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Biology subjects

Sinha, V.

Publications and source records attributed to Sinha, V..

13 recordsLinked to original sources

Mice use predictive environmental cues to adapt predatory behavior in hunting tasks of variable difficulty

Prey capture has long served as a model of complex, multimodal behavior with ethological relevance. In this context, the ability of laboratory animals to capture fleeing prey has been investigated in terms of the sensory systems and neuroanatomical regions required for successful capture. While the ability to hunt is experience-dependent, prey capture is often assumed to be a reflexive behavior, governed by innate neural circuits and a basic drive to obtain food. Whether hunting performance depends on complex learning and cognitive strategy remains largely untested, in part because it is difficult to systematically manipulate the difficulty of predator-prey interactions. To address this, we varied ambient temperature across thousands of trials in which laboratory mice pursued cold-blooded cockroaches, whose movement speed was directly determined by body temperature. As expected, capture difficulty increased as a function of temperature. To test whether mice rely on external cues to adjust their predatory behavior, we then mismatched the ambient temperature and that of the prey, finding that mice learn to use relevant environmental information to inform their hunting strategy. Our results suggest that animals can extract and apply meaningful information from predictive environmental cues to adapt their learned behavior.

animal behavior and cognition↗

Hierarchical control of bacterial growth efficiency by substrate and taxonomy

Heterotrophic bacteria utilize organic carbon as a source of energy and material for growth. The balanced allocation of carbon between these two processes, termed growth efficiency, is a key physiological property of microbes because it links energy and biomass production, and determines the fraction of carbon lost as CO2. However, we do not understand what controls growth efficiency in microbes or how it correlates with taxonomy and resource identity. Here, we develop a quantitative high-throughput method for measuring CO2 production during bacterial growth. For 23 bacterial strains spanning three phyla, grown on two carbon substrates, we quantify growth efficiency by measuring dynamic CO2 production and carbon accumulation in biomass. Intra-phylum comparisons show that glycolytic substrates yield higher efficiency growth (less CO2 produced per biomass carbon) than gluconeogenic substrates. However, growth efficiency varies as much across resources as it does across phyla. A physiological model shows that variation in the growth efficiency depends on the ATP produced per respired CO2 and the ATP needed per biomass on a given substrate, suggesting phylum level differences in energy supply and demand dictate differences in growth efficiency. This theory predicts no global correlation between growth rate and growth efficiency, a finding our data support. Finally, we report phylum-level variation in the dynamics of CO2 production, which we link to the presence of overflow metabolism on glycolytic sub-strates. This study revises our understanding of how taxonomy and substrate identity impact carbon allocation during growth and sets the stage for understanding CO2 production in communities. SignificanceBacteria are responsible for roughly half of all biological CO2 production on the planet, much of it in soils. How organic carbon flows through microbial metabolism sets the rate at which soils return CO2 to the atmosphere. Bacterial growth efficiency, defined by the fraction of consumed carbon that is retained as biomass rather than released as CO2, determines the fluxes of carbon into the atmosphere. At present, we do not understand what controls growth efficiency in bacteria. We develop a new quantitative measurement of growth efficiency which we apply to 23 soil bacteria from three phyla on two substrates. We show that efficiency is structured hierarchically, at the highest level substrate identity defines efficiency which then varies substantially across taxa. So both the identity of species involved and the compounds they consume determine the rate of carbon loss.

microbiology↗

Environmental complexity reveals memory-guided search as the locus of learning in prey capture

Ethological behavior requires the integration of perception, memory, and decision-making, yet standard laboratory tasks are often too simplified to recruit these processes jointly. Here, mice that were expert hunters in a bare arena were challenged to capture live insect prey among objects that occlude vision and afford opportunities to hide. This revealed an axis of learning absent in an empty arena: mice improved not by refining pursuit but by reorganizing how they searched for concealed prey. An agent-based model showed that short-term memory and learning of object-specific values are sufficient to account for the behavior demonstrated by mice. A compact ethogram of object-directed actions revealed that animals selectively acquired the interactions most likely to expose prey. These results show that environmental complexity recruits memory- and value-guided search strategies. Reproducible with inexpensive materials, the paradigm and its open-source analyses offer a sensitive read-out of cognitive processes largely unobservable in conventional assays.

neuroscience↗

Investigation of Double-Stranded DNA Donors and CRISPR-Cas9 RNP for Universal Correction of Mutations Causing Cystic Fibrosis in Human Airway Cells

Cystic fibrosis (CF) is a devastating genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. As morbidity and mortality from CF results from a lack of mucus clearance that leads to chronic bacterial infections and progressive loss of lung function, site-specific insertion of a CFTR cDNA into the endogenous CFTR locus in airway basal stem cells (ABSCs) could prove curative for all disease-causing mutations. This study describes the development of nonviral genome editing reagents, designed to be packaged into nonviral delivery systems. An sgRNA targeting the 5 untranslated region of CFTR was characterized as directing high on-target cutting and displaying a safe off-target profile. Airway cell lines electroporated with chemically-modified (1-Aminohexane - AmC6), linear double-stranded DNA (ldsDNA) constructs were utilized as an Homology Directed Repair (HDR) donor, initially optimized with an mCitrine reporter. Expectedly, when the 780bp mCitrine cDNA was replaced with the 4.4kb CFTR cDNA, integration efficiency dropped significantly. However, 1-2% integration of codon optimized donors was sufficient to restore CFTR expression in the bulk edited population of human bronchial epithelial cell line, 16HBE14o- (16HBE), to levels reaching 50% of wildtype expression as measured by Western Blot. Electrophysiological validation of CFTR ion channel function measured via Ussing Chamber Assay revealed that these bulk edited populations exhibit greater than 40% restoration of the chloride ion currents of the measured wildtype controls. These results demonstrate that low levels of CFTR integration can be made therapeutically relevant by optimizing the designs of gene editing reagents. Importantly, this work utilizes nonviral editing reagents, an essential step towards in vivo gene therapy for CF.

genetics↗

Durable reconstitution of sinonasal epithelium by transplant of CFTR gene corrected airway stem cells

Modulator agents that restore cystic fibrosis transmembrane conductance regulator (CFTR) function have revolutionized outcomes in cystic fibrosis, an incurable multisystem disease. Barriers exist to modulator use, making local CFTR gene and cell therapies attractive, especially in the respiratory tract. We used CRISPR to gene-correct CFTR in upper airway basal stem cells (UABCs) and show durable local engraftment into recipient murine respiratory epithelium. Interestingly, the human cells recapitulate the in vivo organization and differentiation of human sinus epithelium, with little expansion or contraction of the engrafted population over time, while retaining expression of the CFTR transgene. Our results indicate that human airway stem cell transplantation with locoregional restoration of CFTR function is a feasible approach for treating CF and potentially other diseases of the respiratory tract.

bioengineering↗

Lipid Nanoparticles for the Delivery of CRISPR/Cas9 Machinery to Enable Site-Specific Integration of CFTR and Mutation-Agnostic Disease Rescue

We report the engineering of lipid nanoparticles (LNPs) to transport CRISPR/Cas9 payloads, including double-stranded DNA (dsDNA) donor templates, designed for homology directed repair (HDR)-mediated site-specific insertion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to correct cystic fibrosis (CF) in diseased airway epithelium. We screened various nanoparticle formulations, adjusting ratios of Cas9-encoding mRNA, single guide RNAs (sgRNAs), and dsDNA donor templates to optimize gene editing using human bronchial epithelial cells (16HBE14o-) harboring a CF-causing mutation (G542X). Populations of G542X cells edited via LNP delivery of CFTR donors achieved 3 - 3.5% gene integration and yielded comparable CFTR protein expression compared to normal 16HBE14o- controls. These edited populations exhibit restoration of CFTR-dependent Cl- current to ca. 80% of values measured in normal 16HBE14o- cell monolayers. This LNP platform adds capabilities for transporting large gene editing machinery to airway epithelial cells for genomic integration of entire genes, enabling therapeutic solutions that achieve correction of any CF-causing mutation.

bioengineering↗

Temperature-dependent predation predicts a more reptilian future

Vertebrate diversity increases toward the tropics, but the extent to which this pattern varies with thermoregulatory strategy is unknown. A strong divergence, if confirmed, would imply a global restructuring of vertebrate communities with temperature. Here we present evidence for a novel latitudinal and thermal gradient of comparative vertebrate diversity. Synthesizing over 34,000 terrestrial species distributions, we observe a two-orders-of-magnitude shift in comparative richness with temperature, from endothermic dominance in temperate habitats and high elevations, toward parity with ectotherms in the lowland tropics. Next, we provide experimental support for an underlying thermal gradient of predation. Using machine vision tracking in over 4,500 endotherm-ectotherm predation trials, we show that thermally-mediated differences in performance favor endotherm predators in colder conditions and yield theoretically predicted outcomes, including strike count, distance traveled, and time to capture ectotherm prey. Finally, we integrate theory and data to forecast future patterns of diversity, revealing that as the world gets warmer, it will become increasingly reptilian.

ecology↗

Information theory-based approach towards studying anti-coincidence detection via graded amplitude dendritic action potentials.

In contrast to typical all or none action potential, recent discovery of graded amplitude action potentials in cortical neurons enabled the dendrites to perform XOR computation, previously thought to be performed only at network level. Thus, these special neurons can perform anti-coincidence detection at the dendritic level, but a lot is unanswered about this phenomenon. Can such experimentally observed dendritic action potential generating system transmit information about stimuli having varying degrees of temporal overlap? Can the system add to the repertoire of computations performed at dendritic level by enhancing the information transmission about varying amplitude stimuli? In this information theory-based study done in single compartment and two-compartment dendritic models, it is shown that such a system can indeed transmit information about the temporal overlap of stimuli as well as amplitudes of stimuli even at high input noise levels. First, the calculation of mutual information between single stimulus and response i.e. I(S;R) with varying noise showed that the information about temporally overlapping nature of stimuli is precisely transmitted by such a system. Secondly, the time evolution of mutual information was simulated through data from the system and it positively reinforced the above-mentioned result. Next, varying amplitude input stimuli was provided to the system and calculation of mutual information between two stimuli and one response i.e. I(S1,S2;R) with varying noise levels revealed that such a system optimally transmits the information about stimuli even at high noise levels. Finally, calculation of this information measurement with respect to time in an experiment with constant overlap but varying input amplitude again positively reinforced the result. Key PointsO_LIInformation theory-based measurements were employed to assess the role of graded amplitude dendritic action potentials. C_LIO_LIAction potentials (APs) with maximal amplitudes for threshold level stimuli and lower amplitudes for stronger stimuli were modelled with high voltage Ca2+ (HVA like) channels, Ca2+ dependent (BK-like) channel, leak channel and calcium pump in a single compartment model and two compartment dendritic model. C_LIO_LIAnalysis done here, on comparison with control compartment generating constant amplitude AP via standard Hodgkin-Huxley sodium potassium channel revealed that such a compartment shows optimal information transmission about both varying amplitudes input current stimuli as well as varying time overlap stimuli. C_LI

neuroscience↗

Genomic patterns of transcription-replication interactions in mouse primary B cells

Conflicts between transcription and replication machinery are a potent source of replication stress and genome stability; however, no technique currently exists to identify endogenous genomic locations prone to transcription-replication interactions. Here, we report a novel method to identify genomic loci prone to transcription-replication interactions termed transcription-replication immunoprecipitation on nascent DNA sequencing, TRIPn-Seq. TRIPn-Seq employs the sequential immunoprecipitation of RNA polymerase 2 phosphorylated at serine 5 (RNAP2s5) followed by enrichment of nascent DNA previously labeled with bromodeoxyuridine. Using TRIPn-Seq, we mapped 1,009 unique transcription-replication interactions (TRIs) in mouse primary B cells characterized by a bimodal pattern of RNAP2s5, bidirectional transcription, an enrichment of RNA:DNA hybrids, and a high probability of forming G-quadruplexes. While TRIs themselves map to early replicating regions, they exhibit enhanced Replication Protein A association and replication fork termination, marks of replication stress. TRIs colocalize with double-strand DNA breaks, are enriched for deletions, and accumulate mutations in tumors. We propose that replication stress at TRIs induces mutations potentially contributing to age-related disease, as well as tumor formation and development.

genomics↗

Metabolite plasticity drives carbon-nitrogen resource budgeting to enable division of labor in a clonal community

Previously, we discovered that in glucose-limited yeast colonies, metabolic constraints drive cells into groups exhibiting gluconeogenic and glycolytic metabolic states. Here, threshold amounts of trehalose - a limiting, produced resource, controls the emergence and self-organization of the cells exhibiting the glycolytic state, by acting as a carbon source to fuel these metabolic demands (Varahan et al., 2019). We now discover that the plasticity of use of a non-limiting resource, aspartate, controls both resource production and the emergence of heterogeneous cell states, based on differential cellular metabolic budgeting. In gluconeogenic cells, aspartate provides carbon for trehalose production, while in glycolytic cells using trehalose for carbon, aspartate supplies nitrogen to drive nucleotide synthesis. This metabolic plasticity of aspartate enables carbon-nitrogen budgeting, thereby driving the biochemical self-organization of distinct cell states. Through this organization, cells in each state exhibit true division of labor, providing bet-hedging and growth/survival advantages for the whole community.

cell biology↗

Metabolic constraints drive self-organization of specialized cell groups

How phenotypically distinct states in isogenic cell populations appear and stably co-exist remains an unresolved question. We find that within a clonal yeast colony developing in low glucose, cells arrange into metabolically disparate cell groups. Using this system, we model and experimentally identify metabolic constraints sufficient to drive such assembly. Beginning in a gluconeogenic state, cells in a contrary state, exhibiting high pentose phosphate pathway activity, spontaneously appear and proliferate, in a spatially constrained manner. The gluconeogenic cells in the developing colony produce a resource, which we identify as trehalose. At threshold concentrations of trehalose, cells in the new metabolic state emerge and proliferate. A self-organized system establishes, where cells in this new state are sustained by trehalose consumption, which thereby restrains other cells in the trehalose producing, gluconeogenic state. Our work suggests simple physico-chemical principles that determine how isogenic cells spontaneously self-organize into structured assemblies in complimentary, specialized states.

cell biology↗

Variants in regulatory elements of PDE4D associate with Major Mental Illness in the Finnish population

We have previously reported a replicable association between variants at the PDE4D gene and familial schizophrenia in a Finnish cohort. In order to identify the potential functional mutations alluded to by these previous findings, we sequenced the 1.5Mb of the PDE4D genomic locus in 20 families (consisting of 96 individuals, and 79 independent chromosomes), followed by two stages of genotyping across 6,668 individuals from multiple Finnish cohorts for major mental illnesses. We identified 4,570 SNPs across the PDE4D gene, with 380 associated to schizophrenia (p[≤]0.05). Importantly, two of these variants, rs35278 and rs165940, are located at transcription factor binding sites, and displayed replicable association in the two-stage enlargement of the familial schizophrenia cohort, (combined statistics for rs35278 p=0.0012; OR=1.18, 95% CI 1.06-1.32; and rs165940 p=0.0016; OR=1.27, 95% CI 1.13-1.41). Further analysis using additional cohorts and endophenotypes revealed that rs165940 principally associates within the psychosis (p=0.025, OR=1.18, 95% CI 1.07-1.30) and cognitive domains of major mental illnesses (g-score p=0.044, beta=-0.033). Specifically, the cognitive domains represented verbal learning and memory (p=0.0091, beta=-0.044) and verbal working memory (p=0.0062, beta=-0.036). Moreover, expression data from the GTEx database demonstrated that rs165940 significantly correlates with the mRNA expression levels of PDE4D in the cerebellum (p-value=0.04; m-value=0.9), demonstrating a potential functional consequence for this variant. Thus, rs165940 represents the most likely functional variant for major mental illness at the PDE4D locus in the Finnish population, increasing risk broadly to psychotic disorders.

genetics↗

The NDE1 Genomic Locus Affects Treatment of Psychiatric Illness through Gene Expression Changes Related to MicroRNA-484

Genetic studies of familial schizophrenia in Finland have observed significant associations with a group of biologically related genes, DISC1, NDE1, NDEL1, PDE4B and PDE4D, the \"DISC1 network\". Here, we utilize gene expression and psychoactive medication use data to study their biological consequences and potential treatment implications. Gene expression levels were determined in 64 individuals from 18 families, whilst prescription medication information has been collected over a ten-year period for 931 affected individuals. We demonstrate that the NDE1 SNP rs2242549 associates with significant changes in gene expression for 2,908 probes (2,542 genes), of which 794 probes (719 genes) were replicable. A significant number of the genes altered were predicted targets of microRNA-484 (p=3.0x10-8), located on a non-coding exon of NDE1. Variants within the NDE1 locus also displayed significant genotype by gender interaction to early cessation of psychoactive medications metabolized by CYP2C19. Furthermore, we demonstrate that miR-484 can affect the expression of CYP2C19 in a cell culture system. Thus, variation at the NDE1 locus may alter risk of mental illness, in part through modification of miR-484, and such modification alters treatment response to specific psychoactive medications, leading to the potential for use of this locus in targeting treatment.

genetics↗