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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

A simulation-based approach to improve decoded neurofeedback performance

The neural correlates of specific brain functions such as visual orientation tuning and individual finger movements can be revealed using multivoxel pattern analysis (MVPA) of fMRI data. Neurofeedback based on these distributed patterns of brain activity presents a unique ability for precise neuromodulation. Recent applications of this technique, known as decoded neurofeedback, have manipulated fear conditioning, visual perception, confidence judgements and facial preference. However, there has yet to be an empirical justification of the timing and data processing parameters of these experiments. Suboptimal parameter settings could impact the efficacy of neurofeedback learning and contribute to the 'non-responder' effect. The goal of this study was to investigate how design parameters of decoded neurofeedback experiments affect decoding accuracy and neurofeedback performance. Subjects participated in three fMRI sessions: two 'finger localizer' sessions to identify the fMRI patterns associated with each of the four fingers of the right hand, and one 'finger finding' neurofeedback session to assess neurofeedback performance. Using only the localizer data, we show that real-time decoding can be degraded by poor experiment timing or ROI selection. To set key parameters for the neurofeedback session, we used offline simulations of decoded neurofeedback using data from the localizer sessions to predict neurofeedback performance. We show that these predictions align with real neurofeedback performance at the group level and can also explain individual differences in neurofeedback success. Overall, this work demonstrates the usefulness of offline simulation to improve the success of real-time decoded neurofeedback experiments.

neuroscience↗

bioRxiv2018

Pervasive and Dynamic Transcription Initiation in Saccharomyces cerevisiae

Transcription initiation is finely regulated to ensure the proper expression and function of these genes. The regulated transcription initiation in response to various environmental cues in the model organism Saccharomyces cerevisiae has not been systematically investigated. In this study, we generated quantitative maps of transcription start site (TSS) at a single-nucleotide resolution for S. cerevisiae grown in nine different conditions using no-amplification non-tagging Cap analysis of gene expression (nAnT-iCAGE) sequencing. Based on 337 million uniquely mapped CAGE tags, we mapped ~1 million well-supported TSSs, suggesting highly pervasive transcription initiation in the compact genome of yeast. The comprehensive TSS maps allowed us to identify core promoters for ~96% verified protein-coding genes and to revise the predicted translation start codon for 183 genes. We found that 56% of yeast genes have at least two core promoters and alternative usage of different core promoters in a gene is widespread in response to changing environments. More importantly, most core promoter shifts are coupled with differential gene expression, indicating that core promoter shift might play an important role in controlling transcriptional activity of yeast genes. Based on their dynamic activities, we divided yeast core promoters as constitutive core promoters (55%) and inducible core promoters (45%). The two classes of core promoters exhibit distinctive patterns in transcriptional abundance, chromatin structure, promoter shape, and sequence context. In summary, the quantitative TSS maps generated by this study improved the annotation of yeast genome, and revealed a highly pervasive and dynamic nature of transcription initiation in yeast.

genomics↗

bioRxiv2018

Subthalamic nucleus mediates the modulation on cocaine self-administration induced by ultrasonic vocalizations playback in rats

Drug intake is known to be under the influence of social context. We have recently shown that presence of a peer influences drug intake in both rats and humans. Whether or not social acoustic communications between the peers play a role during cocaine or sucrose self-administration (SA) was investigated here, using playback of ultrasonic vocalizations (USV) at 50- and 22-kHz, conveying respectively positive and negative internal affective states in adult rats. To assess the neurobiological substrate of a potential USV influence on drug and food intake, we tested the effects of subthalamic nucleus (STN) lesions, given its role in emotional and motivational processes. In sham-control rats, playback of USV associated with positive affective states induced long-term decreased cocaine consumption, while USV associated with negative affective states induced short-term increase. Interestingly, no effect of USV playback was observed on sucrose intake, whatever the frequency. STN lesions abolished the influence of USV on cocaine intake, highlighting the influence of STN in emotional processes induced by USV emitted by a peer. These results show how acoustic social communication is important to regulate drug intake in rats and how STN modulation could interfere with addiction processes.

neuroscience↗

bioRxiv2018

A Versatile Rapture (RAD-Capture) Platform for Genotyping Marine Turtles

Advances in high-throughput sequencing (HTS) technologies coupled with increased interdisciplinary collaboration is rapidly expanding capacity in the scope and scale of wildlife genetic studies. While existing HTS methods can be directly applied to address some evolutionary and ecological questions, certain research goals necessitate tailoring methods to specific study organisms, such as high-throughput genotyping of the same loci that are comparable over large spatial and temporal scales. These needs are particularly common for studies of highly mobile species of conservation concern like marine turtles, where life history traits, limited financial resources and other constraints require affordable, adaptable methods for HTS genotyping to meet a variety of study goals. Here, we present a versatile marine turtle HTS targeted enrichment platform adapted from the recently developed Rapture (RAD-Capture) method specifically designed to meet these research needs. Our results demonstrate consistent enrichment of targeted regions throughout the genome and discovery of candidate variants in all species examined for use in various conservation genetics applications. Accurate species identification confirmed the ability of our platform to genotype over 1,000 multiplexed samples, and identified areas for future methodological improvement such as optimization for low initial concentration samples. Finally, analyses within green turtles supported the ability of this platform to identify informative SNPs for stock structure, population assignment and other applications over a broad geographic range of interest to management. This platform provides an additional tool for marine turtle genetic studies and broadens capacity for future large-scale initiatives such as collaborative global marine turtle genetic databases.

genomics↗

bioRxiv2018

Choice between 1- and 2-furrow cytokinesis in Caenorhabditis elegans embryos with tripolar spindles

Excess numbers of centrosomes often lead to multipolar spindles, and thus probably to multipolar mitosis and aneuploidy. In Caenorhabditis elegans, approximately 70% of the paternal emb-27APC6 mutant embryonic cells contained more than 2 centrosomes and formed multipolar spindles. However, only 30% of the cells with tripolar spindles formed 2 cytokinetic furrows. The rest formed 1 furrow, like normal cells. To investigate the mechanism how the cells avoided to form 2 cytokinetic furrows even with a tripolar spindle, we conducted live-cell imaging in emb-27APC6 mutant cells. We found that the chromatids were aligned only on 2 of the 3 sides of the tripolar spindle, and the angle of the tripolar spindle relative to the long axis of the cell correlated with the number of cytokinetic furrow. Our numerical modeling showed that the combination of cell shape, cortical pulling forces, and heterogeneity of centrosome size determines whether cells with tripolar spindle form 1 or 2 cytokinetic furrows.

cell biology↗

bioRxiv2018

Drosophila scribble mutant tumors undergo a transition from a growth arrest state to a proliferative state over time

The Drosophila neoplastic tumor suppressor gene (nTSG) mutant tumors have successfully modeled many aspects of human tumor progression. However, the fly nTSG mutant tumors progress rapidly over days. This is in contrast with most human tumors which develop slowly, harbor heterogeneous cell populations for selection and undergo an evolution-like process. Whether the fast-growing fly nTSG mutant tumors have capacity for evolution remains unclear. Through quantitative analysis of the scrib mutant tumor growth, we found that the scrib mutant tumors evolve to display different growth rates and cell cycle profiles over time. Multiple growth-regulatory signaling pathways show quantitative differences in early versus late scrib mutant tumors. These data suggest that the scrib mutant tumors undergo a transition from a growth arrest state to a proliferative state. Through longitudinal single cell RNA (scRNA) data analysis we found that the scrib mutant tumors harbor heterogeneous cell populations likely of distinct proliferative states, which are available for potential selection. This study raises the possibility of studying tumor evolution in a genetically accessible and fast-growing invertebrate tumor model.

developmental biology↗

bioRxiv2018

Ring shape Golden Ratio multicellular structures are algebraically afforded by asymmetric mitosis and one to one cell adhesion

Golden Ratio proportions are found throughout the world of multicellular organisms but the underlying mechanisms behind their appearance and their adaptive value if any remain unknown. The Golden Ratio is a real-valued number but cell population counts are whole numbered. Binet's formula connects the Golden Ratio to the whole numbered Fibonacci sequence (fn+1 = fn + fn-1 where f1 = 1 and f2 = 2), so we seek a cellular mechanism that yields Fibonacci cell kinetics. Drawing on Fibonaccis description of growth patterns in rabbits, we develop a matrix model of Fibonacci cell kinetics based on an asymmetric pause between mitoses by daughter cells. We list candidate molecular mechanisms for asymmetric mitosis such as epigenetically asymmetric chromosomal sorting at anaphase due to cytosine-DNA methylation. A collection of Fibonacci-sized cell groups produced each by mitosis needs to assemble into a larger multicellular structure. We find that the mathematics for this assembly are afforded by a simple molecular cell surface configuration where each cell in each group has four cell to cell adhesion slots. Two slots internally cohere a cell group and two adhere to cells in other cell groups. We provide a notation for expressing each cells participation in dual Fibonacci recurrence relations. We find that single class of cell to cell adhesion molecules suffices to hold together a large assembly of chained Fibonacci groups having Golden Ratio patterns. Specialized bindings between components of various sizes are not required. Furthermore, the notation describes circumstances where chained Fibonacci-sized cell groups may leave adhesion slots unoccupied unless the chained groups anneal into a ring. This unexpected result suggests a role for Fibonacci cell kinetics in the formation of multicellular ring forms such as hollow and tubular structures. In this analysis, a complex molecular pattern behind asymmetric mitosis coordinates with a simple molecular cell adhesion pattern to generate useful multicellular assemblies. Furthermore, this reductively unifies two of the hypothesized evolutionary steps: multicellularity and cellular eusociality.

systems biology↗

bioRxiv2018

On-ratio PDMS bonding for multilayer microfluidic device fabrication

Integrated elastomeric valves, also referred to as Quake valves, enable precise control and manipulation of fluid within microfluidic devices. Fabrication of such valves requires bonding of multiple layers of the silicone polymer polydimethylsiloxane (PDMS). The conventional method for PDMS-PDMS bonding is to use varied base to crosslinking agent ratios between layers, typically 20:1 and 5:1. This bonding technique, known as \"off-ratio bonding,\" provides strong, effective PDMS-PDMS bonding for multi-layer soft-lithography, but it can yield adverse PDMS material properties and can be wasteful of PDMS. Here we demonstrate the effectiveness of on-ratio PDMS bonding for multilayer soft lithography. We show the efficacy of this technique among common variants of PDMS: Sylgard 184, RTV 615, and Sylgard 182.

bioengineering↗

bioRxiv2018

Comparative mode of action of antimicrobial peptide melimine and its derivative Mel4 against Pseudomonas aeruginosa

Melimine and Mel4 are chimeric cationic peptides with broad spectrum antimicrobial activity, and recent investigations have shown that they are highly biocompatible with animal model and human clinical trials. The current study examined the mechanism of action of these two antimicrobial peptides against P. aeruginosa with a series of investigations. Antimicrobial activities were determined by MIC and MBC. Endotoxin neutralization was determined using the LAL assay, effect on the cytoplasmic membrane was evaluated using DiSC(3)-5 and Sytox green stains, and Syto-9 and PI dyes using flow cytometry. Release of cytoplasmic materials (ATP and DNA/RNA) were determined using ATP luminescence and increase in OD260nm. The ability to lyse bacteria was studied by measuring a decrease in OD620nm. The MIC of the peptides remained low against P. aeruginosa strains, which showed efficient neutralization of LPS, indicating their role in the anti-pseudomonas and LPS binding activities. Both AMPs rapidly (starting at 30 seconds) depolarized P. aeruginosa cytoplasmic membrane leading to reduction in viability. Melimine was responsible for more ATP release (75%) compared to Mel4 (36%) (P<0.001) following two minutes exposure. For both peptides, Sytox green entered cells after five minutes of incubation. Flow cytometry demonstrated that both the AMPs permeabilized the cell membrane at 30 minutes and followed by increasing permeability. Similar results were found with DNA/RNA release experiments. Overall, melimine showed higher ability of membrane disruption, cell lysis compared to Mel4 (P<0.001). Knowledge regarding mechanism of action of these two AMPs would be helpful in making them as anti-pseudomonas drug.

microbiology↗

bioRxiv2018

Sonodynamic therapy as an adjunctivw treatment on porphyromonas gingivalis induced periodontitis in rats with diabetes

ObjectivesThe purpose of our research was to examine the effects of Minocycline combined hyaluronic acid (HA)-mediated Ultrasound therapy of infected wound in wister rats.\n\nMethods40 female wister rats were made wound on the two side of the backbone, then infected in Staphylococcus aureus at the comic for three times. then, they are divided into four groups: control group, minocycline combined HA alone, ultasound alone, minocycline combined HA-mediated ultasound group, respective. After 3 times of treatments, the rats were killed and made into specimens. Assessments consisted of visual inspection in the change of the skin, scar formation pathological morphology by hematoxylin and eosin(HE) stain with optical microscopy, IL-1B assaying and TNF-a were performed.\n\nResultCompared with control group, minocycline combined HA alone, ultasound alone, minocycline combined HA-mediated ultasound group all have effect for wound healing, there was a obvious improvement in all parameters over the duration of the experiment(P<0.05). Compared with the control group, minocycline combined HA-mediated ultasound group indicated less inflammation cells (P<0.001) and the reduce of and IL-1B and TNF-a (P<0.001).\n\nConclusionMinocycline combined HA-mediated ultrasound can accelerate tissue regrowth, which exert significant benefits in healing the wounds.

microbiology↗

bioRxiv2018

Factors mediating synergism between vancomycin and chlorhexidine in VanA-type vancomycin-resistant Enterococcus faecium

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens that cause life-threatening infections. To control hospital-associated infections, skin antisepsis and bathing utilizing chlorhexidine is recommended for VRE patients in acute care hospitals. Previously, we reported that exposure to inhibitory chlorhexidine levels induced the expression of vancomycin resistance genes in VanA-type Enterococcus faecium. However, vancomycin susceptibility actually increased for VanA-type E. faecium in the presence of chlorhexidine. Hence, a synergistic effect of the two antimicrobials was observed. In this study, we used multiple approaches to investigate the mechanism of synergism between chlorhexidine and vancomycin in the VanA-type VRE strain E. faecium 1,231,410. We generated clean deletions of 7 of 11 pbp, transpeptidase, and carboxypeptidase genes in this strain (ponA, pbpF, pbpZ, pbpA, ddcP, ldtfm, and vanY). Deletion of ddcP, encoding a membrane-bound carboxypeptidase, altered the synergism phenotype. Furthermore, using in vitro evolution, we isolated a spontaneous synergy escaper mutant and utilized whole genome sequencing to determine that a mutation in pstB, encoding an ATPase of phosphate-specific transporters, also altered synergism. Finally, addition of excess D-lactate, but not D-alanine, enhanced synergism. Overall, our work identified factors that alter chlorhexidine-induced vancomycin resensitization in a model VanA-type VRE strain.

microbiology↗

bioRxiv2018

Werner helicase control of human papillomavirus 16 E1-E2 DNA replication is regulated by SIRT1 deacetylation

Human papillomaviruses (HPV) are double stranded DNA viruses causative in a host of human diseases including several cancers. Following infection two viral proteins, E1 and E2, activate viral replication in association with cellular factors, and stimulate the DNA damage response (DDR) during the replication process. E1-E2 uses homologous replication (HR) to facilitate DNA replication, but an understanding of host factors involved in this process remains incomplete. Previously we demonstrated that the class III deacetylase SIRT1, which can regulate HR, is recruited to E1-E2 replicating DNA and regulates the level of replication. Here we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-E2 replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of WRN, but a reduced ability to interact with E1-E2 replicating DNA. We present a model in which E1-E2 replication turns on the DDR stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN interaction with E1-E2 replicating DNA to control the quantity and fidelity of replication. As well as offering a crucial insight into HPV replication control, this system offers a unique model for investigating the link between SIRT1 and WRN in controlling replication in mammalian cells.\n\nImportanceHPV16 is the major viral human carcinogen, responsible for between 3 and 4% of all cancers worldwide. Following infection this virus activates the DNA damage response (DDR) to promote its life cycle, and recruits DDR proteins to its replicating DNA in order to facilitate homologous recombination during replication. This promotes the production of viable viral progeny. Our understanding of how HPV16 replication interacts with the DDR remains incomplete. Here we demonstrate that the cellular deacetylase SIRT1, which is a part of the E1-E2 replication complex, regulates recruitment of the DNA repair protein WRN to the replicating DNA. We demonstrate that WRN regulates the level and fidelity of E1-E2 replication. Overall the results suggest a mechanism where SIRT1 deacetylation of WRN promotes its interaction with E1-E2 replicating DNA to control the levels and fidelity of that replication.

microbiology↗

bioRxiv2018

Latent developmental potential to form limb-like skeletal structures in zebrafish

The evolution of fins into limbs was a key transition in vertebrate history. A hallmark of this transition is the addition of multiple long bones to the proximal-distal axis of paired appendages. Whereas limb skeletons are often elaborate and diverse, teleost pectoral fins retain a simple endoskeleton. Fins and limbs share many core developmental processes, but how these programs were reshaped to produce limbs from fins during evolution remains enigmatic. Here we identify zebrafish mutants that form supernumerary long bones along the proximal-distal axis of pectoral fins with limb-like patterning. These new skeletal elements are integrated into the fin, as they are connected to the musculature, form joints, and articulate with neighboring bones. This phenotype is caused by activating mutations in previously unrecognized regulators of appendage development, vav2 and waslb, which we show function in a common pathway. We find that this pathway functions in appendage development across vertebrates, and loss of Wasl in developing limbs results in patterning defects identical to those seen in Hoxall knockout mice. Concordantly, formation of supernumerary fin long bones requires the function of hoxall paralogs, indicating developmental homology with the forearm and the existence of a latent functional Hox code patterning the fin endoskeleton. Our findings reveal an inherent limb-like patterning ability in fins that can be activated by simple genetic perturbation, resulting in the elaboration of the endoskeleton.

evolutionary biology↗

bioRxiv2018

Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artefact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artefacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

cell biology↗

bioRxiv2018

Emotional Mirrors in the Rat’s Anterior Cingulate Cortex

AbstractHow do the emotions of others affect us? The human anterior cingulate cortex (ACC) responds while experiencing pain in the self and witnessing pain in others, but underlying cellular mechanisms remain poorly understood. Here we show the ACC (area 24) contains neurons responding when a rat experiences pain and while witnessing another receive footshocks. Most of these do not respond to a fear conditioned tone (CS). Deactivating this region reduces freezing while witnessing footshocks to others but not while hearing the CS. A decoder trained on spike counts while witnessing footshocks can decode the animals own pain intensity when experiencing pain. Mirror-like neurons thus exist in ACC that encode the pain of others in a code shared with pain but not fear in the self.\n\nOne Sentence SummaryACC contains neurons responding selectively when a rat witnesses anothers pain and experiences pain in the self.

neuroscience↗

bioRxiv2018

Influence of nutritional tyrosine on cognition and functional connectivity in healthy old humans

Tyrosine is precursor for monoamine neurotransmitters such as dopamine (DA), which is one of the key neurotransmitters in the frontostriatal network and of crucial relevance for mental disorders. Recent research reported that high dose tyrosine application resulted in increased brain DA synthesis, which is consistent with the observation of positive associations between daily tyrosine intake and cognitive test performance. In the present study, we investigated the associations between working memory (WM) dependent tasks and self-reported nutritional tyrosine intake within a large group of healthy elderly humans (286 subjects) by additionally including brain functional data. We observed a negative correlation between tyrosine intake and resting-state functional connectivity (rsFC) between the striatum (putamen) and the prefrontal cortex. That is to say, we found higher rsFC in individuals consuming less tyrosine per day. At the same time, this increased rsFC or hyperconnectivity was associated with lower WM performance. These findings suggest that lower or insufficient supply of tyrosine might result in dysfunctional connectivity between striatal and frontal regions leading to lower WM capacity in healthy elderly humans.

neuroscience↗

bioRxiv2018

Cellular polarity asymmetrically functionalizes pathogen recognition receptor-mediated intrinsic immune response in human intestinal epithelium cells

Intestinal epithelial cells (IECs) act as a physical barrier separating the commensal-containing intestinal tract from the sterile interior. These cells have found a complex balance allowing them to be prepared for pathogen attacks while still tolerating the presence of bacteria/viral stimuli present in the lumen of the gut. Using primary human IECs, we probed the mechanisms, which allow for such a tolerance. We discovered that viral infection emanating from the basolateral side of IECs elicited a stronger intrinsic immune response as compared to lumenal apical infection. We determined that this asymmetric immune response was driven by the clathrin-sorting adapter AP-1B which mediates the polarized sorting of Toll-like receptor 3 (TLR3) toward the basolateral side of IECs. Mice and human IECs lacking AP-1B showed an exacerbated immune response following apical stimulation. Together these results suggest a model where the cellular polarity program plays an integral role in the ability of IECs to partially tolerate apical commensals while remaining fully responsive against invasive basolateral pathogens.

immunology↗

bioRxiv2018

Re-purposing Ac/Ds transgenic system for CRISPR/dCas9 modulation of enhancers and non-coding RNAs in zebrafish

Due to its genetic amenability coupled with recent advances in genome editing, the zebrafish serves as an excellent model to examine the function of both coding and non-coding elements. Recently, the non-coding genome has gained prominence due to its critical role in development and disease. Here, we have re-purposed the Ac/Ds maize transposition system to reliably screen and efficiently characterise zebrafish enhancers, with or without germline propagation. We further utilised the system to stably express guide RNAs in microinjected embryos enabling tissue-specific CRISPR/dCas9-interference (CRISPRi) knockdown of lncRNA and enhancer activity without disrupting the underlying genetic sequence. Our study highlights the utility of Ac/Ds transposition for transient epigenome modulation of non-coding elements in zebrafish.\n\nSummary statementWe adapted the Ac/Ds transposition system, which enables continuous expression of guide RNAs for CRISPR/dCas9 perturbation, to examine the function of non-coding RNAs and enhancer elements in zebrafish.

developmental biology↗