Biology subjects

Iyengar, R.

Publications and source records attributed to Iyengar, R..

11 recordsLinked to original sources

bioRxiv2018

Geometric principles of second messenger dynamics in dendritic spines.

Dendritic spines are small, bulbous protrusions along dendrites in neurons and play a critical role in synaptic transmission. Dendritic spines come in a variety of shapes that depend on their developmental state. Additionally, roughly 14-19% of mature spines have a specialized endoplasmic reticulum called the spine apparatus. How does the shape of a postsynaptic spine and its internal organization affect the spatio-temporal dynamics of short timescale signaling? Answers to this question are central to our understanding the initiation of synaptic transmission, learning, and memory formation. In this work, we investigated the effect of spine and spine apparatus size and shape on the spatio-temporal dynamics of second messengers using mathematical modeling using reaction-diffusion equations in idealized geometries (ellipsoids, spheres, and mushroom-shaped). Our analyses and simulations showed that in the short timescale, spine size and shape coupled with the spine apparatus geometries govern the spatiotemporal dynamics of second messengers. We show that the curvature of the geometries gives rise to pseudo-harmonic functions, which predict the locations of maximum and minimum concentrations along the spine head. Furthermore, we showed that the lifetime of the concentration gradient can be fine-tuned by localization of fluxes on the spine head and varying the relative curvatures and distances between the spine apparatus and the spine head. Thus, we have identified several key geometric determinants of how the spine head and spine apparatus may regulate the short timescale chemical dynamics of small molecules that control synaptic plasticity.

biophysics↗

bioRxiv2018

Systems therapeutics analyses identify genomic signatures defining responsiveness to allopurinol and combination therapy for lung cancer

The ability to predict responsiveness to drugs in individual patients is limited. We hypothesized that integrating molecular information from databases would yield predictions that could be experimentally tested to develop genomic signatures for sensitivity or resistance to specific drugs. We analyzed TCGA data for lung adenocarcinoma (LUAD) patients and identified a subset where xanthine dehydrogenase expression correlated with decreased survival. We tested allopurinol, a FDA approved drug that inhibits xanthine dehydrogenase on a library of human Non Small Cell Lung Cancer (NSCLC) cell lines from CCLE and identified sensitive and resistant cell lines. We utilized the gene expression profiles of these cell lines to identify six-gene signatures for allopurinol sensitive and resistant cell lines. Network building and analyses identified JAK2 as an additional target in allopurinol-resistant lines. Treatment of resistant cell lines with allopurinol and CEP-33779 (a JAK2 inhibitor) resulted in cell death. The effectiveness of allopurinol alone or allopurinol and CEP-33779 were verified in vivo using tumor formation in NCR-nude mice. We utilized the six-gene signatures to predict five additional allopurinolsensitive NSCLC lines, and four allopurinol-resistant lines susceptible to combination therapy. We found that drug treatment of all cell lines yielded responses as predicted by the genomic signatures. We searched the library of patient derived NSCLC tumors from Jackson Laboratory to identify tumors that would be predicted to be sensitive or resistant to allopurinol treatment. Both patient derived tumors predicted to be allopurinol sensitive showed the predicted sensitivity, and the predicted resistant tumors were sensitive to combination therapy. These data indicate that we can use integrated molecular information from cancer databases to predict drug responsiveness in individual patients and thus enable precision medicine.

systems biology↗

bioRxiv2018

Activated Protein C blocks the inhibitory effect on neurite outgrowth by extracellular histones that mediates its inhibition through a retrograde YB-1 signal

Axonal regeneration in the mature CNS is limited by extracellular inhibitory factors. Triple knockout mice lacking the major myelin-associated inhibitors do not display spontaneous regeneration after injury, indicating the presence of other inhibitors. Searching for such inhibitors we have detected elevated levels of histone H3 in human cerebrospinal fluid (CSF) 24 hours after spinal cord injury. Following dorsal column lesions in mice and optic nerve crushes in rats, elevated levels of extracellular histone H3 were detected at the injury site. Similar to myelin-associated inhibitors, these extracellular histones induced growth cone collapse and inhibited neurite outgrowth. Histones mediate inhibition through the transcription factor YB-1 and Toll-like receptor 2, and these effects are independent of the Nogo receptor. Histone-mediated inhibition can be reversed by the addition of activated protein C (APC) in vitro, and APC treatment promotes axonal regeneration in the crushed optic nerve in vivo. These findings identify extracellular histones as a new class of nerve regeneration-inhibiting molecules within the injured CNS. One sentence summaryProteins typically associated with chromatin structure play an unexpected role in limiting axonal regeneration after injury.

neuroscience↗

bioRxiv2017

Validating Antibodies for Quantitative Western Blot Measurements with Microwestern Array

Western blotting is often considered a semi-quantitative or even qualitative assay for assessing changes in protein or protein post-translational modification levels. Fluorescence-based measurement enables acquisition of quantitative data in principal, but requires determining the linear range of detection for each antibody--a labor-intensive task. Here, we describe the use of a high-throughput western blotting technique called microwestern array to more rapidly evaluate suitable conditions for quantitative western blotting with particular antibodies. We can evaluate up to 192 antibody/dilution/replicate combinations on a single standard size gel with a seven-point, two-fold lysate dilution series (~100-fold range). Pilot experiments demonstrate a surprisingly high proportion of investigated antibodies (17/22) are suitable for quantitative use, and that lack of validity might often be a consequence of lysate composition rather than antibody quality. Linear range for all validated antibodies is at least 8-fold, and in some cases nearly two orders of magnitude. That range could be greater as the presented tests did not find a limit for many antibodies. We find that phospho-specific and total antibodies do not have discernable trend differences in linear range or limit of detection, but total antibodies generally required higher working concentrations, suggesting phospho-specific antibodies may be generally higher affinity. Importantly, we demonstrate that results from microwestern analyses scale to normal \"macro\" western for a subset of antibodies. These data indicate that with initial validation, many antibodies can be readily used quantitatively in a reproducible manner. Antibody validation data and standard operating procedures are available online (www.birtwistlelab.com/protocols and www.dtoxs.org).

biochemistry↗

bioRxiv2017

Curvature regulates subcellular organelle location to control intracellular signal propagation

The shape of the cell is connected to its function; however, we do not fully understand underlying mechanisms by which global shape regulates a cells functional capabilities. Using theory, experiments and simulation, we investigated how physiologically relevant cell shape changes affect subcellular organization, and consequently intracellular signaling, to control information flow needed for phenotypic function. Vascular smooth muscle cells going from a proliferative and motile circular shape to a contractile fusiform shape show changes in the location of the sarcoplasmic reticulum, inter-organelle distances and differential distribution of receptors in the plasma membrane. These factors together lead to the modulation of signals transduced by the M3 muscarinic receptor/Gq/PLC{beta} pathway at the plasma membrane, amplifying Ca2+ dynamics in the cytoplasm and the nucleus resulting in phenotypic changes, as determined by increased activity of myosin light chain kinase in the cytoplasm and enhanced nuclear localization of the transcription factor NFAT. Taken together, our observations show a systems level phenomenon whereby global cell shape affects subcellular organization to modulate signaling that enables phenotypic changes.

cell biology↗

bioRxiv2017

Systems Therapeutics for Axonal Regeneration in the Central Nervous System

There are no known drugs or drug combinations that promote substantial central nervous system axonal regeneration after injury. We used systems pharmacology approaches to model pathways underlying axonal growth and identify a four-drug combination that regulates multiple subcellular processes in the cell body and axon using the optic nerve crush model in rats. We intravitreally injected agonists HU-210 (cannabinoid receptor-1) and IL-6 (interleukin 6 receptor) to stimulate retinal ganglion cells for axonal growth. We applied, in gel foam at the site of nerve injury, Taxol to stabilize growing microtubules, and activated protein C to clear the debris field since computational models predicted that this drug combination regulating two subcellular processes at the growth cone produces synergistic growth. Morphology experiments show that the four-drug combination promotes axonal regrowth to the optic chiasm and beyond. Physiologically, drug treatment restored pattern electroretinograms and some of the animals had detectable visual evoked potentials in the brain and behavioral optokinetic responses. We conclude that spatially targeted drug treatment can promote robust axonal regeneration and can restore limited functional recovery.

neuroscience↗

bioRxiv2017

Dynamic balance between vesicle transport and microtubule growth enables neurite growth

Whole cell responses involve multiple subcellular processes (SCPs). To understand how balance between SCPs controls the dynamics of whole cell responses we studied neurite outgrowth in rat primary cortical neurons in culture. We used a combination of dynamical models and experiments to understand the conditions that permitted growth at a specified velocity and when aberrant growth could lead to the formation of dystrophic bulbs. We hypothesized that dystrophic bulb formation is due to quantitative imbalances between SCPs. Simulations predict redundancies between lower level sibling SCPs within each type of high level SCP. In contrast, higher level SCPs, such as vesicle transport and exocytosis or microtubule growth characteristic of each type need to be strictly coordinated with each other and imbalances result in stalling of neurite outgrowth. From these simulations, we predicted the effect of changing the activities of SCPs involved in vesicle exocytosis or microtubule growth could lead to formation of dystrophic bulbs. siRNA ablation experiments verified these predictions. We conclude that whole cell dynamics requires balance between the higher-level SCPs involved and imbalances can terminate whole cell responses such as neurite outgrowth.

systems biology↗

bioRxiv2017

An extensible ontology for inference of emergent whole cell function from relationships between subcellular processes

Whole cell responses arise from coordinated interactions between diverse human gene products functioning within various pathways underlying sub-cellular processes (SCP). Lower level SCPs interact to form higher level SCPs, often in a context specific manner to give rise to whole cell function. We sought to determine if capturing such relationships enables us to describe the emergence of whole cell functions from interacting SCPs. We developed the \"Molecular Biology of the Cell\" ontology based on standard cell biology and biochemistry textbooks and review articles. Currently, our ontology contains 5,392 genes, 753 SCPs and 19,182 expertly curated gene-SCP associations. Our algorithm to populate the SCPs with genes enables extension of the ontology on demand and the adaption of the ontology to the continuously growing cell biological knowledge. Since whole cell responses most often arise from the coordinated activity of multiple SCPs, we developed a dynamic enrichment algorithm that flexibly predicts SCP-SCP relationships beyond the current taxonomy. This algorithm enables us to identify interactions between SCPs as a basis for higher order function in a context dependent manner, allowing us to provide a detailed description of how SCPs together can give rise to whole cell functions. We conclude that this ontology can, from omics data sets, enable the development of detailed multidimensional SCP networks for predictive modeling of emergent whole cell functions.

systems biology↗

bioRxiv2017

Fragile dynamics enable diverse genomic determinants to influence arrhythmia propensity

Genotype-phenotype relationships are determinants of human diseases. Often, we know little about why so many genes are involved in complex common diseases. We hypothesized that this multigene effect arises from the relationship between genes and physiological dynamics. We tested this hypothesis for arrhythmias as physiological dynamics define this disease. We integrated graph theory analysis of genomic and protein-protein interaction networks with dynamical models of ion channel function to identify the physiological dynamics of genome wide variation for five different arrhythmias. Regulatory networks for the cardiac conduction system and arrhythmias were constructed from GWAS and known disease genes. Electrophysiological models of myocyte action potentials were used to conduct extensive parameter variations to identify robust and fragile kinetic parameters that were then, using regulatory networks, associated with genomic determinants. We find that genome-wide determinants of arrhythmias that represent many cellular processes are selectively associated with fragile physiological dynamics of ion channel kinetics. This association predicts disease propensity. Deep RNA sequencing from human left ventricular tissue of arrhythmia and control subjects confirmed the predictive relationship. Taken together these studies show that the varied multigene effects of arrhythmias arises because of associations with fragile kinetic parameters of cardiac electrophysiology.\n\nSignificance StatementOur understanding of the genetics of common diseases has advanced exponentially over the past decade. We now know that differences and variation in multiple genes contribute to disease susceptibility with significant heterogeneity in the phenotype. However, how genetic variation contributes to disease phenotypes remains unknown. We hypothesized that the relationships between physiological dynamics and genetic architecture is a fundamental determinant of disease susceptibility and genetic heterogeneity. To test our hypothesis, we integrated mathematical models of cardiac electrophysiology with genetic network models of cardiac arrhythmias. We found that disease related genome variants were selectively associated with fragile kinetic parameters that predict disease propensity and identified several novel cellular processes associated with arrhythmogenesis.

systems biology↗

bioRxiv2017

Experience-induced forgetting by WT1 enables learning of sequential tasks.

Under physiological conditions, strength and persistence of memory must be regulated in order to produce behavioral flexibility. In fact, impairments in memory flexibility are associated with pathologies such as post-traumatic stress disorder or autism; however the underlying mechanisms that enable memory flexibility are still poorly understood. Here we identified the transcriptional repressor Wilms Tumor 1 (WT1) as a critical synaptic plasticity regulator that decreases memory strength, promoting memory flexibility. WT1 was activated in the hippocampus following induction of long-term potentiation (LTP) or learning. WT1 knockdown enhanced CA1 neuronal excitability, LTP and long-term memory whereas its over-expression weakened memory retention. Moreover, forebrain WT1-deficient mice showed deficits in both reversal, sequential learning tasks and contextual fear extinction, exhibiting impaired memory flexibility. We conclude that WT1 limits memory strength or promotes memory weakening, thus enabling memory flexibility, a process that is critical for learning from new experience.

neuroscience↗

bioRxiv2017

A Comparison of mRNA Sequencing with Random Primed and 3’-Directed Libraries

Deep mRNA sequencing (mRNAseq) is the state-of-the-art for whole transcriptome measurements. A key step is creating a library of cDNA sequencing fragments from RNA. This is generally done by random priming, creating multiple sequencing fragments along the length of each transcript. A 3 end-focused library approach cannot detect differential splicing, but has potentially higher throughput at lower cost (~10-fold lower), along with the ability to improve quantification by using transcript molecule counting with unique molecular identifiers (UMI) to correct for PCR bias. Here, we compare implementation of such a 3-digital gene expression (3-DGE) approach with \"conventional\" random primed mRNAseq, which has not yet been done. We find that while conventional mRNAseq detects ~15% more genes, the resulting lists of differentially expressed genes and therefore biological conclusions and gene signatures are highly concordant between the two techniques. We also find good quantitative agreement on the level of individual genes between the two techniques in terms of both read counts and fold change between two conditions. We conclude that for high-throughput applications, the potential cost savings associated with the 3-DGE approach are a very reasonable tradeoff for modest reduction in sensitivity and inability to observe alternative splicing, and should enable much larger scale studies focused on not only differential expression analysis, but also quantitative transcriptome profiling. The computational scripts and programs, along with experimental standard operating procedures used in our pipeline presented here, are freely available on our website (www.dtoxs.org).

genomics↗