Biology subjects

Kulkarni, A.

Publications and source records attributed to Kulkarni, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Drivers of phenotypic differentiation across rock outcrop sky islands in Impatiens plants

Habitat specialization can lead to naturally fragmented populations separated by unoccupied habitats that comprise potential barriers to dispersal. Habitat fragmentation can result in phenotypic divergence between populations, and eventual speciation. We investigate trait divergence in a habitat specialist plant species - Impatiens lawii - that is specialized on rock outcrops in the Western Ghats mountains of India. We compare trait divergence in this species with that in a closely related species, I. oppositifolia, which is a continuously distributed habitat generalist that co-occurs with the former species on rock outcrops in addition to being found in intervening regions. As predicted, we found that floral traits were more strongly structured across populations in I. lawii than in I. oppositifolia, supporting the role of habitat specialization. In both species, floral traits were more strongly structured across populations than were vegetative traits. Interestingly, the population pairs that were significantly differentiated for floral traits differed from those differentiated for vegetative traits. Furthermore, some population pairs differed in vegetative traits but not in floral traits. The incongruence in patterns of floral and vegetative trait differentiation suggests the role of selection. There was no association between floral trait variation and geographic distance in either species. Surprisingly, there was a significant correlation between geographic distance and vegetative trait variation in I. oppositifolia, but not in I. lawii (Fig 4D; Table 2), possibly due to genetic drift. Overall, a combination of habitat specificity, drift and selection best explains trait variation in I. lawii.

evolutionary biology↗

bioRxiv2023

FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement

Long-range glutamatergic inputs from the cortex and thalamus are critical for motor and cognitive processing in the striatum. Transcription factors that orchestrate the development of these inputs are largely unknown. We investigated the role of a transcription factor and high-risk autism-associated gene, FOXP1, in the development of glutamatergic inputs onto spiny projection neurons (SPNs) in the striatum. We find that FOXP1 robustly drives the strengthening and maturation of glutamatergic input onto dopamine receptor 2-expressing SPNs (D2 SPNs) but has a comparatively milder effect on D1 SPNs. This process is cell-autonomous and is likely mediated through postnatal FOXP1 function at the postsynapse. We identified postsynaptic FOXP1-regulated transcripts as potential candidates for mediating these effects. Postnatal reinstatement of FOXP1 rescues electrophysiological deficits, reverses gene expression alterations resulting from embryonic deletion, and mitigates behavioral phenotypes. These results provide support for a possible therapeutic approach for individuals with FOXP1 syndrome.

neuroscience↗

bioRxiv2023

WELPCR: Low-cost polymerase chain reaction (PCR) thermal cycler for nucleic acid amplification and sensing

The COVID-19 pandemic highlighted the need for sensitive and cost-effective diagnostics. The gold standard test to diagnose infectious diseases such as COVID-19 is quantitative polymerase chain reaction (qPCR). Although there have been multiple advances in nucleic acid testing, PCR thermocyclers are expensive and typically not affordable for offering hands-on training to PCR in instructional laboratory courses in undergraduate colleges. We introduce a low-cost, portable, and automated real-time PCR device designed at the Wadhwani Electronics Lab (WEL) at IIT Bombay, christened WELPCR. WELPCR is an open-source design, and capable of reverse transcription PCR (RT-PCR) for complementary DNA synthesis, as well as end-point PCR of small amount of DNA in a test sample. The WELPCR design enables ramp rate up to 2 {degrees}C/s, weighs less than 2 kg, and is easy to assemble. The built-in LED display and keypad provides an intuitive and seamless user interface to configure and set up WELPCR. With bill of materials (BOM) cost less than INR Rs. 10 000, WELPCR is an attractive solution for bringing PCR technology to instructional and research laboratories in a cost-effective manner.

molecular biology↗

bioRxiv2023

Identification of resistance mechanisms to small-molecule inhibition of TEAD-regulated transcription

The Hippo tumour suppressor pathway controls transcription by regulating nuclear abundance of YAP and TAZ, which activate transcription with the TEAD1-TEAD4 DNA-binding proteins. Recently, several small-molecule inhibitors of YAP and TEADs have been reported, with some now entering clinical trials for different cancers. Here, we investigated the cellular response to TEAD palmitoylation inhibitors, using a combination of genomic and genetic strategies. Genome-wide CRISPR/Cas9 screens identified genes that modulate the cellular response to TEAD inhibition, including members of the Hippo, MAPK and JAK-STAT signaling pathways. By exploring gene expression programs of mutant cells, we found that MAPK pathway hyperactivation confers resistance to TEAD inhibition by reinstating expression of a subset of YAP/TEAD target genes. Consistent with this, combined inhibition of TEAD and the MAPK protein MEK, synergistically blocked proliferation of several mesothelioma and lung cancer cell lines and more potently reduced the growth of patient-derived lung cancers in vivo. Collectively, we reveal mechanisms by which cells can overcome small-molecule inhibition of TEADs and potential strategies to enhance the anti-tumor activity of emerging Hippo pathway targeted therapies.

cancer biology↗

bioRxiv2023

Compensation between FOXP transcription factors maintains proper striatal function

Spiny projection neurons (SPNs) of the striatum are critical in integrating neurochemical information to coordinate motor and reward-based behavior. Mutations in the regulatory transcription factors expressed in SPNs can result in neurodevelopmental disorders (NDDs). Paralogous transcription factors Foxp1 and Foxp2, which are both expressed in the dopamine receptor 1 (D1) expressing SPNs, are known to have variants implicated in NDDs. Utilizing mice with a D1-SPN specific loss of Foxp1, Foxp2, or both and a combination of behavior, electrophysiology, and cell-type specific genomic analysis, loss of both genes results in impaired motor and social behavior as well as increased firing of the D1-SPNs. Differential gene expression analysis implicates genes involved in autism risk, electrophysiological properties, and neuronal development and function. Viral mediated re-expression of Foxp1 into the double knockouts was sufficient to restore electrophysiological and behavioral deficits. These data indicate complementary roles between Foxp1 and Foxp2 in the D1-SPNs.

neuroscience↗

bioRxiv2023

Multiprotein Assemblies, Phosphorylation and Dephosphorylation in Neuronal Cytoskeleton

Filament systems are comprised of fibrous and globular cytoskeletal proteins and are key elements regulating cell shape, rigidity, and dynamics. The cellular localization and assembly of neurofilaments depend on phosphorylation by kinases. The involvement of the BRCA1 (Breast cancer associated protein 1)/BARD1 (BRCA1-associated RING domain 1) pathways in Alzheimer disease (AD) is suggested by colocalization studies. In particular, BRCA1 accumulation within neurofibrillary tangles and colocalization with tau aggregates in the cytoplasm of AD patients implicates the involvement of mutant forms of BRCA1/BARD1 proteins in disease pathogenesis. The purpose of this study is to show that the location of mutations in the translated BARD1, specifically within ankyrin repeats, has strong correlation with the Cdk5 motifs for phosphorylation. Mapping of the mutation sites on the proteins three-dimensional structure and estimation of the backbone dihedral angles show transitions between the canonical helical and extended conformations of the tetrapeptide sequence of ankyrin repeats. Clustering of mutations in BARD1 ankyrin repeats near the N-termini of the helices with T/SXXH motifs provides a basis for conformational transitions that might be necessary to ensure the compatibility of the substrate with active site geometry and accessibility of the substrate to the kinase. Ankyrin repeats are interaction sites for phosphorylation-dependent dynamic assembly of proteins including those involved in transcription regulation and signaling, and present potential targets for the design of new drugs.

biochemistry↗

bioRxiv2023

ACTIVATION OF CYCLIN-DEPENDENT KINASE 5 BROADENS ACTION POTENTIALS IN HUMAN SENSORY NEURONS

Chronic pain is one of the most devastating and unpleasant conditions, associated with many pathological conditions. Tissue or nerve injuries induce comprehensive neurobiological plasticity in nociceptive neurons, which leads to chronic pain. Recent studies suggest that cyclin-dependent kinase 5 (CDK5) in primary afferents is a key neuronal kinase that modulates nociception through phosphorylation-dependent manner under pathological conditions. However, the impact of the CDK5 on nociceptor activity especially in human sensory neurons are not known. To determine the CDK5-mediated regulation of human dorsal root ganglia (hDRG) neuronal properties, we have performed the whole-cell patch clamp recordings in neurons dissociated from hDRG. CDK5 activation induced by overexpression of p35 depolarized the resting membrane potential and reduced the rheobase currents as compared to the uninfected neurons. CDK5 activation evidently changed the shape of the action potential (AP) by increasing AP rise time, AP fall time, and AP half width. The application of a prostaglandin E2 (PG) and bradykinin (BK) cocktail in uninfected hDRG neurons induced the depolarization of RMP and the reduction of rheobase currents along with increased AP rise time. However, PG and BK applications failed to induce any further significant changes in addition to the aforementioned changes of the membrane properties and AP parameters in the p35-overexpressing group. We conclude that CDK5 activation through the overexpression of p35 in dissociated hDRG neurons broadens AP in hDRG neurons and that CDK5 may play important roles in the modulation of AP properties in human primary afferents under pathological conditions, contributing to chronic pain.

neuroscience↗

bioRxiv2023

Using CRISPR/Cas9 to identify genes required for mechanosensory neuron development and function

Tunicates are marine, non-vertebrate chordates that comprise the sister group to the vertebrates. Most tunicates have a biphasic lifecycle that alternates between a swimming larva and a sessile adult. Recent advances have shed light on the neural basis for the tunicate larvas ability to sense a proper substrate for settlement and initiate metamorphosis. Work in the highly tractable laboratory model tunicate Ciona robusta suggests that sensory neurons embedded in the anterior papillae of transduce mechanosensory stimuli to trigger larval tail retraction and initiate the process of metamorphosis. Here, we take advantage of the low-cost and simplicity of Ciona by using tissue-specific CRISPR/Cas9-mediated mutagenesis to screen for genes potentially involved in mechanosensation and metamorphosis, in the context of an undergraduate "capstone" research course. This small screen revealed at least one gene, Vamp1/2/3, that appears crucial for the ability of the papillae to trigger metamorphosis. We also provide step-by-step protocols and tutorials associated with this course, in the hope that it might be replicated in similar CRISPR-based laboratory courses wherever Ciona are available.

developmental biology↗

bioRxiv2023

Reservoir Dogs: Consequences of variable contact network structures for disease spread in free-ranging dogs

The risk of disease spread is contingent on not only the biological properties of causal agents, such as viruses and bacteria, but also the socio-ecological context of the outbreak. Therefore, researchers need to incorporate the variation in animal-animal interactions in models of disease spread. Much research has focused on how ecological factors (seasonality, mating systems) can influence the variation in animal interactions, but heterogeneity in interactions may arise due to human action, and this has received little attention. We hypothesised that social-mediated differences in the landscape can have consequences for interactions for free-roaming dogs and consequently for disease spread. We used GPS collared data from 31 free-ranging dogs present along a farm-village gradient to build social contact networks to examine differences in interactions. Additionally, we simulated scenarios of rabies infection of two types - dumb and furious - on the contact network structure of village and farm dogs. We found significant differences in the node degree between village and farm contact networks, but the node betweenness was similar. Our simulations showed that infections arising in village dogs are likely to spread to that network, but infections arising in farm dogs get are less likely to spread. Interestingly, this was independent of the type of rabies infection. We discuss our results in light of control strategies for managing rabies in the tropics.

ecology↗

bioRxiv2023

An extra-circadian function for human CLOCK in the neocortex

Core circadian-related proteins such as the transcription factor CLOCK are ubiquitously expressed and important for regulating molecular pathways underlying circadian rhythms. Previous work has suggested that CLOCK has evolved human neocortex-specific gene regulation and therefore may have extra-circadian functions. To test this in vivo, we generated a mouse model that recapitulates human cortical expression of CLOCK. The CLOCK humanized (HU) mice show enhanced cognitive flexibility, which is associated with the alteration in spatiotemporal expression of CLOCK. Cell type specific genomic profiling of HU mice identified upregulated genes related to dendritic growth and spine formation in excitatory neurons. Consistent with this result, we found that excitatory neurons in HU mice have increased complexity of dendritic branching and spine density, as well as a greater frequency of excitatory postsynaptic currents, suggesting an increase in neural connectivity. In contrast, CLOCK knockout in human induced pluripotent stem cell-induced neurons showed reduced complexity of dendrites and lower density of presynaptic puncta. Together, our data demonstrate that CLOCK evolved extra-circadian gains of function via altered spatiotemporal gene expression and these functions may underlie human brain specializations.

neuroscience↗

bioRxiv2022

A single-cell trajectory atlas of striatal development

The striatum integrates dense neuromodulatory inputs from many brain regions to coordinate complex behaviors. This integration relies on the coordinated responses from distinct striatal cell types. While previous studies have characterized the cellular and molecular composition of the striatum using single-cell RNA-sequencing at distinct developmental timepoints, the molecular changes spanning embryonic through postnatal development at the single-cell level have not been examined. Here, we combine published mouse striatal single-cell datasets from both embryonic and postnatal timepoints to analyze the developmental trajectory patterns and transcription factor regulatory networks within striatal cell types. Using this integrated dataset, we found that dopamine receptor-1 expressing spiny projection neurons have an extended period of postnatal development with greater transcriptional complexity compared to dopamine receptor-2 expressing neurons. Moreover, we found the transcription factor, FOXP1, exerts indirect changes to oligodendrocytes. These data can be accessed and further analyzed through an interactive website (https://cells-test.gi.ucsc.edu/?ds=mouse-striatal-dev).

neuroscience↗

bioRxiv2022

oskar acts with the transcription factor Creb to regulate long-term memory in crickets

Novel genes have the potential to drive the evolution of new biological mechanisms, or to integrate into pre-existing regulatory circuits and contribute to the regulation of older, conserved biological functions. One such gene, the novel insect-specific gene oskar, was first identified based on its role in establishing the Drosophila melanogaster germ line. We previously showed that this gene likely arose through an unusual domain transfer event involving bacterial endosymbionts, and played a somatic role before evolving its well-known germ line function. Here, we provide empirical support for this hypothesis in the form of evidence for a novel neural role for oskar. We show that oskar is expressed in the adult neural stem cells of a hemimetabolous insect, the cricket Gryllus bimaculatus. In these stem cells, called neuroblasts, oskar is required together with the ancient animal transcription factor Creb to regulate long-term (but not short-term) olfactory memory. We provide evidence that oskar positively regulates Creb, which plays a conserved role in long-term memory across animals, and that oskar in turn may be a direct target of Creb. Together with previous reports of a role for oskar in nervous system development and function in crickets and flies, our results are consistent with the hypothesis that oskars original somatic role may have been in the insect nervous system. Moreover, its co-localization and functional cooperation with the conserved pluripotency gene piwi in the nervous system may have facilitated oskars later co-option to the germ line in holometabolous insects.

animal behavior and cognition↗

bioRxiv2022

FOXP1 orchestrates neurogenesis in human cortical basal radial glial cells

During cortical development, human basal radial glial cells (bRGCs) are highly capable of sustained self-renewal and neurogenesis. Selective pressures on this cell type may have contributed to the evolution of the human neocortex, leading to an increase in cortical size. bRGCs have enriched expression for Forkhead Box P1 (FOXP1), a transcription factor implicated in neurodevelopmental disorders such as autism spectrum disorder. However, the cell type-specific roles of FOXP1 in bRGCs during cortical development remain unexplored. Here, we examine the requirement for FOXP1 gene expression regulation underlying the production of bRGCs using human brain organoids. We examine a developmental time point when FOXP1 expression is highest in the cortical progenitors, and the bRGCs, in particular, begin to actively produce neurons. With the loss of FOXP1, we show a reduction in the number of bRGCs, as well as reduced proliferation and differentiation of the remaining bRGCs, all of which lead to reduced numbers of excitatory cortical neurons over time. Using single-nuclei RNA sequencing and cell trajectory analysis, we uncover a role for FOXP1 in directing cortical progenitor proliferation and differentiation by regulating key signaling pathways related to neurogenesis and neurodevelopmental disorders. Together, these results demonstrate that FOXP1 regulates human-specific features in early cortical development.

neuroscience↗

bioRxiv2022

Locomotion control during curb descent: Bilateral ground reaction variables covary consistently during the double support phase regardless of future foot placement constraints

During community ambulation, anticipatory adaptations in gait are key for navigating built, populated and natural environments. It has been argued that some instability in gait can be functionally beneficial in situations demanding high maneuverability, and while the mechanisms utilized to maintain locomotor balance are well understood, relatively less is known about how the control of gait stability changes to facilitate upcoming maneuvers in challenging environments. The double support phase may be important in this regard; since both feet can push off the ground simultaneously, there is greater control authority over the bodys movement during this phase. Our goal was to identify how this control authority is exploited to prepare for upcoming maneuvers in challenging environments. We used synergy indices to quantify the degree of coordination between the ground reaction forces and moments under the two feet for stabilizing the resultant force and moment on the body during the double support phase of curb descent. In contrast to our expectations, we observed that the kinetic synergy indices during curb descent were minimally influenced by expected foot targeting maneuvers for the subsequent step. Only the resultant moment in the frontal plane showed reduced stability when targeting was required, but the synergy index was still high, indicating that the resultant moment was stable. Furthermore, the synergy indices indicated that the main function of the ground reaction variables is to maintain stability of whole-body rotations during double support, and this prerogative was minimally influenced by the subsequent foot targeting tasks, likely because the cost of losing balance while descending a curb would be higher than the cost of mis-stepping on a visual target. Our work demonstrates the salience of stabilizing body rotations during curb negotiation and improves our understanding of locomotor control in challenging environments.

bioengineering↗

bioRxiv2022

Step length and the extrapolated center of mass co-vary to stabilize the margin of stability in the anterior-posterior direction during unobstructed and obstructed walking

In human gait, the bodys mechanical energy at the end of one step is reused to achieve forward progression during the subsequent step, thereby reducing the required muscle work. During the single stance phase, humans rely on the largely uncontrolled passive inverted pendular motion of the body to perpetuate forward motion. These passive body dynamics, while improving walking efficiency, also indicate that lower passive dynamic stability in the anterior direction since the individual will be less able to withstand a forward external perturbation. Here we test the novel hypothesis that humans manipulate passive anterior-posterior (AP) stability via active selection of step length to either achieve energy-efficient gait or to improve stability when it is threatened. We computed the AP margin of stability, which quantifies the passive dynamic stability of gait, for multiple steps as healthy young adults (N=20) walked on a clear and on an obstructed walkway. Participants used passive dynamics to achieve energy-efficient gait for all but one step; when crossing the obstacle with the leading limb, AP margin of stability was increased. This increase indicated caution to offset the greater risk of falling after a potential trip. Furthermore, AP margin of stability increased while approaching the obstacle, indicating that humans proactively manipulate the passive dynamics to meet the demands of the locomotor task. Finally, the step length and the center of mass motion co-varied to maintain the AP margin of stability for all steps in both tasks at the specific values for each step. We conclude that humans actively regulate step length to maintain specific levels of passive dynamic stability for each step during unobstructed and obstructed gait.

bioengineering↗

bioRxiv2022

An electrophysiological and behavioral model of Paramecium, the "swimming neuron"

Paramecium is a large unicellular organism that swims in fresh water using cilia. When stimulated by various means (mechanically, chemically, optically, thermally), it often swims backward then turns and swims forward again in a new direction: this is called the avoiding reaction. This reaction is triggered by a calcium-based action potential. For this reason, several authors have called Paramecium the "swimming neuron". Here we present an empirically constrained model of its action potential based on electrophysiology experiments on live immobilized paramecia, together with simultaneous measurement of ciliary beating using particle image velocimetry. Using these measurements and additional behavioral measurements of free swimming, we extend the electrophysiological model by coupling calcium concentration to kinematic parameters, turning it into a swimming model. In this way, we obtain a model of autonomously behaving Paramecium. Finally, we demonstrate how the modeled organism interacts with an environment, can follow gradients and display collective behavior. This work provides a modeling basis for investigating the physiological basis of autonomous behavior of Paramecium in ecological environments. Author SummaryBehavior depends on a complex interaction between a variety of physiological processes, the body and the environment. We propose to examine this complex interaction in an organism consisting of a single excitable and motile cell, Paramecium. The behavior of Paramecium is based on trial and error: when it encounters an undesirable situation, it backs up and changes direction. This avoiding reaction is triggered by an action potential. Here we developed an empirically constrained biophysical model of Parameciums action potential, which we then coupled to its kinematics. We then demonstrate the potential of this model in investigating various types of autonomous behavior, such as obstacle avoidance, gradient-following and collective behavior.

neuroscience↗

bioRxiv2021

An instance-specific causal framework for learning intercellular communication networks that define microenvironments of individual tumors

Cells within a tumor microenvironment (TME) dynamically communicate and influence each others cellular states through an intercellular communication network (ICN). In cancers, intercellular communications underlie immune evasion mechanisms of individual tumors. We developed an individualized causal analysis framework for discovering tumor-specific ICNs. Using head and neck squamous cell carcinoma (HNSCC) tumors as a testbed, we first mined single-cell RNA-sequencing data to discover gene expression modules (GEMs) that reflect the states of transcriptomic processes within tumor and stromal single cells. By deconvoluting bulk transcriptomes of HNSCC tumors profiled by The Cancer Genome Atlas (TCGA), we estimated the activation states of these transcriptomic processes in individual tumors. Finally, we applied individualized causal network learning to discover an ICN within each tumor. Our results show that cellular states of cells in TMEs are coordinated through ICNs that enable multi-way communications among epithelial, fibroblast, endothelial, and immune cells. Further analyses of individual ICNs revealed structural patterns that were shared across subsets of tumors, leading to the discovery of 4 different subtypes of networks that underlie disparate TMEs of HNSCC. Patients with distinct TMEs exhibited significantly different clinical outcomes. Our results show that the capability of estimating individual ICNs reveals heterogeneity of ICNs and sheds light on the importance of intercellular communication in impacting disease development and progression.

cancer biology↗

bioRxiv2021

Prime-boost vaccination regimens with INO-4800 and INO-4802 augment and broaden immune responses against SARS-CoV-2 in nonhuman primates

The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens.

immunology↗