evidence of mutational order. To elucidate the clonal framework of myeloid malignancies, we performed single cell mutational profiling on 146 samples from 123 patients. We found AML is most commonly comprised of a small number of dominant clones, which in many cases harbor co-occurring mutations in epigenetic regulators. Conversely, mutations in signaling genes often occur more than once in distinct subclones consistent with increasing clonal diversity. We also used these data to map the clonal trajectory of each patient and found that specific mutation combinations (FLT3-ITD + NPM1c) synergize to promote clonal expansion and dominance. We combined cell surface protein expression with single cell mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our studies of clonal architecture at a single cell level provide novel insights into the pathogenesis of myeloid transformation and how clonal complexity contributes to disease progression.

cancer biology↗

bioRxiv2019

Ongoing repair of migration-coupled DNA damage allows stem cells to reach wound sites

The impact of mechanical stress during cell migration may be a previously unappreciated source of genome instability [1-3], but to what extent this happens in vivo remains unknown. Here we consider an in vivo system where the adult stem cells of planarian flatworms are required to migrate to a distal wound site [4]. We observe a relationship between adult stem cell migration and ongoing DNA damage and repair during tissue regeneration. Migrating planarian stem cells undergo changes in nuclear shape and increased levels of DNA damage. Increased DNA damage levels resolve once stem cells reach the wound site and stop migrating. Stem cells in which DNA damage is induced prior to wounding take longer to initiate migration suggesting migration activity is sensitive to DNA damage. Migrating stem cells populations are more sensitive to further DNA damage than stationary stem cells, providing evidence that levels of migration-coupled-DNA-damage (MCDD) are significant. RNAi mediated knockdown of DNA repair pathway components blocks normal stem cell migration, confirming that DNA repair pathways are required to allow successful migration to a distal wound site. Together these lines of evidence demonstrate that migration leans to DNA damage in vivo and requires DNA repair mechanisms. Our findings reveal that migration of stem cells represents an unappreciated source of damage, that could be a significant source of mutations in animals during development or during long term tissue homeostasis.

developmental biology↗

bioRxiv2019

Self-Organization of Spindle-Like Microtubule Structures

Microtubule self-organization is an important physical process underlying a number of essential cellular functions including cell division. In cell division, the dominant organization is the mitotic spindle, a football-shaped microtubule organization. We are interested in the underlying fundamental principles behind the self-organization of the spindle shape. Prior biological works have hypothesized that motor proteins are required for the proper formation of the spindle. Many of these motor proteins are also microtubule-crosslinkers, so it is unclear if the important aspect is the motor activity or the crosslinking. In this study, we seek to address this question by examining the self-organization of microtubules using crosslinkers alone. We use a minimal system composed of tubulin, an antiparallel microtubule-crosslinking protein, and a crowding agent to explore the phase space of organizations as a function of tubulin and crosslinker concentration. We find that the concentration of the antiparallel crosslinker, MAP65, has a significant effect on the organization and resulted in spindle-like organizations at relatively low concentration without the need for motor activity. Surprisingly, the length of the microtubules only moderately affects the equilibrium organization. We characterize both the shape and dynamics of these spindle-like organizations. We find that they are birefringent homogeneous tactoids. The microtubules have slow mobility, but the crosslinkers have fast mobility within the tactoids. These structures represent a first step in the recapitulation of self-organized spindles of microtubules that can be used as initial structures for further biophysical and active matter studies relevant to the biological process of cell division.

biophysics↗

bioRxiv2019

Countries are out of step with international recommendations for tuberculosis testing, treatment, and care: Findings from a 29-country survey of policy adoption and implementation

BackgroundTuberculosis (TB) poses a global health crisis requiring robust international and country-level action. Adopting and implementing TB policies from the World Health Organization (WHO) is essential to meeting global targets for reducing TB burden. However, many high TB burden countries lag in implementing WHO recommendations. Assessing the progress of implementation at national level can identify key gaps that must be addressed to expand and improve TB care. MethodsIn 2016/2017, Medecins Sans Frontieres and the Stop TB Partnership conducted a survey on adoption and implementation of 47 WHO TB policies in the national TB programs of 29 countries. Here we analyze a subset of 23 key policies in diagnosis, models of care, treatment, prevention, and drug regulation to provide a snapshot of national TB policy adoption and implementation. We examine progress since an analogous 2015 survey of 23 of the same countries. ResultsAt the time of the survey, many countries had not yet aligned their national guidelines with all WHO recommendations, although some progress was seen since 2015. For diagnosis, about half of surveyed countries had adopted the WHO-recommended initial rapid test (Xpert MTB/RIF). A majority of countries had adopted decentralized models of care, although one-third of them still required hospitalization for drug-resistant (DR-)TB. Recommended use of the newer drugs bedaquiline (registered in only 6 high-burden TB countries) and delamanid (not registered in any high-burden country) was adopted by 23 and 18 countries, respectively, but short-course (9-month) and newer pediatric regimens by only 13 and 14 countries, respectively. Guidelines in all countries included preventive treatment of latent TB infection for child TB contacts and people living with HIV/AIDS, but only four extended this to adult contacts. ConclusionTo reach global TB targets, greater political will is needed to rapidly adopt and implement internationally recognized care guidelines. KEY MESSAGESO_ST_ABSWhat is already known?C_ST_ABSO_LICountries may be slow to adopt and implement updated World Health Organization (WHO) Tuberculosis (TB) testing, treatment, and prevention recommendations. C_LIO_LIImplementing updated TB guidelines from WHO is a fundamental first step to honoring international commitments, made through the United Nations (UN) Sustainable Development Goals (SDGs) and UN High-Level Meeting on TB Political Declaration, to end TB by 2030. C_LI What are the new findings?O_LIOf 29 mostly high TB burden countries, none had fully aligned their national guidelines with WHO recommendations, although some progress has been made since 2015. C_LIO_LIA lack of alignment with WHO recommendations was found across all policy areas surveyed, including prevention, diagnosis, treatment, models of care and drug regulation, particularly regarding uptake of newer, faster, more effective approaches. C_LI What do the new findings imply?O_LITo reach global TB targets, greater political will is needed to adopt and implement internationally recognized care guidelines more rapidly, and specifically, to keep up with the latest recommendations. C_LIO_LIPeriodic surveys of progress at the national level are a valuable way to identify specific areas where countries or regions have fallen behind and that require specific policy and/or programmatic attention. C_LI

epidemiology↗

bioRxiv2018

Morphofunctional deficits in the cerebral cortex of NeuroD2 mutant mice are associated with autism/schizophrenia-like behaviors

We identified seven families associating NEUROD2 pathogenic mutations with ASD and intellectual disability. To get insight into the pathophysiological mechanisms, we analyzed cortical development in Neurod2 KO mice. Cortical projection neurons (CPNs) over-migrated during embryogenesis, inducing abnormal thickness and laminar positioning of cortical layers. At juvenile ages, dendritic spine turnover and intrinsic excitability were increased in L5 CPNs. Differentially expressed genes in Neurod2 KO mice were enriched for voltage-gated ion channels, and the human orthologs of these genes were strongly associated with ASD. Furthermore, adult Neurod2 KO mice exhibited core ASD-like behavioral abnormalities. Finally, by generating Neurod2 conditional mutant mice we demonstrate that forebrain excitatory neuron-specific Neurod2 deletion recapitulates cellular and behavioral ASD phenotypes found in full KO mice. Our findings demonstrate crucial roles for Neurod2 in cortical development and function, whose alterations likely account for ASD and related symptoms in the newly defined NEUROD2 mutation syndrome.

neuroscience↗

bioRxiv2017

Correct laminar positioning in the neocortex influences proper dendritic and synaptic development

The neocortex is a six-layered laminated structure with a precise anatomical and functional organization ensuring proper function. Laminar positioning of cortical neurons, as determined by termination of neuronal migration, is a key determinant of their ability to assemble into functional circuits. However, the exact contribution of laminar placement to dendrite morphogenesis and synapse formation remains unclear. Here we manipulated the laminar position of cortical neurons by knocking down Dcx, a crucial effector of migration, and show that misplaced neurons fail to properly form dendrites, spines and functional glutamatergic synapses. We further show that knocking down Dcx in properly positioned neurons induces similar but milder defects, suggesting that the laminar misplacement is the primary cause of altered neuronal development. Thus, the specific laminar environment of their fated layers is crucial for the maturation of cortical neurons, and influences their functional integration into developing cortical circuits.

neuroscience↗

bioRxiv2017

An essential post-developmental role for Lis1 in mice

LIS1 mutations cause lissencephaly (LIS), a severe developmental brain malformation. Much less is known about its role in the mature nervous system. LIS1 regulates the microtubule motor cytoplasmic dynein 1 (dynein), and as LIS1 and dynein are both expressed in the adult nervous system, Lis1 could potentially regulate dynein-dependent processes such as axonal transport. We therefore knocked out Lis1 in adult mice using tamoxifen-induced, Cre-ER-mediated recombination. When an actin promoter was used to drive Cre-ER expression (Act-Cre-ER), heterozygous Lis1 KO caused no obvious change in viability or behavior, despite evidence of widespread recombination by a Cre reporter three weeks after tamoxifen exposure. In contrast, homozygous Lis1 KO caused the rapid onset of neurological symptoms in both male and female mice. One tamoxifen-dosing regimen caused prominent recombination in the midbrain/hindbrain, PNS, and cardiac/skeletal muscle within a week; these mice developed severe symptoms in that time frame and were euthanized. A different tamoxifen regimen resulted in delayed recombination in midbrain/hindbrain, but not in other tissues, and also delayed the onset of symptoms. This indicates that Lis1 loss in the midbrain/hindbrain causes the severe phenotype. In support of this, brainstem regions known to house cardiorespiratory centers showed signs of axonal dysfunction in KO animals. Transport defects, neurofilament alterations, and varicosities were observed in axons in cultured DRG neurons from KO animals. Because no symptoms were observed when a cardiac specific Cre-ER promoter was used, we propose a vital role for Lis1 in autonomic neurons and implicate defective axonal transport in the KO phenotype.\n\nSIGNIFICANCE STATEMENTMammalian Lis1 is best known for its role in brain development. Lis1 binds to and regulates the microtubule motor, cytoplasmic dynein. We show that Lis1 function is needed post-developmentally and provide evidence that loss of Lis1 in the hindbrain leads to death. The effect is dose dependent in mice, as loss of only one allele does not produce an overt phenotype. However, since LIS1 haploinsufficiency causes lissencephaly (LIS) in humans, our study raises the possibility that post-developmental axonal transport defects could contribute to worsening symptoms in children with LIS1 mutations. Our data are consistent with the hypothesis is that Lis1 regulates dynein-dependent axon transport in the mature nervous system.

neuroscience↗

bioRxiv2017

The Abrogation Of Condensin Function Provides Independent Evidence For Defining The Self-Renewing Population Of Pluripotent Stem Cells

Heterogeneity of planarian neoblast stem cells has been categorised on the basis of single cell expression analyses and subsequent experiments to demonstrate lineage relationships. Some data suggest that despite gene expression heterogeneity amongst cells in the cell cycle, in fact only one sub-population, known as sigma neoblasts, can self-renew. Without the tools to perform live in vivo lineage analysis, we instead took an alternative approach to provide independent evidence for defining the self-renewing stem cell population. We exploited the role of highly conserved condensin proteins to functionally assay neoblast self-renewal properties. Condensins are involved in forming properly condensed chromosomes to allow cell division to proceed during mitosis, and their abrogation inhibits mitosis and can lead to repeated endoreplication of the genome in cells that make repeated attempts to divide. We find that planarians possess only the condensin I complex, and that this is required for normal stem cell function. Abrogation of condensin function led to rapid stem cell depletion accompanied by the appearance of giant cells with increased DNA content. Using previously discovered markers of heterogeneity we show that enlarged cells are always from the sigma-class of the neoblast population and we never observe evidence for endoreplication for the other neoblast subclasses. Overall, our data establish that condensins are essential for stem cell maintenance and provide independent evidence that only sigma-neoblasts are capable of multiple rounds of cell division and hence self-renewal.

developmental biology↗

bioRxiv2016

Analyses of the core eukaryotic protein subunit of telomerase support extensive adaptation to different evolutionary and life histories in the Metazoa

Most animals employ telomerase, which consists of a catalytic subunit known as the telomerase reverse transcriptase (TERT) and an RNA template, to maintain telomere ends. Given the importance of TERT and the apparent importance of telomere biology in core metazoan life history traits like ageing and the control of somatic cell proliferation, we hypothesised that TERT would have patterns of sequence and regulatory evolution reflecting adaptations to diverse evolutionary and life histories across the Animal Kingdom. To test this, we performed a complete investigation of the evolutionary history of TERT across animals. We show that although TERT is almost ubiquitous across Metazoa, it has undergone substantial sequence evolution in canonical motifs. Beyond the known canonical motifs, we also identify and compare regions that are highly variable between lineages, but for which conservation exists within phyla. Recent data have highlighted the importance of alternate splice forms of TERT in non-canonical functions in some animals. Although animals may share some conserved introns, we find that the selection of exons for alternative splicing appears to be highly variable, and regulation by alternative splicing appears to be a very dynamic feature of TERT evolution. We show that even within a closely related group of triclad flatworms, where alternative splicing of TERT was previously correlated with reproductive strategy, we observe highly diverse alternative splicing patterns. Our work establishes that the evolutionary history and structural evolution of TERT involves previously unappreciated levels of change, supporting the view that this core eukaryotic protein has adapted to the requirements of diverse animal life histories.

evolutionary biology↗