Biology subjects

Kim, T.

Publications and source records attributed to Kim, T..

At least 145 records · Page 8Linked to original sources

bioRxiv2019

The network structure and eco-evolutionary dynamics of CRISPR-induced immune diversification

As a heritable sequence-specific adaptive immune system, CRISPR-Cas is a powerful force shaping strain diversity in host-virus systems. While the diversity of CRISPR alleles has been explored, the associated structure and dynamics of host-virus interactions have not. We explore the role of CRISPR in mediating the interplay between host-virus interaction structure and eco-evolutionary dynamics in a computational model and compare results with three empirical datasets from natural systems. We show that the structure of the networks describing who infects whom and the degree to which strains are immune, are respectively modular (containing groups of hosts and viruses that interact strongly) and weighted-nested (specialist hosts are more susceptible to subsets of viruses that in turn also infect the more generalist hosts with many spacers matching many viruses). The dynamic interplay between these networks influences transitions between dynamical regimes of virus diversification and host control. The three empirical systems exhibit weighted-nested protection networks, a pattern our theory shows is indicative of hosts able to suppress virus diversification. Previously missing from studies of microbial host-pathogen systems, the protection network plays a key role in the coevolutionary dynamics.

evolutionary biology↗

bioRxiv2019

Single-molecule study of the dynamic organization of Vascular Endothelial Growth Factor Receptor 2 on the endothelial cell surface

The nanoscale organization of cell surface receptors plays an important role in signaling. We determined this organization and its relation to receptor activation for VEGF Receptor-2 (VEGFR-2), a critical receptor tyrosine kinase in endothelial cells (ECs), by combining live-cell single-molecule imaging of endogenous VEGFR-2 with rigorous computational analysis. We found that surface VEGFR-2 can be mobile or immobile/confined, and monomeric or non-monomeric, with a complex interplay between the two. The mobility and interaction heterogeneity of VEGFR-2 in the basal state led to heterogeneity in the sequence of steps leading to VEGFR-2 activation by VEGF. Specifically, we found that VEGF can bind to both monomeric and non-monomeric VEGFR-2, and, when binding to monomeric VEGFR-2, promotes dimer formation but only for immobile/confined receptors. Overall, our study highlights the dynamic and heterogeneous nature of cell surface receptor organization and its complex relationships with receptor activation and signaling.

biophysics↗

bioRxiv2019

Autoencoder-based cluster ensembles for single-cell RNA-seq data analysis

BackgroundSingle-cell RNA-sequencing (scRNA-seq) is a transformative technology, allowing global transcriptomes of individual cells to be profiled with high accuracy. An essential task in scRNA-seq data analysis is the identification of cell types from complex samples or tissues profiled in an experiment. To this end, clustering has become a key computational technique for grouping cells based on their transcriptome profiles, enabling subsequent cell type identification from each cluster of cells. Due to the high feature-dimensionality of the transcriptome (i.e. the large number of measured genes in each cell) and because only a small fraction of genes are cell type-specific and therefore informative for generating cell type-specific clusters, clustering directly on the original feature/gene dimension may lead to uninformative clusters and hinder correct cell type identification.\n\nResultsHere, we propose an autoencoder-based cluster ensemble framework in which we first take random subspace projections from the data, then compress each random projection to a low-dimensional space using an autoencoder artificial neural network, and finally apply ensemble clustering across all encoded datasets for generating clusters of cells. We employ four evaluation metrics to benchmark clustering performance and our experiments demonstrate that the proposed autoencoder-based cluster ensemble can lead to substantially improved cell type-specific clusters when applied with both the standard k-means clustering algorithm and a state-of-the-art kernel-based clustering algorithm (SIMLR) designed specifically for scRNA-seq data. Compared to directly using these clustering algorithms on the original datasets, the performance improvement in some cases is up to 100%, depending on the evaluation metrics used.\n\nConclusionsOur results suggest that the proposed framework can facilitate more accurate cell type identification as well as other downstream analyses. The code for creating the proposed autoencoder-based cluster ensemble framework is freely available from https://github.com/gedcom/autoencoder_cluster_ensemble

bioinformatics↗

bioRxiv2019

Time-resolved proteomic profile of Amblyomma americanum tick saliva during feeding

Amblyomma americanum ticks transmit more than a third of human tick-borne disease (TBD) agents in the United States. Tick saliva proteins are critical to success of ticks as vectors of TBD agents, and thus might serve as targets in tick antigen-based vaccines to prevent TBD infections. We describe a systems biology approach to identify, by LC-MS/MS, saliva proteins (tick=1182, rabbit=335) that A. americanum ticks likely inject into the host every 24 h during the first 8 days of feeding, and towards the end of feeding using two different sample preparation approaches (in-gel and in-solution). The in-gel approach determined molecular identification of predominant protein bands in tick saliva, and the in-solution added depth to discovery of proteins. Searching against entries in GenBank grouped tick and rabbit proteins in this study into 27 and 25 functional categories. Aside from housekeeping-like proteins, majority of tick saliva proteins belong to the tick-specific (no homology to non-tick organisms: 32%), protease inhibitors (13%), proteases (8%), glycine-rich proteins (6%) and lipocalins (4%) categories. Global secretion dynamics analysis suggests that majority (74%) of proteins in this study are associated with regulating initial tick feeding functions and transmission of pathogens as they are secreted within 24-48 h of tick attachment. Comparative analysis of the A. americanum tick saliva proteome to five other tick saliva proteomes identified 284 conserved tick saliva proteins: we speculate that these regulate critical tick feeding functions and might serve as tick vaccine antigens. We discuss our findings in the context of understanding A. americanum tick feeding physiology as a means through which we can find effective targets for a vaccine against tick feeding.\n\nAuthor SummaryThe lone star tick, Amblyomma americanum, is a medically important species in US that transmits 5 of the 16 reported tick-borne disease agents. Most recently, bites of this tick were associated with red meat allergies in humans. Vaccination of animals against tick feeding has been shown to be a sustainable and effective alternative to current acaricide based tick control method which has several limitations. The pre-requisite to tick vaccine development is to understand the molecular basis of tick feeding physiology. Toward this goal, this study has identified proteins that A. americanum ticks inject into the host at different phases of its feeding cycle. This data set has identified proteins that A. americanum inject into the host within 24-48 h of feeding before it starts to transmit pathogens. Of high importance, we identified 284 proteins that are present in saliva of other tick species, which we suspect regulate important role(s) in tick feeding success and might represent rich source target antigens for a tick vaccine. Overall, this study provides a foundation to understand the molecular mechanisms regulating tick feeding physiology.

systems biology↗

bioRxiv2019

Modeling the control of bacterial infections via antibiotic-induced proviruses

Most bacteria and archaea are infected by latent viruses that change their physiology and responses to environmental stress. We use a population model of the bacteria-phage relationship to examine the role that latent phage play on the bacterial population over time in response to antibiotic treatment. We demonstrate that the stress induced by antibiotic administration, even if bacteria are resistant to killing by antibiotics, is sufficient to control the infection under certain conditions. This work expands the breadth of understanding of phage-antibiotic synergy to include both temperate and chronic viruses persisting in their latent form in bacterial populations.\n\nImportanceAntibiotic-resistance is a growing concern for management of common bacterial infections. Here we show that antibiotics can be effective at sub-inhibitory levels when bacteria carry latent phage. Our findings suggest that specific treatment strategies based on the identification of latent viruses in individual bacterial strains may be an effective personalized medicine approach to antibiotic stewardship.

ecology↗

bioRxiv2019

High diagnostic yield and clinical utility of WES for patients with undiagnosed genetic disorder by automating variant interpretation

PurposeEVIDENCE, an automated interpretation system, has been developed to facilitate the entire process of whole exome sequencing (WES) analyses. This study investigated the diagnostic yield of EVIDENCE in patients suspected genetic disorders. MethodsDNA from 330 probands (age range, 0-68 years) with suspected genetic disorders were subjected to WES. Candidate variants were identified by EVIDENCE and confirmed by testing family members and/or clinical reassessments. ResultsThe average number of overlapping organ categories per patient was 4.5 {+/-} 5.0. EVIDENCE reported a total 244 variants in 215 (65.1%) of the 330 probands. After clinical reassessment and/or family member testing, 196 variants were identified in 171 probands (51.8%), including 115 novel variants. These variants were confirmed as being responsible for 146 genetic disorders. One hundred-seven (54.6%) of the 196 variants were categorized as pathogenic or likely pathogenic before, and 146 (74.6%) after, clinical assessment and/or family member testing. Factors associated with a variant being confirmed as causative include rules, such as PVS1, PS1, PM1, PM5, and PP5, and similar symptom scores between that variant and a patients phenotype. ConclusionThis new, automated variant interpretation system facilitated the diagnosis of various genetic diseases with a 51% improvement in diagnostic yield.

genetics↗

bioRxiv2019

Medial prefrontal cortex guides goal-directed decision-making about hunger and thirst

Decision-making guided by self-evaluation of bodily need states (interoception) is thought to be important for cognitive control over eating or drinking1. However, interoception of body states is notoriously unreliable2-4 because hunger and thirst have similar motivational characteristics5. Consequently, individuals may inaccurately assess their need state and consume food when dehydrated, leading some healthcare professionals to advise overweight patients to drink water before eating6-8. Neuroimaging in humans9-11 and recordings in rodents12-15 indicate medial prefrontal frontal cortex (mPFC) involvement in need state-dependent decisions about hunger and thirst, but mPFC surgical lesions16,17 and electrical activity perturbations have little influence on eating or drinking18,19. To investigate need-state dependent decision-making as well as the function of mPFC in hunger and thirst, we developed an instrumental foraging task for mice that mimics key elements of human decisions. When homeostatic need state was variable, mice did not show intrinsic knowledge of hunger or thirst state but, instead, rapidly identified their need after consumption of small portions of food and water (outcome evaluation). We observed a food-seeking bias, even in thirsty mice, that required outcome evaluation for mice to correctly seek water. mPFC was required for need state-dependent decisions about hunger and thirst, specifically under variable need state conditions. Food-seeking or water-seeking choices were controlled by multiple decision-making processes, and mPFC was involved in goal-directed decisions about the identity of need states. Thus, we have discovered a role for mPFC in decision-making about hunger and thirst, which is relevant for human behaviors that contribute to obesity.

neuroscience↗

bioRxiv2019

Tetraploidy in rodent cardiac stem cells confers enhanced biological properties

Ploidy for cardiomyocytes is well described but remains obscure in cardiac interstitial cells (CICs). Ploidy of c-kit+CICs were assessed using a combination of confocal, karyotypic, and flow cytometric assessments coupled with molecular and bioinformatic analyses. Fundamental differences were found between cultured rodent (rat, mouse) c-kit+CICs possessing mononuclear tetraploid (4n) content versus large mammal (human, swine) with mononuclear diploid (2n) content. In-situ analysis, confirmed with fresh isolates, revealed diploid content in c-kit+CICs from human and a mixture of diploid and tetraploid nuclei in mouse. Molecular assessment of the p53 signaling pathway provides a plausible explanation for escape from replicative senescence in rodent but not human ckit+CICs. Single cell transcriptional profiling reveals distinctions between diploid versus tetraploid populations in mouse ckit+CICs, alluding to functional divergences. Collectively, these data reveal fundamental species-specific biological differences in c-kit+CICs that could account for challenges in extrapolation of myocardial preclinical studies from rodent to large animal models.

cell biology↗

bioRxiv2018

Mechanical Interactions between a Cell and an Extracellular Environment Facilitate Durotactic Cell Migration

Cell migration is a fundamental process in biological systems, playing an important role for diverse physiological processes. Cells often exhibit directed migration in a specific direction in response to various types of cues. In particular, cells are able to sense the rigidity of surrounding environments and then migrate towards stiffer regions. To understand this mechanosensitive behavior called durotaxis, several computational models have been developed. However, most of the models made phenomenological assumptions to recapitulate durotactic behaviors, significantly limiting insights provided from these studies. In this study, we developed a computational biomechanical model without any phenomenological assumption to illuminate intrinsic mechanisms of durotactic behaviors of cells migrating on a two-dimensional substrate. The model consists of a simplified cell generating contractile forces and a deformable substrate coarse-grained into an irregular triangulated mesh. Using the model, we demonstrated that durotactic behaviors emerge from purely mechanical interactions between the cell and the underlying substrate. We investigated how durotactic migration is regulated by biophysical properties of the substrate, including elasticity, viscosity, and stiffness profile.

biophysics↗

bioRxiv2018

A role for long-range, through-lattice coupling in microtubule catastrophe

Microtubules are cylindrical polymers of {beta}-tubulin that play critical roles in fundamental processes like chromosome segregation and vesicular transport. Microtubules display dynamic instability, switching stochastically between growing and rapid shrinking as a consequence of GTPase activity in the lattice. The molecular mechanisms behind microtubule catastrophe, the switch from growing to rapid shrinking, remain poorly defined. Indeed, two-state stochastic models that seek to describe microtubule dynamics purely in terms of the biochemical properties of GTP- and GDP-bound {beta}-tubulin incorrectly predict the concentration-dependence of microtubule catastrophe. Recent studies provided evidence for three distinct conformations of {beta}-tubulin in the lattice that likely correspond to GTP, GDP.Pi, and GDP. The incommensurate lattices observed for these different conformations raises the possibility that in a mixed nucleotide state lattice, neighboring tubulin dimers might modulate each others conformations and hence their biochemistry. We explored whether incorporating a GDP.Pi state or the likely effects of conformational accommodation can improve predictions of catastrophe. Adding a GDP.Pi intermediate did not improve the model. In contrast, adding neighbor-dependent modulation of tubulin biochemistry improved predictions of catastrophe. Conformational accommodation should propagate beyond nearest-neighbor contacts, and consequently our modeling demonstrates that long-range, through-lattice effects are important determinants of microtubule catastrophe.

cell biology↗

bioRxiv2018

Direct observation of individual tubulin dimers binding to growing microtubules

The biochemical basis of microtubule growth has remained elusive for over thirty years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make the first direct, single-molecule measurements of tubulin on- and off-rates. We detect two populations of transient dwell times, and determine via binding-interface mutants that they are separated by the formation of inter-protofilament bonds. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data, and furthermore predict plus-end tapering. Overall, we provide the most direct and complete quantification of how microtubules grow to date.\n\nSIGNIFICANCEMicrotubule polymerization dynamics are fundamental to cell migration and cell division, where they are targets for chemotherapy drugs. Despite significant progress, the precise structural and biochemical events occurring at growing microtubule tips are not well defined, and better understanding is necessary for discriminating mechanisms of microtubule dynamics regulation in cells. Here, we visualize individual tubulin subunits reversibly and irreversibly interacting with dynamic microtubule tips, and thereby directly measure tubulin on- and off-rates. By analyzing plus-tip residence times of wild-type and mutant tubulin, we characterize the relative contributions of longitudinal (along protofilaments) and lateral (between protofilaments) bond energies to microtubule growth. This work provides insights into microtubule tip structure and potential modes of microtubule dynamics regulation.

biophysics↗

bioRxiv2018

40 Hz acoustic stimulation decreases amyloid beta and modulates brain rhythms in a mouse model of Alzheimers disease

IntroductionThe accumulation of amyloid-beta (A{beta}) is one of the neuropathologic hallmarks of Alzheimers disease (AD) and abnormal gamma band oscillations and brain connectivity have been observed. Recently, a therapeutic potential of gamma entrainment of the brain was reported by Iaccarino et al. However, the affected areas were limited to hippocampus and visual cortex. Therefore, we sought to test the effects of acoustic stimulation in a mouse model of AD.\n\nMethodsFreely moving 6-month-old 5XFAD mice with electroencephalogram (EEG) electrodes were treated with daily two-hour acoustic stimulation at 40Hz for 2 weeks. A{beta} and microglia were evaluated by immunohistochemistry and ELISA. Evoked and spontaneous gamma power were analyzed by wavelet analysis. Coherence, phase locking value (PLV), and cross-frequency coupling were analyzed.\n\nResultsThe number of A{beta} plaques decreased in the pre-and infralimbic (PIL) and hippocampus regions and soluble A{beta}-40 and A{beta}-42 peptides in PIL in the acoustic stimulation group. We also found that the number of microglia increased in PIL and hippocampus. In EEG analysis, evoked gamma power was decreased and spontaneous gamma power was increased. Gamma coherence and phase locking value did not show significant changes. Cross-frequency coupling was shifted from gamma-delta to gamma-theta rhythm.\n\nConclusionIn summary, we found that acoustic stimulation at 40Hz can reduce A{beta} in the brain and restore the gamma band oscillations and the frontoparietal connectivity. Our data suggest that acoustic stimulation might alter the natural deterioration processes of AD and have a therapeutic potential in AD.

neuroscience↗

bioRxiv2018

Information-rich localization microscopy through machine learning

While current single-molecule localization microscopy (SMLM) methods often rely on the target-specific alteration of the point spread function (PSF) to encode the multidimensional contents of single fluorophores, we argue that the details of the PSF in an unmodified microscope already contain rich, multidimensional information. We introduce a data-driven approach in which artificial neural networks (ANNs) are trained to make a direct link between an experimental PSF image and its underlying parameters. To demonstrate this concept in real systems, we decipher in fixed cells both the colors and the axial positions of single molecules in regular SMLM data.

biophysics↗

bioRxiv2018

A smartphone-based tool for rapid, portable, and automated wide-field retinal imaging

PurposeHigh-quality, wide-field retinal imaging is a valuable method to screen preventable, vision-threatening diseases of the retina. Smartphone-based retinal cameras hold promise for increasing access to retinal imaging, but variable image quality and restricted field of view can limit their utility. We developed and clinically tested a smartphone-based system that addresses these challenges with automation-assisted imaging.\n\nMethodsThe system was designed to improve smartphone retinal imaging by combining automated fixation guidance, photomontage, and multi-colored illumination with optimized optics, user-tested ergonomics, and touch-screen interface. System performance was evaluated from images of ophthalmic patients taken by non-ophthalmic personnel. Two masked ophthalmologists evaluated images for abnormalities and disease severity.\n\nResultsThe system automatically generated 100-degree retinal photomontages from five overlapping images in under 1 minute at full resolution (52.3 pixels per retinal degree) fully on-phone, revealing numerous retinal abnormalities. Feasibility of the system for DR screening using the retinal photomontages was performed in 71 diabetics by masked graders. DR grade matched perfectly with dilated clinical examination in 55.1% of eyes and within 1 severity level for 85.2% of eyes. For referral-warranted DR, average sensitivity was 93.3% and specificity 56.8%.\n\nConclusionsAutomation-assisted imaging produced high-quality, wide-field retinal images that demonstrate the potential of smartphone-based retinal cameras to be used for retinal disease screening.\n\nTranslational RelevanceEnhancement of smartphone-based retinal imaging through automation and software intelligence holds great promise for increasing the accessibility of retinal screening.

bioengineering↗

bioRxiv2018

In situ transcriptome characteristics are lost following culture adaptation of adult cardiac stem cells

Regenerative therapeutic approaches for myocardial diseases often involve adoptive transfer of stem cells expanded ex vivo. Prior studies indicate that cell culture conditions affect functional and phenotypic characteristics, but relationship(s) of cultured cells derived from freshly isolated populations and the heterogeneity of the cultured population remain poorly defined. Functional and phenotypic characteristics of adoptively donated cells will determine outcomes of interventional treatment for disease, necessitating characterization of the impact that ex vivo expansion has upon isolated stem cell populations. Single-cell RNA-Seq profiling (scRNA-Seq) was performed to determine consequences of culture expansion upon adult cardiac progenitor cells (CPCs) as well as relationships with other cell populations. Bioinformatic analyses reveal loss of identity marker genes in cultured CPCs while simultaneously acquiring thousands of additional genes. Cultured CPCs exhibited decreased transcriptome variability within their population relative to their freshly isolated cells. Findings were validated by comparative analyses using scRNA-Seq datasets of various cell types generated by multiple scRNA-Seq technology. Increased transcriptome diversity and decreased population heterogeneity in the cultured cell population relative to freshly isolated cells may help account for reported outcomes associated with experimental and clinical use of CPCs for treatment of myocardial injury.

bioinformatics↗

bioRxiv2018

Cohesin interacts with a panoply of splicing factors required for cell cycle progression and genomic organization

The cohesin complex regulates sister chromatid cohesion, chromosome organization, gene expression, and DNA repair. Here we report that endogenous human cohesin interacts with a panoply of splicing factors and RNA binding proteins, including diverse components of the U4/U6.U5 tri-snRNP complex and several splicing factors that are commonly mutated in cancer. The interactions are enhanced during mitosis, and the interacting splicing factors and RNA binding proteins follow the cohesin cycle and prophase pathway of regulated interactions with chromatin. Depletion of cohesin-interacting splicing factors results in stereotyped cell cycle arrests and alterations in genomic organization. These data support the hypothesis that splicing factors and RNA binding proteins control cell cycle progression and genomic organization via regulated interactions with cohesin and chromatin.\n\nOne Sentence SummaryEndogenous tagging reveals that cohesin interacts with diverse chromatin-bound splicing factors that regulate cell cycle progression and genomic organization in human cells.

cell biology↗

bioRxiv2017

Optical excitation and detection of neuronal activity

Optogenetics has emerged as an exciting tool for manipulating neural activity, which in turn, can modulate behavior in live organisms. However, detecting the response to the optical stimulation requires electrophysiology with physical contact or fluorescent imaging at target locations, which is often limited by photobleaching and phototoxicity. In this paper, we show that phase imaging can report the intracellular transport induced by optogenetic stimulation. We developed a multimodal instrument that can both stimulate cells with subcellular spatial resolution and detect optical pathlength changes with nanometer scale sensitivity. We found that optical pathlength fluctuations following stimulation are consistent with active organelle transport. Furthermore, the results indicate a broadening in the transport velocity distribution, which is significantly higher in stimulated cells compared to optogenetically inactive cells. It is likely that this label-free, contactless measurement of optogenetic response will provide an enabling approach to neuroscience.\n\nO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Quantitative phase imaging enables label-free detection of enhanced intracellular activities induced by optogenetic stimulation.\n\nC_FIG_DISPLAY

bioengineering↗

bioRxiv2017

Free Aerial Imagery as a Resource to Monitor Compliance with the Endangered Species Act

Compliance monitoring is an integral part of law and policy implementation. A lack of compliance monitoring for the Endangered Species Act (ESA), driven in part by resource limitations, may be undercutting efforts to recover threatened and endangered species. Here we evaluate the utility of freely available satellite and aerial imagery as a cost-efficient component of ESA compliance monitoring. Using data on actions authorized by the U.S. Fish and Wildlife Service (FWS) under section 7 of the ESA, we show that approximately 40% of actions can be found in remotely sensed imagery. Some types of actions, such as residential and commercial development, roadwork, and forestry, show substantially higher observability. Based on our results and the requirements of compliance monitoring, we recommend FWS standardize data collection requirements for consultations; record and publish terms and conditions of consultations; and encourage their staff to use technology such as remotely sensed data as a central part of their workflow for implementing the ESA.

ecology↗