Biology subjects

Thomas, C.

Publications and source records attributed to Thomas, C..

68 records · Page 4Linked to original sources

bioRxiv2020

An optimal set of inhibitors for Reverse Engineering via Kinase Regularization.

We present a comprehensive resource of 257 kinase inhibitor profiles against 365 human protein kinases using gold-standard kinase activity assays. We show the utility of this dataset with an improved version of Kinome Regularization (KiR) to deconvolve protein kinases involved in a cellular phenotype. We assayed protein kinase inhibitors against more than 70% of the human protein kinome and chose an optimal subset of 58 inhibitors to assay at ten doses across four orders of magnitude. We demonstrate the effectiveness of KiR to identify key kinases by using a quantitative cell migration assay and updated machine learning methods. This approach can be widely applied to biological problems for which a quantitative phenotype can be measured and which can be perturbed with our set of kinase inhibitors.

systems biology↗

bioRxiv2020

O-GlcNAc regulates gene expression by controlling detained intron splicing

Intron detention in precursor RNAs serves to regulate expression of a substantial fraction of genes in eukaryotic genomes. How detained intron (DI) splicing is controlled is poorly understood. Here we show that a ubiquitous post-translational modification called O-GlcNAc, which is thought to integrate signaling pathways as nutrient conditions fluctuate, controls detained intron splicing. Using specific inhibitors of the enzyme that installs O-GlcNAc (O-GlcNAc transferase, or OGT) and the enzyme that removes O-GlcNAc (O-GlcNAcase, or OGA), we first show that O-GlcNAc regulates splicing of the highly conserved detained introns in OGT and OGA to control mRNA abundance in order to buffer O-GlcNAc changes. We show that OGT and OGA represent two distinct paradigms for how DI splicing can control gene expression. We also show that when DI splicing of the O-GlcNAc-cycling genes fails to restore O-GlcNAc homeostasis, there is a global change in detained intron levels. Strikingly, almost all detained introns are spliced more efficiently when O-GlcNAc levels are low, yet other alternative splicing pathways change minimally. Our results demonstrate that O-GlcNAc controls detained intron splicing to tune system-wide gene expression, providing a means to couple nutrient conditions to the cells transcriptional regime.

cell biology↗

bioRxiv2020

Pharmacologic activation of the mitochondrial phosphoenolpyruvate cycleenhances islet function in vivo

The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle is an anaplerotic-cataplerotic mitochondrial shuttle utilizing mitochondrial PEPCK (PCK2) and pyruvate kinase (PK). PEP cycling stimulates insulin secretion via OxPhos-independent lowering of ADP by PK. We assess in vivo whether islet PCK2 is necessary for glucose sensing and if speeding the PEP cycle via pharmacological PK activators amplifies insulin secretion. Pck2-/- mice had severely impaired insulin secretion during islet perifusion, oral glucose tolerance tests and hyperglycemic clamps. Acute and chronic pharmacologic PK activator therapy improved islet insulin secretion from normal, high-fat diet (HFD) fed, or Zucker diabetic fatty (ZDF) rats, and glucolipotoxic or diabetic humans. A similar improvement in insulin secretion was observed in regular chow and HFD rats in vivo. Insulin secretion and cytosolic Ca2+ during PK activation were dependent on PCK2. These data provide a preclinical rationale for strategies, such as PK activation, that target the PEP cycle to improve glucose homeostasis. HighlightsO_LILoss of mitochondrial phosphoenolpyruvate (PEP) impairs insulin release in vivo. C_LIO_LIPyruvate kinase (PK) activators stimulate beta-cells in preclinical diabetes models. C_LIO_LIPEP cycling in vivo depends on PK and mitochondrial PEPCK (PCK2) for insulin release. C_LIO_LIAcute and 3-week oral PK activator amplifies insulin release during hyperglycemia. C_LI eTOC BlurbAbudukadier et al. show that small molecule pyruvate kinase activation in vivo and in vitro increases insulin secretion in rodent and human models of diabetes. The phosphoenolpyruvate (PEP) cycling mechanism and its amplification are dependent on mitochondrial PEPCK (PCK2).

physiology↗

bioRxiv2019

Cortical neuron response selectivity derives from strength in numbers of synapses

Single neocortical neurons are driven by populations of excitatory inputs, forming the basis of neural selectivity to features of sensory input. Excitatory connections are thought to mature during development through activity-dependent Hebbian plasticity1, whereby similarity between presynaptic and postsynaptic activity selectively strengthens some synapses and weakens others2. Evidence in support of this process ranges from measurements of synaptic ultrastructure to slice and in vivo physiology and imaging studies3,4,5,6,7,8. These corroborating lines of evidence lead to the prediction that a small number of strong synaptic inputs drive neural selectivity, while weak synaptic inputs are less correlated with the functional properties of somatic output and act to modulate activity overall6,7. Supporting evidence from cortical circuits, however, has been limited to measurements of neighboring, connected cell pairs, raising the question of whether this prediction holds for the full profile of synapses converging onto cortical neurons. Here we measure the strengths of functionally characterized excitatory inputs contacting single pyramidal neurons in ferret primary visual cortex (V1) by combining in vivo two-photon synaptic imaging and post hoc electron microscopy (EM). Using EM reconstruction of individual synapses as a metric of strength, we find no evidence that strong synapses play a predominant role in the selectivity of cortical neuron responses to visual stimuli. Instead, selectivity appears to arise from the total number of synapses activated by different stimuli. Moreover, spatial clustering of co-active inputs, thought to amplify synaptic drive, appears reserved for weaker synapses, further enhancing the contribution of the large number of weak synapses to somatic response. Our results challenge the role of Hebbian mechanisms in shaping neuronal selectivity in cortical circuits, and suggest that selectivity reflects the co-activation of large populations of presynaptic neurons with similar properties and a mixture of strengths.

neuroscience↗

bioRxiv2019

Spatial distribution and research trend of illegal activities and the factors associated with wild mammal population declines in protected areas

Illegal activities are a persistent problem in many protected areas, but an overview of the extent of this problem and its impact is lacking. We review 35 years (1980-1914) of research across the globe to examine the spatial distribution of research and socio-ecological factors influencing population decline within protected areas under illegal activities pressure. From 92 papers reporting 1048 species/site combinations, more than 350 species comprising mammals, reptiles, birds, fishes and molluscs were reported to have been extracted illegally from 146 protected areas across four continents. Research in illegal activities has increased substantially during the review period but also shows strong taxonomic and geographic biases towards large wild mammals and African continent respectively, suggesting persistent poaching pressures on wild mammals in African protected areas. Population declines were most frequent i) where there was commercial poaching as opposed to subsistence poaching alone, ii) in countries with a low human development index particularly in strict protected areas and iii) for species with a body mass over 100 kg. Habitat loss associated with greater land use change had an additional significant impact on population decline, particularly in the less-strict categories (IUCN III-VI) of protected area across the continents. Overall, these findings provide evidence that illegal activities are most likely to cause species declines of large-bodied animals in protected areas in resource-poor countries regardless of protected area conservation status (i.e. IUCN category). Given the mounting pressures of illegal activities, additional conservation effort such as improving anti-poaching strategies and conservation resources in terms of improving funding and personnel directed at this problem is a growing priority.

zoology↗

bioRxiv2019

A Homogeneous SIRPα-CD47 Cell-Based, Ligand-Binding Assay: Utility for Small Molecule Drug Development in Immuno-oncology.

CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRP. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRP receptors, that block the SIRP-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRP and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRP variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. The LSC assay also confirmed published studies, such as the inhibition of amino-terminal pyroglutamate formation in CD47 disrupts SIRP binding. SIRP-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRP-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign.

immunology↗

bioRxiv2019

Synchronous chromosome segregation in mouse oocytes is ensured by biphasic securin destruction and cyclin B1-Cdk1

Successful cell division relies on the faithful segregation of chromosomes. If chromosomes segregate prematurely the cell is at risk of aneuploidy. Alternatively, if cell division is attempted in the absence of complete chromosome segregation, non-segregated chromosomes can become trapped within the cleavage furrow and the cell can lose viability. Securin plays a key role in this process, acting as a pseudosubstrate to inhibit the protease separase that functions to cleave the cohesin rings that hold chromosomes together. Consequently, securin must be depleted ahead of anaphase, ensuring chromosome segregation occurs in time with the anaphase trigger. Here we find that MI mouse oocytes contain a large excess of securin over separase and reveal the existence of a novel mechanism that functions to promote the destruction of excess securin in prometaphase. Critically, this mechanism relies on key residues that are only exposed when securin is not bound to separase. We suggest that the majority of non-separase bound securin is removed by this mechanism, allowing for separase activity to be protected until just before anaphase. In addition, we further demonstrate the importance of complementary mechanisms of separase inhibition by directly measuring cleavage activity in live oocytes, confirming that both securin and inhibition by cyclin B1-Cdk1 are independently sufficient to prevent premature separase activation.

cell biology↗

bioRxiv2019

The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability

Microtubules are bistable cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage 1. Growing microtubules are thought to be stabilized by a GTP cap at their ends 2-5. The nature of this cap, however, is still poorly understood. How GTP hydrolysis determines the properties of the GTP cap and hence microtubule stability is unclear. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends 6. Whether these regulatory platforms at growing microtubule ends are identical to the GTP cap is not known. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate in microscopy-based in vitro reconstitutions that EB proteins bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps and consequently hyper-stable microtubules. Single molecule experiments reveal that the microtubule conformation gradually changes in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability; and establish that the GTP cap coincides with the EB-binding regulatory hub that modulates microtubule cytoskeleton function in cells.

biochemistry↗

bioRxiv2019

Kin recognition and genetic variation for competitive ability in the annual legume Medicago minima

Knowing which mechanisms drive the outcome of intraspecific interactions is highly relevant for understanding diversity maintenance. Plant species that exhibit strong genetic substructure over small spatial scales may be exposed to frequent interactions with closely related individuals. Predictions of how genetic similarity may drive the outcome of intraspecific interactions are based on two contrasting theories: the resource partitioning hypothesis and kin selection theory. The first predicts that competition will be stronger among closely related conspecific (i.e. kin) because similar genotypes have similar resource requirements. The second predicts instead that competition will be reduced among kin, in order to maximize the inclusive fitness. Although efforts have been made to reconcile these two theories as non-mutually exclusive, the outcomes of intraspecific interaction studies are frequently interpreted as the results of either one or the other. We experimentally tested the hypothesis that intraspecific interactions may be driven by both genetic variation for competitive ability and reduced competition among kin. We used an annual legume, Medicago minima, to conduct two greenhouse experiments testing changes in root behaviour, above-ground growth and biomass in response to neighbour identity. We found evidence of both genetic variation for competitive ability and reduced competition among kin in some genotypes. Reduced competitive growth towards kin was found in the most competitive genotypes, suggesting that kin avoidance and competitive ability were simultaneously affecting plant behaviour and growth. With presence of both kin competition avoidance and variation for competitive ability, the outcome of intraspecific interactions will strongly depend on the local spatial genetic substructure. This is highly relevant to predict how intraspecific competition affect diversity maintenance.

ecology↗

bioRxiv2018

Early detection of impending cryostorage tank failure using a weight-based monitoring system

ObjectTo study the ability of custom-built, web-enabled scales to monitor liquid nitrogen (LN2) levels in cryostorage dewars.\n\nDesignLaboratory study\n\nSettingA large academic fertility center in New York City.\n\nInterventionsCryostorage dewars were placed on top of the custom-engineered scales with continuous real-time monitoring, and weight and temperature data were recorded in the setting of slow, medium, and fast rate-loss of LN2 designed to mimic models of tank failures.\n\nMain Outcome MeasuresWeights were continuously monitored and recorded, with a calculated alarm trigger set at 10% weight loss. Temperature within the tanks was simultaneously monitored with probes placed near the top of the tanks, with calculated alarms using a -185 {degrees}C as the threshold. For the \"slow rate-loss\" simulations, tanks were left intact and closed in usual operating conditions, and LN2 was allowed to evaporate at the normal rate. For the \"medium rate-loss\" simulation, the foam core of the tank neck was removed and the insulating vacuum was eliminated by making a 1/16 inch hole in the outer tank wall. For the \"fast rate-loss\" simulation, a 1/16\" hole was made through the outer tank wall and LN2 was released at a rate of 0.15 L/second. All simulations were performed in duplicate.\n\nResultsWith an intact and normally functioning tank, a 10% loss in LN2 occurred in 4.2-4.9 days. Warming to -185 {degrees}C occurred in 37.8 - 43.7 days, over 30 days after the weight-based alarm was triggered. Full evaporation of LN2required 36.8 days. For the medium rate-loss simulation, a 10% loss in LN2 occurred in 0.8 h. Warming to -185 {degrees}C occurred in 3.7 - 4.8 hours, approximately 3 hours after the weight-based alarm was triggered. For the fast rate-loss simulation, a 10% weight loss occurred within 15 seconds and tanks were completely depleted in under 3 minutes. Tank temperatures began to rise immediately and at a relatively constant rate of 43.9 {degrees}C/hour and 51.6 {degrees}C/hour. Temperature alarms would have sounded within 0.37 and 0.06 hours after the breech.\n\nConclusionsThis study demonstrates that a weight-based, automated alarm system can detect tank failures prior to a temperature-based alarm system, in some cases over a month in advance. In combination with existing safety mechanisms such as temperature probes, a weight-based monitoring system could serve as a redundant safety mechanism for added protection of cryopreserved reproductive tissues.

developmental biology↗

bioRxiv2018

Limits to anatomical accuracy of diffusion tractography using modern approaches

Diffusion MRI fiber tractography is widely used to probe the structural connectivity of thebrain, with a range of applications in both clinical and basic neuroscience. Despite widespread use, tractography has well-known pitfalls that limits the anatomical accuracy of this technique. Numerous modern methods have been developed to address these shortcomings through advances in acquisition, modeling, and computation. To test whether these advances improve tractography accuracy, we organized the ISBI 2018 3D Validation of Tractography with Experimental MRI (3D-VoTEM) challenge. We made available three unique independent tractography validation datasets - a physical phantom and two ex vivo brain specimens - resulting in 176 distinct submissions from 9 research groups. By comparing results over a wide range of fiber complexities and algorithmic strategies, this challenge provides a more comprehensive assessment of tractographys inherent limitations than has been reported previously. The central results were consistent across all sub-challenges in that, despite advances in tractography methods, the anatomical accuracy of tractography has not dramatically improved in recent years. Taken together, our results independently confirm findings from decades of tractography validation studies, demonstrate inherent limitations in reconstructing white matter pathways using diffusion MRI data alone, and highlight the need for alternative or combinatorial strategies to accurately map the fiber pathways of the brain.

neuroscience↗

bioRxiv2018

Incomplete penetrance for isolated congenital asplenia in humans with mutations in translated and untranslated RPSA exons

Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here eleven new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that six of eighteen (33%) protein-coding mutations and the two (100%) 5-UTR mutations display incomplete penetrance. Three mutations were identified in 2 independent kindreds, due to a hotspot or a founder effect. Lastly, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.

genetics↗

bioRxiv2017

Spectrin is a mechanoresponsive protein shaping the architecture of intercellular invasion

Spectrin is a membrane skeletal protein best known for its structural role in maintaining cell shape and protecting cells from mechanical damage1-3. Here, we report that spectrin dynamically accumulates and dissolves at the fusogenic synapse, where an attacking fusion partner mechanically invades its receiving partner with actin-propelled protrusions to promote cell-cell fusion4-7. Using genetics, cell biology, biophysics and mathematical modeling, we demonstrate that unlike myosin II that responds to dilation deformation, spectrin exhibits a mechanosensitive accumulation in response to shear deformation, which is highly elevated at the fusogenic synapse. The accumulated spectrin forms an uneven network, which functions as a \"sieve\" to constrict the invasive fingerlike protrusions, thus putting the fusogenic synapse under high mechanical tension to promote cell membrane fusion. Taken together, our study has revealed a previously unrecognized function of spectrin as a dynamic mechanoresponsive protein that shapes the architecture of intercellular invasion. These findings have general implications for understanding spectrin function in other dynamic cellular processes beyond cell-cell fusion.

cell biology↗

bioRxiv2017

MicroRNA-451a regulates colorectal cancer radiosensitivity

Colorectal cancer (CRC) is a leading cause of cancer-related death. The responses of CRC to standard of care adjuvant therapies such as radiation or chemotherapy are poorly understood. MicroRNAs (miRs) are small non-coding RNAs that affect gene expression programs in cells by downregulating specific mRNAs. In this study, we discovered a set of microRNAs upregulated rapidly in response to a single 2 Gy dose fraction of {gamma}-radiation in a mouse colorectal carcinoma xenograft model. The most upregulated candidate in our signature, miR-451a inhibits tumor cell proliferation and attenuated surviving fraction in longer-term cultures. Conversely, inhibition of miR-451a increased proliferation, tumorsphere formation and surviving fraction of tumor cells. Using a bioinformatics approach, we identified four genes-CAB39, EMSY, MEX3C and EREG as targets of miR-451a. Transfection of miR-451a decreased both mRNA and protein levels of these targets. Importantly, we found miR-451a expression was decreased with tumor stage in a small subset of CRC patients. Finally, analysis of a TCGA colorectal cancer dataset reveals that the CAB39 and EMSY are upregulated at the protein level in a significant number of CRC patients and correlates with poorer overall survival. Taken together, our data indicates miR-451a influences the radiation sensitivity of colorectal carcinomas.

cancer biology↗