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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 973 records · Page 54Linked to original sources

T-SNE visualization of large-scale neural recordings.

Electrophysiology is entering the era of Big Data. Multiple probes, each with hundreds to thousands of individual electrodes, are now capable of simultaneously recording from many brain regions. The major challenge confronting these new technologies is transforming the raw data into physiologically meaningful signals, i.e. single unit spikes. Sorting the spike events of individual neurons from a spatiotemporally dense sampling of the extracellular electric field is a problem that has attracted much attention [22, 23], but is still far from solved. Current methods still rely on human input and thus become unfeasible as the size of the data sets grow exponentially.\n\nHere we introduce the t-student stochastic neighbor embedding (t-sne) dimensionality reduction method [27] as a visualization tool in the spike sorting process. T-sne embeds the n-dimensional extracellular spikes (n = number of features by which each spike is decomposed) into a low (usually two) dimensional space. We show that such embeddings, even starting from different feature spaces, form obvious clusters of spikes that can be easily visualized and manually delineated with a high degree of precision. We propose that these clusters represent single units and test this assertion by applying our algorithm on labeled data sets both from hybrid [23] and paired juxtacellular/extracellular recordings [15]. We have released a graphical user interface (gui) written in python as a tool for the manual clustering of the t-sne embedded spikes and as a tool for an informed overview and fast manual curration of results from other clustering algorithms. Furthermore, the generated visualizations offer evidence in favor of the use of probes with higher density and smaller electrodes. They also graphically demonstrate the diverse nature of the sorting problem when spikes are recorded with different methods and arise from regions with different background spiking statistics.

neuroscience

Electroceutical disinfection strategies impair the motility of pathogenic Pseudomonas aeruginosa and Escherichia coli

Electrotaxis or galvanotaxis refers to the migration pattern of cells induced in response to electrical potential. Although it has been extensively studied in mammalian cells, electrotaxis has not been explored in detail in bacterial cells; information regarding the impact of current on pathogenic bacteria is severely lacking. Therefore, we designed a series of single and multi-cue experiments to assess the impact of varying currents on bacterial motility dynamics in pathogenic multi-drug resistant (MDR) strains of Pseudomonas aeruginosa and Escherichia coli using a microfluidic platform. Motility plays key roles in bacterial migration and the colonization of surfaces during the formation of biofilms, which are inherently recalcitrant to removal and resistant to traditional disinfection strategies (e.g. antibiotics). Use of the microfluidic platform allows for exposure to current, which can be supplied at a range that is biocidal to bacteria, yet physiologically safe in humans (single cue). This system also allows for multi-cue experiments where acetic acid, a relatively safe compound with anti-fouling/antimicrobial properties, can be combined with current to enhance disinfection. These strategies may offer substantial therapeutic benefits, specifically for the treatment of biofilm infections, such as those found in the wound environment. Furthermore, microfluidic systems have been successfully used to model the unique microfluidic dynamics present in the wound environment, suggesting that these investigations could be extended to more complex biological systems. Our results showed that the application of current in combination with acetic acid has profound inhibitory effects on MDR strains of P. aeruginosa and E. coli, even with brief applications. Specifically, E. coli motility dynamics and cell survival were significantly impaired starting at a concentration of 125 A DC and 0.31% acetic acid, while P. aeruginosa was impaired at 70 A and 0.31% acetic acid. As these strains are relevant wound pathogens, it is likely that this strategy would be effective against similar strains in vivo and could represent a new approach to hasten wound healing.

bioengineering

Ubiquity and quantitative significance of bacterioplankton lineages inhabiting the oxygenated hypolimnion of deep freshwater lakes

Freshwater bacterioplankton in the oxygenated hypolimnion are reportedly dominated by specific members that are distinct from those in the epilimnion. However, no consensus exists regarding the ubiquity and abundance of these bacterioplankton, which is necessary to evaluate their ecological importance. The present study investigated the bacterioplankton community in the oxygenated hypolimnia of 10 deep freshwater lakes. Despite the broad geochemical characteristics of the lakes, 16S rRNA gene sequencing demonstrated that many predominant lineages in the hypolimnion were shared by several lakes and consisted of members occurring in the entire water layer and members specific to the hypolimnion. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) revealed that representative hypolimnion-specific lineages, CL500-11 (Chloroflexi), CL500-3, CL500-37, CL500-15 (Planctomycetes), and the MGI group (Thaumarchaeota), together accounted for 1.5-32.9% of all bacterioplankton in the hypolimnion of the lakes. Furthermore, an analysis of micro-diversification based on single-nucleotide variation in the partial 16S rRNA gene sequence (oligotyping) suggested the presence of hypolimnion-specific ecotypes among the lineages occurring in the entire water layer (e.g., acI and Limnohabitans). Collectively, these results demonstrate the uniqueness, ubiquity, and quantitative significance of bacterioplankton in the oxygenated hypolimnion, motivating future studies to focus on their eco-physiological characteristics.

ecology

G protein-coupled estrogen receptor regulates heart rate in zebrafish embryos

Estrogens act by binding to estrogen receptors alpha and beta (ER, ER{beta}), ligand-dependent transcription factors that play crucial roles in sex differentiation, tumor growth and cardiovascular physiology. Estrogens also activate the G protein-coupled estrogen receptor (GPER), however the function of GPER in vivo is less well understood. Here we find that GPER is required to maintain normal heart rate in zebrafish embryos. Acute exposure to estrogens increased heart rate in wildtype and in ER and ER{beta} mutant embryos but not in GPER mutants. Nuclear estrogen receptor signaling remained normal in GPER mutant embryos, however GPER mutant embryos exhibited reduced basal heart rate while heart rate was normal in ER and ER{beta} mutants. We detected GPER transcript in discrete regions of the brain but not in the heart. In the brain, we observed gper expression in cells lacking nuclear estrogen receptor activity, suggesting that GPER acts in the brain to regulate heart rate independently of nuclear estrogen receptor signaling. Our results demonstrate that estradiol plays a previously unappreciated role in the acute modulation of heart rate during zebrafish embryonic development and that GPER functions as an autonomous estrogen receptor in vivo to regulate basal heart rate.

pharmacology and toxicology

SINCERITIES: Inferring gene regulatory networks fromtime-stamped single cell transcriptional expression profiles

Recent advances in single cell transcriptional profiling open up a new avenue in studying the functional role of cell-to-cell variability in physiological processes such as stem cell differentiation. In this work, we developed a novel algorithm called SINCERITIES (SINgle CEll Regularized Inference using TIme-stamped Expression profileS), for the inference of gene regulatory networks (GRNs) from single cell transcriptional expression data. In particular, we focused on time-stamped cross-sectional expression data, a common type of dataset generated from transcriptional profiling of single cells collected at multiple time points after cell stimulation. SINCERITIES recovers the regulatory (causal) relationships among genes by employing regularized linear regression, particularly ridge regression, using temporal changes in the distributions of gene expressions. Meanwhile, the modes of the gene regulations (activation and repression) come from partial correlation analyses between pairs of genes. We demonstrated the efficacy of SINCERITIES in inferring GRNs using simulated time-stamped in silico single cell expression data and single transcriptional profiling of THP-1 monocytic human leukemia cell differentiation. The case studies showed that SINCERITIES could provide accurate GRN predictions, significantly better than other GRN inference algorithms such as TSNI, GENIE3 and JUMP3. Meanwhile, SINCERITIES has a low computational complexity and is amenable to problems of extremely large dimensionality.

systems biology

The NCA-1 ion channel functions downstream of Gq and Rho to regulate locomotion in C. elegans

The heterotrimeric G protein Gq positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of Gq that acts in parallel to the canonical Gq effector phospholipase C. Here we examine how Trio and Rho act to stimulate neuronal activity downstream of Gq in the nematode Caenorhabditis elegans. Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the Gq/Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of Gq in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans. Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal Gq-Rho signaling in C. elegans.

genetics

Summary of muscle parameters for Hill-based muscle modeling in the human lower limb

A summary is presented of five mechanical parameters from human lower limb skeletal muscles critical for Hill-based muscle modeling: the optimal fiber length, the fiber pennation angle, the physiological cross-sectional area (PCSA), the unloaded tendon length, and the fast-twitch fiber fraction. The data presented are drawn from a total of 29 publications including human cadaver studies, in vivo imaging studies of live humans, musculoskeletal modeling studies, and combinations of these methods. Where possible, parameter values were adjusted from the referenced data to present them with consistent definitions (normalization of measured fiber lengths to optimal sarcomere length, and calculation of PCSA as the ratio of fiber volume to fiber length). It is seen that within a specific muscle, optimal fiber lengths are fairly consistent between studies, pennation angles and PCSAs vary widely between studies, and data for unloaded tendon length are comparatively sparse. Few studies have reported fiber type fractions for a large number of muscles. Guidelines for implementing these parameter values in muscle modeling and musculoskeletal modeling are suggested.\n\nUpdate HistoryO_LIDecember 2, 2016: original submission\nC_LIO_LIDecember 3, 2016: discussion on maximum isometric force and specific tension added\nC_LIO_LIDecember 6, 2016: minor edits for typos, clarity, and missing references\nC_LIO_LIDecember 7, 2016: Tirrell et al. (2012) study added\nC_LI

bioengineering

How Cancer Genomics Drives Cancer Biology: Does Synthetic Lethality Explain Mutually Exclusive Oncogenic Mutations?

Large-scale analyses of cancer genomes are revealing patterns of mutations that suggest biologically significant ideas about many aspects of cancer, including carcinogenesis, classification, and preventive and therapeutic strategies. Among those patterns is \"mutual exclusivity\", a phenomenon observed when two or more mutations that are commonly observed in samples of a type of cancer are not found combined in individual tumors. We have been studying a striking example of mutual exclusivity: the absence of co-existing mutations in the KRAS and EGFR proto-oncogenes in human lung adenocarcinomas, despite the high individual frequencies of such mutations in this common type of cancer. Multiple lines of evidence suggest that toxic effects of the joint expression of KRAS and EGFR mutant oncogenes, rather than loss of any selective advantages conferred by a second oncogene that operates through the same signaling pathway, are responsible for the observed mutational pattern. We discuss the potential for understanding the physiological basis of such toxicity, for exploiting it therapeutically, and for extending the studies to other examples of mutual exclusivity.

cancer biology

Validating Regulatory Predictions from Diverse Bacteria with Mutant Fitness Data

Although transcriptional regulation is fundamental to understanding bacterial physiology, the targets of most bacterial transcription factors are not known. Comparative genomics has been used to identify likely targets of some of these transcription factors, but these predictions typically lack experimental support. Here, we used mutant fitness data, which measures the importance of each gene for a bacteriums growth across many conditions, to validate regulatory predictions from RegPrecise, a curated collection of comparative genomics predictions. Because characterized transcription factors often have correlated fitness with one of their targets (either positively or negatively), correlated fitness patterns provide support for the comparative genomics predictions. At a false discovery rate of 3%, we identified significant cofitness for at least one target of 158 TFs in 107 ortholog groups and from 24 bacteria. Thus, high-throughput genetics can be used to identify a high-confidence subset of the sequence-based regulatory predictions.

microbiology

No evidence for genome editing of the endogenous DNA in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo).

A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5- phosphorylated guide DNA (gDNA) from 2.5 ng/l to 50 ng/l and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that we failed to edit using NgAgo.

molecular biology

The Evolution of Primate Body Size: Left-skewness, Maximum Size, and Cope’s Rule

Body size is a key physiological, ecological, and evolutionary characteristic of species. Within most major clades, body size distributions follow a right-skewed pattern where most species are relatively small while a few are orders of magnitude larger than the median size. Using a novel database of 742 extant and extinct primate species sizes over the past 66 million years, we find that primates exhibit the opposite pattern: a left-skewed distribution. We investigate the long-term evolution of this distribution, first showing that the initial size radiation is consistent with plesiadapiformes (an extinct group with an uncertain ancestral relationship to primates) being ancestral to modern primates. We calculate the strength of Copes Rule, showing an initial tendency for descendants to increase in size relative to ancestors until the trend reverses 40 million years ago. We explore when the primate size distribution becomes left-skewed and study correlations between body size patterns and climactic trends, showing that across Old and New World radiations the body size distribution initially exhibits a right-skewed pattern. Left-skewness emerged early in Old World primates in a manner consistent with a previously unidentified possible maximum body size, which may be mechanistically related to primates encephalization and complex social groups.

evolutionary biology

Variability and stability of large-scale cortical oscillation patterns

Individual differences in brain organization exist at many spatial and temporal scales, contributing to the substantial heterogeneity underlying human thought and behavior. Oscillatory neural activity is crucial for these behaviors, but how such rhythms are expressed across the cortex within and across individuals has not been thoroughly characterized. Combining electroencephalography (EEG) with representational similarity and multivariate classification techniques, we provide a systematic characterization of brain-wide activity across frequency bands and oscillatory features during rest and task performance. Results indicate that oscillatory profiles exhibit sizable group-level correspondences, indicating the presence of common templates of oscillatory organization. At the same time, we observed well-defined subject-specific network profiles that were discernible above and beyond the structure shared across individuals. These individualized patterns were sufficiently stable over time to allow successful classification of individuals several months later. Finally, our findings indicate that the network structure of rhythmic activity varies considerably across distinct oscillatory frequencies and features, suggesting the existence of multiple, parallel information processing streams embedded in distributed electrophysiological activity. Together, these findings affirm the richness of spatiotemporal EEG signals and emphasize the utility of multivariate network analyses for understanding the role of brain oscillations in physiology and behavior.

neuroscience

Fractone bulbs derive from ependymal cells and their laminin composition affects cell proliferation in the subventricular zone

Fractones are extracellular matrix structures in the neural stem cell niche of the subventricular zone (SVZ), where they appear as round deposits named bulbs or thin branching lines called stems. Their cellular origin and what determines their localization at this site is poorly studied and it remains unclear whether they influence neural stem and progenitor cells formation, proliferation and/or maintenance. To address these questions, we analyzed whole mount preparations of the lateral ventricle by confocal microscopy using different extracellular matrix and cell markers. We found that bulbs are rarely connected to stems and that they contain laminin 5 and 2 chains, respectively. Fractone bulbs were profusely distributed throughout the SVZ and appeared associated with the center of pinwheels, a critical site for adult neurogenesis. We demonstrate that bulbs appear at the apical membrane of ependymal cells at the end of the first week after birth. The use of transgenic mice lacking laminin 5 gene expression (Lama5) in endothelium and in FoxJ1-expressing ependymal cells, revealed ependymal cells as the source of laminin 5-containing fractone bulbs. Loss of laminin 5 from bulbs correlated with a 60% increase in cell proliferation, as determined by PH3 staining, and with a selective reduction in the number of quiescent neural stem cells in the SVZ. These results indicate that fractones are a key component of the SVZ and suggest that laminin 5 modulates the physiology of the neural stem cell niche.\n\nSignificance StatementOur work unveils key aspects of fractones, extracellular matrix structures present in the SVZ that still lack a comprehensive characterization. We show that fractones extensively interact with neural stem cells, whereas some of them are located precisely at pinwheel centers, which are hotspots for adult neurogenesis. Our results also demonstrate that fractones increase in size during aging and that their interactions with NSPCs become more complex in old mice. Lastly, we show that fractone bulbs are produced by ependymal cells and that their laminin content regulates neural stem cells.

neuroscience

Quantitative Analysis of Dopamine Neuron Subtypes Generated from Mouse Embryonic Stem Cells

Dopamine (DA) neuron subtypes modulate specific physiological functions and are involved in distinct neurological disorders. Embryonic stem cell (ESC) derived DA neurons have the potential to aid in the study of disease mechanisms, drug discovery, and possibly cell replacement therapies. DA neurons can be generated from ESCs in vitro, but the subtypes of ESC-derived DA neurons have not been investigated in detail despite the diversity of DA neurons observed in vivo. Due to cell culture heterogeneity, sampling methods applied to ESC-derived cultures can be ambiguous and potentially biased. Therefore, we developed a quantification method to capture the depth of DA neuron production in vitro by estimating the error associated with systematic random sampling. Using this method, we quantified calbindin+ and calretinin+ subtypes of DA neurons generated from mouse ESCs. We found a higher production of the calbindin+ subtype (11-27%) compared to the calretinin+ subtype (2-13%) of DA neuron; in addition, DA neurons expressing neither subtype marker were also generated. We then examined whether exogenous sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) affected subtype generation. Our results demonstrate that exogenous SHH and FGF8 did not alter DA neuron subtype generation in vitro. These findings suggest that a deeper understanding DA neuron derivation inclusive of mechanisms that govern the in vitro subtype specification of ESC-derived DA neurons is required.\n\nNoteAll research was planned and conducted while members were at Brown University\n\nResearch fundingNIH/NCRR/NIGMS RI Hospital COBRE Center for Stem Cell Biology (8P20GM103468-04) (MZ) Brown Institute for Brain Science Pilot Grant (4-63662) (MZ/DHK)

neuroscience

Genome-wide analysis of phased nucleosomal arrays reveals the functional characteristic of the nucleosome remodeler ACF

Regular successions of positioned nucleosomes - phased nucleosome arrays (PNAs) - are predominantly known from transcriptional start sites (TSS). It is unclear whether PNAs occur elsewhere in the genome. To generate a comprehensive inventory of PNAs for Drosophila, we applied spectral analysis to nucleosome maps and identified thousands of PNAs throughout the genome. About half of them are not near TSS and strongly enriched for a novel sequence motif. Through genome-wide reconstitution of physiological chromatin in Drosophila embryo extracts we uncovered the molecular basis of PNA formation. We identified Phaser, an unstudied zinc finger protein that positions nucleosomes flanking the new motif. It also revealed how the global activity of the chromatin remodeler CHRAC/ACF, together with local barrier elements, generates islands of regular phasing throughout the genome. Our work demonstrates the potential of chromatin assembly by embryo extracts as a powerful tool to reconstitute chromatin features on a global scale in vitro.

molecular biology

Critical minimum temperature limits xylogenesis and maintains treelines on the Tibetan Plateau

Physiological and ecological mechanisms that define treelines are still debated. It is suggested that the absence of trees above the treeline is caused by the low temperature that limits growth. Thus, we raise the hypothesis that there is a critical minimum temperature (CTmin) preventing xylogenesis at treeline. We tested this hypothesis by examining weekly xylogenesis across three and four growing seasons in two natural Smith fir (Abies georgei var. smithii) treeline sites on the south-eastern Tibetan Plateau. Despite differences in the timing of cell differentiation among years, minimum air temperature was the dominant climatic variable associated with xylem growth; the critical minimum temperature (CTmin) for the onset and end of xylogenesis occurred at 0.7{+/-}0.4 {degrees}C. A process-based-modeled chronology of tree-ring formation using this CTmin was consistent with actual tree-ring data. This extremely low CTmin permits Smith fir growing at treeline to complete annual xylem production and maturation and provides both support and a mechanism for treeline formation.

ecology

Incorporating the Rate of Transcriptional Change Improves Construction of Transcription Factor Regulatory Networks

Transcriptional regulatory networks (TRNs) can be developed by computational approaches that infer regulator-target gene interactions from transcriptional assays. Successful algorithms that generate predictive, accurate TRNs enable the identification of regulator-target relationships in conditions where experimentally determining regulatory interactions is a challenge. Improving the ability of TRNs to successfully predict known regulator-target relationships in model species will enhance confidence in applying these approaches to determine regulator-target interactions in non-model species where experimental validation is challenging. Many transcriptional profiling experiments are performed across multiple time points; therefore we sought to improve regulator-target predictions by adjusting how time is incorporated into the network. We created ExRANGES, which incorporates Expression in a Rate-Normalized GEne Specific manner that adjusts how expression data is provided to the network algorithm. We tested this on a two different network construction approaches and found that ExRANGES prioritizes targets differently than traditional expression and improves the ability of these networks to accurately predict known regulator targets. ExRANGES improved the ability to correctly identify targets of transcription factors in large data sets in four different model systems: mouse, human, Arabidopsis, and yeast. Finally, we examined the performance of ExRANGES on a small data set from field-grown Oryza sativa and found that it also improved the ability to identify known targets even with a limited data set.\n\nAuthor SummaryTo understand how organisms can turn a collection of genes into a physiological response, we need to understand how certain genes are turned on and off. In model organisms, the ability to identify direct targets of transcription factors via ChIP-Seq in a high-throughput manner has advanced our understanding of transcriptional regulatory networks and how organisms regulate gene expression. However, for non-model organisms, it remains a challenge to identify TF-target relationships through experimental approaches such as ChIP-Seq. Without this information, the ability to understand regulatory control is limited. Computational approaches to identify regulator-target relationships in silico from easily attainable transcriptional data offer a solution. Several approaches exist for identifying gene regulatory networks, including many that take advantage of time series data. Most of these approaches weigh the relationship between regulators and putative targets at all time points equally. However, many regulators may control a single target in response to different inputs. In our approach, we focus on the association between regulators and targets primarily at times when there is a significant change in expression. ExRANGES essentially weights the expression value of each time point by the slope change after that time point so that relationships between regulators and targets are emphasized at the time points when the transcript levels are changing. This change in input into network identification algorithms improves the ability to predict regulator-target interactions and could be applied to many different algorithms We hope this improvement will increase the ability to identify regulators of interest in non-model species.

bioinformatics

Exosomes purified from a single cell type have diverse morphology and composition

Extracellular vesicles (EVs) are produced by all known organisms and have important roles in cell communication and physiology. Exosomes are known in the literature to be small round EVs (40 to 100 nm in diameter) and are commonly purified with a serial ultracentrifugation protocol followed by density gradient floatation. Great morphological diversity has been described before regarding EVs found in body fluids such as blood plasma, breast milk and ejaculate. However, a detailed morphological analysis has never been performed on exosomes purified from a single cell type.\n\nTherefore, the aim of this study was to analyze and quantify via multiple electron microscopy techniques the morphology of exosomes purified from the human mast cell line HMC-1. The results revealed a novel spectrum of diversity in exosomes, which suggests that subpopulations of exosomes with different and specific functions might also exist. Our findings therefore argue that a new and more efficient way of defining exosome subpopulations is necessary. A system was proposed where exosomes were classified into nine different categories according to their size and shape. Three additional morphological features could also be found in exosomes regardless of their classification.\n\nThese findings show that morphological diversity is found among exosomes purified from a single cell line, similarly to what was previously observed for EVs in body fluids. This knowledge can help improving the interpretation of experimental results and widening our general understanding of the biological functions of exosomes.

cell biology