title: Scattering-driven PPG signal model

abstract: This article discusses the origin of photoplethysmographic (PPG) signals. Two plausible hypotheses that could explain the phenomenon of pulsatile optical signals are analyzed: the volumetric hypothesis in which changes in the arterial blood volume are responsible for the observed signal pulsation, and a model wherein changes in the measured signal are driven by the size fluctuations of red blood cell (RBC) aggregates. The theoretical approximation where the size of scattering particles representing RBC aggregates varies as a function of pulsatile changes in blood flow is elaborated. Within this models framework, the gamma coefficient used in pulse oximetry was calculated for the volumetric-related and aggregation-related models. Two pairs of wavelengths, (670 nm, 940 nm) and (590 nm, 940 nm), were selected to determine gamma. As a function of aggregate size, the gamma behavior was simulated for these pairs and the two hypotheses. To verify the model predictions experimentally, the PPG signals at the fingertip were measured using reflection geometry. Two combinations of light-emitting diodes with two pairs of wavelengths were utilized as light sources. To manipulate the length of aggregates in the blood, external pressure was applied to the fingertip, presumably reducing the blood flow velocity. The gamma values were determined. The derived results fully agree with the theoretical predictions of the aggregation-driven PPG signal model. In addition, using a pressure sensor, the oscillometric signal amplitude in the fingertip and the PPG signal amplitude were simultaneously measured. The comparison results of oscillometric and optical signals at elevated external pressure values are not consistent with the volumetric hypothesis. All of the foregoing experimental results strongly support the argument favoring the incorporation of the proposed aggregation mechanism into the generic PPG signal model.


title: Interface Refinement of Low-to-Medium Resolution Cryo-EM Complexes using HADDOCK2.4

abstract: A wide range of cellular processes require the formation of multimeric protein complexes. The rise of cryo-electron microscopy (cryo-EM) has enabled the structural characterization of these protein assemblies. The produced density maps can, however, still suffer from limited resolution, impeding the process of resolving structures at atomic resolution. In order to solve this issue, monomers can be fitted into low-to-medium resolution maps. Unfortunately, the produced models frequently contain atomic clashes at the protein-protein interfaces (PPIs) as intermolecular interactions are typically not considered during monomer fitting. Here, we present a refinement approach based on HADDOCK2.4 to remove intermolecular clashes and optimize PPIs. A dataset of 14 cryo-EM complexes was used to test eight protocols. The best performing protocol, consisting of a semi-flexible simulated annealing refinement with restraints on the centroids of the monomers, was able to decrease intermolecular atomic clashes by 98% without significantly deteriorating the quality of the cryo-EM density fit.

category: bioinformatics


title: Exploring cell surface-nanopillar interactions with 3D super-resolution microscopy

abstract: Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used 3D nanostructures such as nanopillars to imprint well-defined membrane curvatures (the "nano-bio interface"). In these studies, proteins and their interactions were probed by 2D fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano-bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone oil immersion objective and 3D double-helix point-spread function microscopy. We carefully optimize the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano-bio interface using 3D SR microscopy.

category: biophysics


title: PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

abstract: Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

category: cell biology


title: Leveraging Allele-Specific Expression for Therapeutic Response Gene Discovery in Glioblastoma.

abstract: Glioblastoma is the most prevalent primary malignant brain tumor in adults and is characterized by poor prognosis and universal tumor recurrence. Effective glioblastoma treatments are lacking, in part due to somatic mutations and epigenetic reprogramming that alter gene expression and confer drug resistance. Here, we interrogated allele-specific expression (ASE) in 43 patient-derived glioblastoma stem cells (GSCs) to identify recurrently dysregulated genes in glioblastoma. We identified 118 genes with recurrent ASE preferentially found in GSCs compared to normal tissues. These genes were enriched for apoptotic regulators, including Schlafen Family Member 11 (SLFN11). Loss of SLFN11 gene expression was associated with aberrant promoter methylation and conferred resistance to chemotherapy and poly ADP ribose polymerase inhibition. Conversely, low SLFN11 expression rendered GSCs susceptible to the oncolytic flavivirus Zika, which suggests a potential alternative treatment strategy for chemotherapy resistant GBMs.

category: cancer biology


title: Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

abstract: We report that the recently introduced commercial strain of V. natriegens (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 non-canonical -amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than10-fold higher than those achieved using two different genomically recoded E. coli strains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.{Delta}A.opt and C321.{Delta}A.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of mis-incorporated natural -amino acids at the UAG-programmed position, especially in cases where the ncAA is an imperfect substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve specific activity of highly desirable but imperfect ncAA substrates.

category: synthetic biology


title: Point-of-care analyte quantification and digital readout via lysate-based cell-free biosensors interfaced with personal glucose monitors

abstract:
Field-deployable diagnostics based on cell-free systems have advanced greatly, but on-site quantification of target analytes remains a challenge. Here we demonstrate that Escherichia coli lysate-based cell-free biosensors coupled to a personal glucose monitor (PGM) can enable on-site analyte quantification, with the potential for straightforward reconfigurability to diverse types of analytes. We show that analyte-responsive regulators of transcription and translation can modulate production of the reporter enzyme {beta}-galactosidase, which in turn converts lactose into glucose for PGM quantification. Because glycolysis is active in the lysate and would readily deplete converted glucose, we decoupled enzyme production and glucose conversion to increase endpoint signal output. This lysate metabolism did, however, allow for one-pot removal of glucose present in complex samples (like human serum) without confounding target quantification. Taken together, we show that integrating lysate-based cell-free biosensors with PGMs enables accessible target detection and quantification at the point of need.

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title: Gut Microbiota predicts Healthy Late-life Aging in Male Mice

abstract: Calorie restriction (CR) extends lifespan and retards age-related chronic diseases in most species. There is growing evidence that the gut microbiota has a pivotal role in host health and age-related pathological conditions. Yet, it is still unclear how CR and the gut microbiota are related to healthy aging. Here we report findings from a small longitudinal study of male C57BL/6 mice maintained on either ad libitum or mild (15%) CR diets from 21 months of age and tracked until natural death. We demonstrate that CR results in a significant reduction in frailty index (FI), a well-established indicator of aging. We observed significant alterations in bacterial load, diversity, and compositional patterns of the mouse gut microbiota during the aging process. Interrogating the FI-related microbial features using machine learning techniques, we show that gut microbial signatures from 21-month-old mice can predict the healthy aging of 30-month-old mice with reasonable accuracy. This study deepens our understanding of the links between CR, gut microbiota, and frailty in the aging process of mice.

category: microbiology


title: Why Environmental Biomarkers Work: Transcriptome-Proteome Correlations and Modeling of Multi-Stressor Experiments in the Marine Bacterium Trichodesmium

abstract: Ocean microbial communities are important contributors to the global biogeochemical reactions that sustain life on Earth. The factors controlling these communities are being increasingly explored through the use of metatranscriptomic and metaproteomic environmental biomarkers, despite ongoing uncertainty about the coherence between RNA and protein signals. Using published proteomes and transcriptomes from the abundant colony-forming cyanobacterium Trichodesmium (strain T. erythraeum IMS101) grown under varying Fe and/or P limitation and/or co-limitation in low and high CO2, we observed robust correlations of stress induced proteins and RNAs (i.e., those involved in transport and homeostasis) that can yield useful information on nutrient status under low and/or high CO2. Conversely, transcriptional and translational correlations of many other central metabolism pathways exhibit broad discordance. A cellular RNA and protein production/degradation model demonstrates how biomolecules with small initial inventories, such as environmentally responsive proteins, can achieve large increases in fold-change units, as opposed to those with higher basal expression and inventory such as metabolic systems. Microbial cells, due to their close proximity to the environment, tend to show large adaptive responses to environmental stimuli in both RNA and protein that result in transcript-protein correlations. These observations and model results demonstrate a multi-omic coherence for environmental biomarkers and provide the underlying mechanism for those observations, supporting the promise for global application in detecting responses to environmental stimuli in a changing ocean.


title: Probing interplays between human XBP1u translational arrest peptide and 80S ribosome

abstract: The ribosome stalling mechanism is a crucial biological process; yet its atomistic underpinning is still elusive. In this framework, the XBP1u translational arrest peptide (AP) plays a central role in regulating the Unfolded Protein Response (UPR) in eukaryotic cells. Here, we report multi-microseconds all atom molecular dynamics simulations designed to probe the interactions between the XBP1u AP and the mammalian ribosome exit tunnel, both for the wild type AP and for four mutant variants of different arrest potency. Enhanced sampling simulations allow investigating the AP release process of the different variants shedding light on this complex mechanism. The present outcomes are in qualitative/quantitative agreement with available experimental data. In conclusion, we provide an unprecedented atomistic picture of this biological process and clear-cut insights into the key AP-ribosome interactions.

category: molecular biology


title: Linker histone dH1K27 dimethylation marks Drosophila heterochromatin independently of H3K9 methylation

abstract: Post-translational modifications (PTMs) of histones are important epigenetic determinants and specific core histones PTMs correlate with functional chromatin states. However, despite linker histone H1s are heavily post-translationally modified, little is known about the genomic distribution of H1s PTMs and their association with epigenetic chromatin states. Here, we address this question in Drosophila that encodes a single somatic linker histone, dH1. We previously reported that dH1 is dimethylated at K27 (dH1K27me2). Here, we show that dH1K27me2 is a major PTM of Drosophila heterochromatin. At mitosis, dH1K27me2 accumulates at pericentromeric heterochromatin, while, in interphase cells, it is also detected at intercalary heterochromatin. ChIPseq experiments show that dH1K27me2 enriched regions cluster at both the assembled and unassembled heterochromatin regions of all four Drosophila chromosomes. More than 98% of the dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. We also show that dH1K27me2 is independent of H3K9 methylation, as it is equally detected in flies carrying a H3K9R mutation. Moreover, dH1K27me2 is not affected by depletion of Su(var)3-9, HP1a and Su(var)4-20. Altogether these results suggest that dH1K27me2 is a novel epigenetic mark of Drosophila heterochromatin that acts upstream of the major Su(var)3-9/HP1a pathway of heterochromatin formation.


title: Mechanistic target of rapamycin complex 1 (mTORC1) activity occurs predominantly in the periphery of human skeletal muscle fibers, in close proximity to focal adhesion complexes, following anabolic stimuli

abstract: Following anabolic stimuli (e.g. mechanical loading and/or amino acid provision) the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1 activity occurs prior to or following this translocation. We therefore aimed to determine the cellular location of mTORC1 activity in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25g/kg protein, 0.75g/kg carbohydrate) alone (n=7, 23{+/-}5yrs, 76.8{+/-}3.6kg, 13.6{+/-}3.8%BF, FED) or following a whole-body resistance exercise bout (n=7, 22{+/-}2yrs, 78.1{+/-}3.6kg, 12.2{+/-}4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300min following anabolic stimuli. The spatial regulation of mTORC1 activity was assessed through immunofluorescent staining of p-RPS6Ser240/244, an mTORC1-specific phosphorylation event. p-RPS6Ser240/244 measured by immunofluorescent staining or immunoblot was positively correlated (r=0.76, p<0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120min (~54% and ~138% respectively, p<0.05) but was greater in EXFED at both post-stimuli time points (p<0.05). The peripheral-central ratio of p-RPS6240/244 staining was displayed a similar pattern, suggesting mTORC1 activity occurs predominantly in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120min irrespective of stimulus (p=0.006) before returning to PRE at 300min. These data confirm that mTORC1 activity occurs in the region of human muscle fibers to which mTORC1 translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 activation in vivo.


title: Multiple modes of regulation control dynamic transcription patterns during the mitosis-G1 transition

abstract: Following cell division, genomes must reactivate gene expression patterns that reflect the identity of the cell. Here, we use PRO-seq to examine the mechanisms that reestablish transcription patterns after mitosis. We uncover regulation of the transcription cycle at multiple steps including initiation, promoter-proximal pause positioning and escape, poly-A site cleavage and termination during the mitotic-G1 transition. During mitosis, RNA polymerase activity is retained at initiation sites, albeit shifted in position relative to non-mitotic cells. This activity is strongly linked to maintenance of local chromatin architecture during mitosis and is more predictive of rapid gene reactivation than histone modifications previously associated with bookmarking. These molecular bookmarks, combined with sequence-specific transcription factors, direct expression of select cell growth and cell specific genes during mitosis followed by reactivation of functional gene groups with distinct kinetics after mitosis. This study details how dynamic regulation of transcription at multiple steps contributes to gene expression during the cell cycle.

category: genomics


title: Engineering 3D Vascularized Adipose Tissue Construct using a Decellularized Lung Matrix

abstract: Critically sized defects in subcutaneous white adipose tissue result in extensive disfigurement and dysfunction and remain a reconstructive challenge for surgeons; as larger defect sizes are correlated with higher rates of complications and failure due to insufficient vascularization following implantation. Our study demonstrates for the first-time a method to engineer perfusable, pre-vascularized, high-density adipose grafts that combine patient-derived adipose cells with a decellularized lung matrix (DLM). The lung is one of the most vascularized organs with high flow, low resistance, and a large blood-alveolar interface separated by a thin basement membrane. For our work, the large volume capacity within the alveolar compartment was repurposed for high-density adipose cell filling, while the acellular vascular bed provided efficient graft perfusion throughout. Both adipocytes and hASCs were successfully delivered and remained in the alveolar space even after weeks of culture. While adipose derived cells maintained their morphology and functionality in both static and perfusion DLM cultures, perfusion culture offered enhanced outcomes over static culture. Furthermore, we demonstrate that endothelial cells seamlessly integrate into the acellular vascular tree of the DLM with adipocytes. These results support that the DLM is a unique platform for creating vascularized adipose tissue grafts for large defect filling.

category: bioengineering


title: A method for three-dimensional single-cell chronic electrophysiology from developing brain organoids

abstract: Human induced pluripotent stem cell-derived brain organoids have shown great potential for studies of human brain development and neurological disorders. However, quantifying the evolution and development of electrical functions in brain organoids is currently limited by measurement techniques that cannot provide long-term stable three-dimensional (3D) bioelectrical interfaces with brain organoids during development. Here, we report a cyborg brain organoid platform, in which 2D progenitor or stem cell sheets can fold "tissue-like" stretchable mesh nanoelectronics through organogenesis, distributing stretchable electrode arrays across 3D organoids. The tissue-wide integrated stretchable electrode arrays show no interruption to neuronal differentiation, adapt to the volume and morphological changes during organogenesis, and provide long-term stable electrical contacts with neurons within brain organoids during development. The seamless and non-invasive coupling of electrodes to neurons enables a 6-month continuous recording of the same brain organoids and captures the emergence of single-cell action potentials from early-stage brain organoid development.


title: A-type lamins are critical for the recruitment of RPA and RAD51 to stalled replication forks to maintain fork stability

abstract: Lamins provide a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. The mechanisms whereby lamins regulate genome stability remain poorly understood. Here, we demonstrate a crucial role for A-type lamins preserving the integrity of the replication fork (RF) during replication stress (RS). We find that lamins bind to nascent DNA strands, especially during RS, and ensure the recruitment of fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, play crucial roles in the stabilization, remodeling and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamins depletion elicits replication fork instability (RFI) depicted by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced accumulation of DNA damage, and increased sensitivity to replication inhibitors. Importantly, in contrast to cells deficient in various homology recombination repair proteins, the RFI phenotype of lamins-depleted cells is not linked to RF reversal. This suggests that the point of entry of nucleases is not the reversed fork, but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamins-depleted cells is rescued by forced elevation of the heterotrimeric RPA complex or RAD51. These data unveil a clear involvement of structural nuclear proteins in the protection of ssDNA from the action of nucleases during RS by warranting proper recruitment of ssDNA binding proteins RPA and RAD51 to stalled RFs. In support of this model, we show physical interaction between RPA and lamins. Our study also suggests that RS is a major source of genomic instability in laminopathies and in lamins-depleted tumors.


title: Parvalbumin interneuron dendrites enhance gamma oscillations

abstract: Dendrites are important determinants of the input-output relationship of single neurons, but their role in network computations is not well understood. Here, we used a combination of dendritic patch-clamp recordings and in silico modeling to determine how dendrites of parvalbumin (PV)-expressing basket cells contribute to network oscillations in the gamma frequency band. Simultaneous soma-dendrite recordings from PV basket cells in the dentate gyrus revealed that the slope, or gain, of the dendritic input-output relationship is exceptionally low, thereby reducing the cells sensitivity to changes in its input. By simulating gamma oscillations in detailed network models, we demonstrate that the low gain is key to increase spike synchrony in PV neuron assemblies when cells are driven by spatially and temporally heterogeneous synaptic input. These results highlight the role of dendritic computations in synchronized network oscillations.

category: neuroscience


title: Evidence for widespread alterations in cortical microstructure after 32 hours of sleep deprivation

abstract: Cortical microstructure is influenced by circadian rhythm and sleep deprivation, yet the precise underpinnings of these effects remain unclear. The ratio between T1-weighted and T2-weighted magnetic resonance images (T1w/T2w ratio) has been linked to myelin levels and dendrite density and may offer novel insight into the intracortical microstructure of the sleep deprived brain. Here, we examined intracortical T1w/T2w ratio in 41 healthy young adults (26 women) before and after 32 hours of either sleep deprivation (n = 18) or a normal sleep-wake cycle (n = 23). Linear models revealed significant group differences in T1w/T2w ratio change after 32 hours in four clusters, including bilateral effects in the insular, cingulate, and superior temporal cortices, comprising regions involved in attentional, auditory and pain processing. Across clusters, the sleep deprived group showed an increased T1w/T2w ratio, while the normal sleep-wake group exhibited a reduced ratio. These changes were not explained by in-scanner head movement, and 95% of the effects across clusters remained significant after adjusting for cortical thickness and hydration. Compared with a normal sleep-wake cycle, 32 hours of sleep deprivation yields intracortical T1w/T2w ratio increases. While the intracortical changes detected by this study could reflect alterations in myelin or dendritic density, or both, histological analyses are needed to clarify the precise underlying cortical processes.


title: Norepinephrine Potentiates and Serotonin Depresses Visual Cortical Responses by Transforming Eligibility Traces

abstract: Reinforcement allows organisms to learn which stimuli predict subsequent biological relevance. Hebbian mechanisms of synaptic plasticity are insufficient to account for reinforced learning because neuromodulators signaling biological relevance are delayed with respect to the neural activity associated with the stimulus. A theoretical solution is the concept of eligibility traces (eTraces), silent synaptic processes elicited by activity which upon arrival of a neuromodulator are converted into a lasting change in synaptic strength. Previously we demonstrated in visual cortical slices the Hebbian induction of eTraces and their conversion into LTP and LTD by the retroactive action of norepinephrine and serotonin Here we show in vivo in V1 that the induction of eTraces and their conversion to LTP/D by norepinephrine and serotonin respectively potentiates and depresses visual responses. We also show that the integrity of this process is crucial for ocular dominance plasticity, a canonical model of experience-dependent plasticity.


title: Prevalence of white matter pathways coming into a single diffusion MRI voxel orientation: the bottleneck issue in tractography

abstract: Characterizing and understanding the limitations of diffusion MRI fiber tractography is a prerequisite for methodological advances and innovations which will allow these techniques to accurately map the connections of the human brain. The so-called "crossing fiber problem" has received tremendous attention and has continuously triggered the community to develop novel approaches for disentangling distinctly oriented fiber populations. Perhaps an even greater challenge occurs when multiple white matter bundles converge within a single voxel, or throughout a single brain region, and share the same parallel orientation, before diverging and continuing towards their final cortical or sub-cortical terminations. These so-called "bottleneck" regions contribute to the ill-posed nature of the tractography process, and lead to both false positive and false negative estimated connections. Yet, as opposed to the extent of crossing fibers, a thorough characterization of bottleneck regions has not been performed. The aim of this study is to quantify the prevalence of bottleneck regions. To do this, we use diffusion tractography to segment known white matter bundles of the brain, and assign each bundle to voxels they pass through and to specific orientations within those voxels (i.e. fixels). We demonstrate that bottlenecks occur in greater than 50-70% of fixels in the white matter of the human brain. We find that all projection, association, and commissural fibers contribute to, and are affected by, this phenomenon, and show that even regions traditionally considered "single fiber voxels" often contain multiple fiber populations. Together, this study shows that a majority of white matter presents bottlenecks for tractography which may lead to incorrect or erroneous estimates of brain connectivity or quantitative tractography (i.e., tractometry), and underscores the need for a paradigm shift in the process of tractography and bundle segmentation for studying the fiber pathways of the human brain.


title: A binocular synaptic network supports interocular response alignment in visual cortical neurons

abstract: In the visual system, signals from the two eyes are combined to form a coherent representation through the convergence of synaptic input populations onto individual cortical neurons. As individual synapses originate from either monocular (representing one eye) or binocular (representing both eyes) cortical networks, it has been unclear how these inputs are integrated coherently. Here, we imaged dendritic spines on layer 2/3 binocular cells in ferret visual cortex with in vivo two-photon microscopy to examine how monocular and binocular synaptic networks contribute to the interocular alignment of orientation tuning. We found that binocular synapses varied in degree of  congruency, namely response correlation between left and right eye visual stimulation. Binocular congruent inputs were functionally distinct from binocular noncongruent and monocular inputs, exhibiting greater tuning selectivity and connection specificity. Using correlative light and electron microscopy, we found no difference in ultrastructural anatomy and instead, observed strength in numbers using a simple model simulating aggregate synaptic input. This model demonstrated a predominate contribution of binocular congruent inputs in sculpting somatic orientation preference and interocular response alignment. Our study suggests that, in layer 2/3 cortical neurons, a binocular network is responsible for forming a coherent representation in individual neurons through recurrent intracortical interactions.


title: Regulatory approved monoclonal antibodies contain framework mutations predicted from human antibody repertoires

abstract: Monoclonal antibodies (mAbs) are an important class of therapeutics used to treat cancer, inflammation, and infectious diseases. Identifying highly developable mAb sequences in silico could greatly reduce the time and cost required for therapeutic mAb development. Here, we present position-specific scoring matrices (PSSMs) for antibody framework mutations developed using natural human antibody repertoire sequences. Our analysis shows that natural human antibody repertoire-based PSSMs are consistent across individuals and demonstrate high correlations between related germlines. We show that mutations in existing therapeutic antibodies can be accurately predicted solely from natural human antibody sequence data. mAbs developed using humanized mice had more human-like FR mutations than mAbs originally developed by hybridoma technology. A quantitative assessment of entire framework regions of therapeutic antibodies revealed that there may be potential for improving the properties of existing therapeutic antibodies by incorporating additional mutations of high frequency in natural human antibody repertoires. In addition, high frequency mutations in natural human antibody repertoires were predicted in silico to reduce immunogenicity in therapeutic mAbs due to the removal of T cell epitopes. Several therapeutic mAbs were identified to have common, universally high-scoring framework mutations, and molecular dynamics simulations revealed the mechanistic basis for the evolutionary selection of these mutations. Our results suggest that natural human antibody repertoires may be useful as predictive tools to guide mAb development in the future.

category: immunology


title: Distinct MUNC lncRNA structural domains regulate transcription of different promyogenic factors

abstract: Many lncRNAs have been discovered using transcriptomic data, however, it is broadly unclear what fraction of lncRNAs are functional and what structural properties affect their phenotype. MUNC lncRNA, also known as DRReRNA, stimulates skeletal muscle differentiation. The prevailing hypothesis is that MUNC stimulates the Myod1 gene in cis as an enhancer RNA and stimulates expression of several other promyogenic genes in trans by recruiting the cohesin complex to their promoters. Experimental probing of the RNA structure revealed that MUNC contains multiple structural domains not detected by RNA structure prediction algorithms in the absence of experimental information. We discovered that these specific and structurally distinct domains are required for induction of different promyogenic genes, for binding at different genomic sites to regulate the expression of adjacent genes, and for binding the cohesin complex. Moreover, we found that induction of Myod1 or interaction with cohesin comprise only a subset of the broad regulatory impact of this lncRNA. Our study thus reveals unexpectedly complex, structure-driven functions for the MUNC lncRNA and emphasizes the importance of experimentally determined structures for understanding structure-function relationships in lncRNAs.


title: Transcription Factor Activation Profiles (TFAP) identify compounds promoting differentiation of Acute Myeloid Leukemia cell lines

abstract: Repurposing of drugs for new therapeutic use has received considerable attention for its potential to reduce time and cost of drug development. Here we present a new strategy to identify chemicals that are likely to induce differentiation of leukemic cells. As Acute Myeloid Leukemia (AML) is the result of a block in myeloid differentiation, finding new drugs that are capable of inducing blast terminal maturation is considered a valuable strategy. We used data from the Connectivity Map (CMap) to identify drugs that could be repositioned for their potential to activate transcription factors that mediate myeloid differentiation. Compounds promoting the activation of transcription factors that play a positive role in myeloid differentiation were considered candidate pro-differentiation drugs. This approach yielded a list of chemicals ranked according to the potential to activate transcription factors that induce differentiation of leukemic progenitor cells. Drugs that are already used in differentiation therapy, such as for instance all-trans retinoic acid (ATRA) are in the top positions of this ranked list. To validate our strategy, we tested the in vitro differentiation potential of 22 candidate compounds using the HL-60 human cell line as a myeloid differentiation model. Ten out of 22 compounds, ranking high in the inferred list, were confirmed to induce significant differentiation of HL-60. Some of these compounds are known to trigger the DNA damage response, thus identifying this process as a target to modulate myeloid differentiation. These results underscore the potential of our approach to accelerate the drug discovery process. The method that we have developed is highly versatile and it can be adapted to different drug repurposing projects.


title: Single-cell Chromatin Accessibility and Lipid Profiling Reveals a Metabolic Shift in Adipocytes Induced by Bariatric Surgery

abstract: Obesity promotes type 2 diabetes and cardiometabolic pathologies. Vertical sleeve gastrectomy (VSG) is used to treat obesity resulting in long-term weight loss and health improvements that precede weight loss; however, the mechanisms underlying the immediate benefits remain incompletely understood. Because adipose plays a crucial role in energy homeostasis and utilization, we hypothesized that VSG exerts its influences, in part, by modulating adipose functional states. We applied single-cell ATAC sequencing and lipid profiling to inguinal and epididymal adipose depots from mice that received sham surgery or VSG. We observed depot-specific cellular composition and chromatin accessibility patterns that were altered by VSG. Specifically, accessibility at Scd1, a fatty acid desaturase, was substantially reduced after VSG in mature adipocytes of inguinal but not epididymal depots. This was accompanied by reduced accumulation of SCD1-produced unsaturated fatty acids. Given these findings and reports that reductions in Scd1 attenuate obesity and insulin resistance and that unsaturated fatty acids stimulate glucose uptake, storage, and oxidation, our results suggest VSG exerts its beneficial effects through modifications of fatty acid profiles mediated by Scd1.


title: Witnessing the Structural Evolution of an RNA Enzyme

abstract: An RNA polymerase ribozyme that has been the subject of extensive directed evolution efforts has attained the ability to synthesize complex functional RNAs, including a full-length copy of its own evolutionary ancestor. During the course of evolution, the catalytic core of the ribozyme has undergone a major structural rearrangement, resulting in a novel tertiary structural element that lies in close proximity to the active site. Through a combination of site-directed mutagenesis, structural probing, and deep sequencing analysis, the trajectory of evolution was seen to involve the progressive stabilization of the new structure, which provides the basis for improved catalytic activity of the ribozyme. Multiple paths to the new structure were explored by the evolving population, converging upon a common solution. Tertiary structural remodeling of RNA is known to occur in nature, as evidenced by the phylogenetic analysis of extant organisms, but this type of structural innovation had not previously been observed in an experimental setting. Despite prior speculation that the catalytic core of the ribozyme had become trapped in a narrow local fitness optimum, the evolving population has broken through to a new fitness locale, suggesting that further improvement of polymerase activity may be achievable.

category: biochemistry


title: Assembled Cell-Decorated Collagen (AC-DC) bioprinted implants mimic musculoskeletal tissue properties and promote functional recovery

abstract:
Musculoskeletal tissue injuries, including the damage and rupture of ligaments and tendons, and volumetric muscle loss (VML), are exceptionally commonplace and often lead to permanent disability and deformation. We developed an advanced biomanufacturing platform producing cellularized collagen microfiber implants to facilitate functional repair and regeneration of musculoskeletal soft tissues. This Assembled Cell-Decorated Collagen (AC-DC) bioprinting process rapidly and reproducibly forms 3D implants using clinically relevant cells and strong, microfluidic extruded collagen fibers. Quantitative analysis showed that the directionality and distribution of cells throughout AC-DC implants mimic the cellular properties of native musculoskeletal tissue. AC-DC bioprinted implants further approximate or exceed the strength and stiffness of human tendons and ligaments and exceeded the properties of commonplace collagen hydrogels by orders of magnitude. The regenerative potential of AC-DC implants was also assessed in vivo in a rodent VML model. A critically sized muscle injury in the hindlimb was created and repaired, and limb torque generation potential was measured over 12 weeks. Both acellular and cellular implants were found to promote functional recovery compared to the unrepaired group, with AC-DC implants containing therapeutic muscle progenitor cells promoting the highest degree of recovery. Histological analysis and automated image processing of explanted muscle cross-sections revealed increased total muscle fiber count, median muscle fiber size, and increased cellularization for injuries repaired with cellularized implants. These studies introduce the tremendous potential of an advanced bioprinting method for generating tissue analogs with near-native biological and biomechanical properties with the potential to repair numerous challenging musculoskeletal injuries.

One Sentence SummaryBioprinted collagen microfiber-based implants mimic musculoskeletal tissue properties in vitro and promote functional recovery in vivo.


title: Super.Complex: A supervised machine learning pipeline for molecular complex detection in protein-interaction networks

abstract: Characterization of protein complexes, i.e. sets of proteins assembling into a single larger physical entity, is important, as such assemblies play many essential roles in cells such as gene regulation. From networks of protein-protein interactions, potential protein complexes can be identified computationally through the application of community detection methods, which flag groups of entities interacting with each other in certain patterns. Most community detection algorithms tend to be unsupervised and assume that communities are dense network subgraphs, which is not always true, as protein complexes can exhibit diverse network topologies. The few existing supervised machine learning methods are serial and can potentially be improved in terms of accuracy and scalability by using better-suited machine learning models and parallel algorithms. Here, we present Super.Complex, a distributed, supervised AutoML-based pipeline for overlapping community detection in weighted networks. We also propose three new evaluation measures for the outstanding issue of comparing sets of learned and known communities satisfactorily. Super.Complex learns a community fitness function from known communities using an AutoML method and applies this fitness function to detect new communities. A heuristic local search algorithm finds maximally scoring communities, and a parallel implementation can be run on a computer cluster for scaling to large networks. On a yeast protein-interaction network, Super.Complex outperforms 6 other supervised and 4 unsupervised methods. Application of Super.Complex to a human protein-interaction network with ~8k nodes and ~60k edges yields 1,028 protein complexes, with 234 complexes linked to SARS-CoV-2, the COVID-19 virus, with 111 uncharacterized proteins present in 103 learned complexes. Super.Complex is generalizable with the ability to improve results by incorporating domain-specific features. Learned community characteristics can also be transferred from existing applications to detect communities in a new application with no known communities. Code and interactive visualizations of learned human protein complexes are freely available at: https://sites.google.com/view/supercomplex/super-complex-v3-0.


title: DNA Mutations via Chern-Simons Current

abstract: We test the validity of a possible schematization of DNA structure and dynamics based on the Chern-Simons theory, that is a topological field theory mostly considered in the context of effective gravity theories. By means of the expectation value of the Wilson Loop, derived from this analogue gravity approach, we find the point-like curvature of genomic strings in KRAS human gene and COVID-19 sequences, correlating this curvature with the genetic mutations. The point-like curvature profile, obtained by means of the Chern-Simons currents, can be used to infer the position of the given mutations within the genetic string. Generally, mutations take place in the highest Chern-Simons current gradient locations and subsequent mutated sequences appear to have a smoother curvature than the initial ones, in agreement with a free energy minimization argument.


title: Modelling the spatial and temporal constrains of the GABAergic influence on neuronal excitability

abstract:
GABA ({gamma}-amino butyric acid) is an inhibitory neurotransmitter in the adult brain that can mediate depolarizing responses during development or after neuropathological insults. Under which conditions GABAergic membrane depolarizations are sufficient to impose excitatory effects is hard to predict, as shunting inhibition and GABAergic effects on spatiotemporal filtering of excitatory inputs must be considered. To evaluate at which reversal potential a net excitatory effect was imposed by GABA (EGABAThr), we performed a detailed in-silico study using simple neuronal topologies and distinct spatiotemporal relations between GABAergic and glutamatergic inputs.

These simulations revealed for GABAergic synapses located at the soma an EGABAThr close to action potential threshold (EAPThr), while with increasing dendritic distance EGABAThr shifted to positive values. The impact of GABA on AMPA-mediated inputs revealed a complex temporal and spatial dependency. EGABAThr depends on the temporal relation between GABA and AMPA inputs, with a striking negative shift in EGABAThr for AMPA inputs appearing after the GABA input. The spatial dependency between GABA and AMPA inputs revealed a complex profile, with EGABAThr being shifted to values negative to EAPThr for AMPA synapses located proximally to the GABA input, while for distally located AMPA synapses the dendritic distance had only a minor effect on EGABAThr. For tonic GABAergic conductances EGABAThr was negative to EAPThr over a wide range of gGABAtonic values. In summary, these results demonstrate that for several physiologically relevant situations EGABAThr is negative to EAPThr, suggesting that depolarizing GABAergic responses can mediate excitatory effects even if EGABA did not reach EAPThr.

Author summaryThe neurotransmitter GABA mediates an inhibitory action in the mature brain, while it was found that GABA provokes depolarizations in the immature brain of after neurological insults. It is, however, not clear to which extend these GABAergic depolarizations con contribute to an excitatory effect. In the present manuscript we approached this question with a computational model of a simplified neurons to determine which amount of a GABAergic depolarizing effect, which we quantified by the so called GABA reversal potential (EGABA), was required to turn GABAergic inhibition to excitation. The results of our simulations revealed that if GABA was applied alone a GABAergic excitation was induced when EGABA was around the action potential threshold. When GABA was applied together with additional excitatory inputs, which is the physiological situation in the brain, only for spatially and temporally correlated inputs EGABA was close to the action potential threshold. For situations in which the additional excitatory inputs appear after the GABA input or are distant to the GABA input, an excitatory effect of GABA could be observed already at EGABA substantially negative to the action potential threshold. This results indicate that even slightly depolarizing GABA responses, which may be induced during or after neurological insults, can potentially turn GABAergic inhibition into GABAergic excitation.


title: The C-terminal PARP domain of the long ZAP isoform contributes essential effector functions for CpG-directed antiviral activity

abstract:
The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNA and, through the recruitment of TRIM25, KHNYN and other cellular RNA degradation machinery, target them for degradation or prevent their translation. ZAPs activity requires the N-terminal RNA binding domain that selectively binds CpG-containing RNA. However, much less is known about the functional contribution of the remaining domains. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain of the long ZAP isoform (ZAP-L) is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the inactive catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks CpG-dependent antiviral activity despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-Ls CpG-directed antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in the ZAP-L-mediated antiviral activity.

Author summaryCell-intrinsic antiviral factors, such as the zinc-finger antiviral protein (ZAP), provide a first line of defence against viral pathogens. ZAP acts by selectively binding CpG dinucleotide-rich RNAs, which are more common in some viruses than their vertebrate hosts, leading to their degradation. Here, we show that the ability to target these foreign elements is not only dependent on ZAPs N-terminal RNA-binding domain, but additional determinants in the central and C-terminal regions also regulate this process. The PARP domain and its associated CaaX box, are crucial for ZAPs CpG-specific activity and required for optimal binding to cofactors TRIM25 and KHNYN. Furthermore, a CaaX box, known to mediate post-translational modification by a hydrophobic S-farnesyl group, caused re-localization of ZAP from the cytoplasm and increased its association with intracellular membranes. This change in ZAPs distribution was essential for inhibition of both a ZAP-sensitized HIV-1 and SARS-CoV-2. Our work unveils how the determinants outside the CpG RNA-binding domain assist ZAPs antiviral activity and highlights the role of S-farnesylation and membrane association in this process.


title: Structurally conserved domains between flavivirus and alphavirus fusion glycoproteins contribute to replication in mammals and infectious virion production.

abstract:
Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants including an envelope glycoprotein variant in {beta}-strand c (V114M) of domain II. We have previously shown that the analogous {beta}-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E {beta}-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice, and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that structurally analogous residues in the alphavirus and flavivirus class II fusion proteins contribute to virus infection in vivo and highlight these shared domains as targets for broad-spectrum arbovirus therapies.

Author SummaryArboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge on the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.


title: Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV 1/SIVcpz lentiviral lineage

abstract:
The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy - termed the "R-clamp" - that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.

AUTHOR SUMMARYViral replication depends on interactions with a plethora of host cell proteins. Cellular protein interactions are typically mediated by specialized binding modules, such as the SH3 domain. To gain access to host cell regulation viruses have evolved to contain SH3 domain binding sites in their proteins, a notable example of which is the HIV-1 Nef protein. Here we show that during the primate lentivirus evolution the structural strategy that underlies the avid binding of Nef to cellular SH3 domains, which we have dubbed the R-clamp, has been generated via alternative but functionally interchangeable molecular designs. These patterns of SH3 recognition depend on the amino acid combinations at the positions corresponding to residues 83 and 120 in the consensus HIV-1 Nef sequence, and are distinctly different in Nef proteins from SIVs of Eastern and Central chimpanzees, gorillas, and the four groups of HIV-1 that have independently originated from the latter two. These results highlight the evolutionary plasticity of viral proteins, and have implications on therapeutic development aiming to interfere with SH3 binding of Nef.


title: A Hypoxia Sensitive to Hypoxia Resistant Transformation in Long-Lived Germline Mutants

abstract:
Signals from the germline play a significant role in determining longevity in numerous animal models. In C. elegans, ablation of the germline leads to long life span and various other types of stress resistance. It has been reported that mutations that block oogenesis or an upstream step in germline development confer strong resistance to hypoxia. We report here that the hypoxia resistance of sterile mutants is dependent on developmental stage and age. In just a 12-hour period, sterile animals transform from hypoxia sensitive L4 larvae into highly hypoxia resistant adults. Since this transformation occurs in animals with no germline, the physiological programs that determine hypoxia sensitivity must occur independently of germline signals and instead rely on developmental signals from somatic tissues. Furthermore, we found two distinct mechanisms of hypoxia resistance in long-lived germline deficient animals. First, a DAF-16/FoxO independent mechanism that occurs in all hypoxia resistant sterile adults and, second, a DAF-16/FoxO dependent mechanism that confers an added layer of resistance, or "super-resistance", to animals with no germline as they age past day 1 of adulthood. RNAseq data showed that nearly all genes involved in both cytosolic and mitochondrial protein translation, as well as in mitochondrial protein import, are repressed in germline deficient adults and further repressed as they age. The hypoxia super-resistance of aging germline deficient animals was suppressed by dual mutation of ncl-1 and larp-1, two regulators of nucleolar biology and protein translation, demonstrating that the hypoxia super-resistance mechanism involves reduced protein translation. These studies provide novel insight into a profound physiological transformation that takes place in germline mutants during development, showing that some of the unique physiological properties of these long-lived animals are dependent on developmental repression of genes involved in protein translation, which operate independently of germline signals.

AUTHOR SUMMARYIn addition to being extremely long lived, germline deficient animals have other extraordinary properties, such as robust resistance to oxygen deprivation. Here we provide new insight into the mechanisms of hypoxia resistance in germline deficient animals. We demonstrate that, in just a 12-hour period, germline mutants transform from hypoxia sensitive larvae into highly hypoxia resistant adults. Therefore, hypoxia resistance is not a general property of germline ablated animals, but is instead "switched on" only in adult animals. We have found two distinct mechanisms of hypoxia resistance in germline deficient animals and both mechanisms are mediated by signals from somatic tissues and do not require the germline. We have determined that reduced transcription of genes involved in protein translation is one of the mechanisms of hypoxia resistance. Like hypoxia resistance, repression of protein translation genes only occurs in adults. Our findings establish that the unique physiological properties of germline-deficient animals are "switched" on in adults and therefore must be mediated by developmental signals from somatic tissues. We conclude that the L4/adult developmental switch in germline ablated animals presents an excellent system for investigating the longevity and hypoxia resistance of germline deficient animals.

category: genetics


title: Sequential sampling from memory underlies action selection during abstract decision making

abstract: The study of perceptual decision making in monkeys has provided insights into the process by which sensory evidence is integrated towards a decision. When monkeys make decisions with the knowledge of the motor actions the decisions bear upon, the process of evidence integration is instantiated by neurons involved in the selection of said actions. It is less clear how monkeys make decisions when unaware of the actions required to communicate their choice--what we refer to as  abstract decisions. We investigated this by training monkeys to associate the direction of motion of a noisy random-dot display with the color of two targets. Crucially, the targets were displayed at unpredictable locations after the motion stimulus was extinguished. We found that monkeys postponed decision formation until the targets were revealed. Neurons in the parietal association area LIP represented the integration of evidence leading to a choice, but as the stimulus was no longer visible, the samples of evidence must have been retrieved from short-term memory. Our results imply that when decisions are temporally unyoked from the motor actions they bear upon, decision formation is protracted until they can be framed in terms of motor actions.


title: Rapid Ensemble Measurement of Protein Diffusion, Probe Blinking and Photobleaching Dynamics in the Complex Cellular Space

abstract: We present a fluorescence fluctuation image correlation analysis method that can rapidly and simultaneously measure the diffusion coefficient, photoblinking rates, and fraction of diffusing particles of fluorescent molecules in cells. Unlike other image correlation techniques, we demonstrated that our method could be applied irrespective of a non-uniformly distributed, immobile blinking fluorophore population. This allows us to measure blinking and transport dynamics in complex cell morphologies, a benefit for a range of super-resolution fluorescence imaging approaches that rely on probe emission blinking. Furthermore, we showed that our technique could be applied without directly accounting for photobleaching. We successfully employed our technique on several simulations with realistic EMCCD noise and photobleaching models, as well as on Dronpa-C12 labeled beta-actin in living NIH/3T3 and HeLa cells. We found that the diffusion coefficients measured using our method were consistent with previous literature values. We further found that photoblinking rates measured in the live HeLa cells varied as expected with changing excitation power.


title: Forward genetics combined with unsupervised classifications identified zebrafish mutants affecting biliary system formation.

abstract: Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested that these 24 mutants affect 24 different genes. We applied unsupervised clustering algorithms to classify the recovered mutants into three classes unbiasedly. Further computational analyses revealed that each of the recovered mutations in these three classes shows a unique effect on node subtype composition and connection property distribution of the intrahepatic biliary network. Besides, we found that most recovered mutations are viable. In those mutant fish, biliary network phenotypes persist into adulthood, which themselves are good animal models to study chronic cholestatic liver diseases. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases.


title: A multimodal iPSC platform for cystic fibrosis drug testing

abstract: Cystic fibrosis (CF) is a monogenic lung disease caused by dysfunction of the cystic fibrosis transmembrane regulator (CFTR) anion channel, resulting in significant morbidity and mortality. The progress in elucidating the role of the CFTR channel using established animal and cell-based models led to the recent discovery of effective CFTR modulators for most individuals with CF. However, a subset of individuals with CF do not respond to these modulators and there is an urgent need to develop novel therapeutic strategies. In this study, we assembled a panel of iPSCs derived from individuals with common or rare variants representative of three distinct classes of CFTR dysfunction. To measure CFTR function in patient-specific iPSCs we adapted two established in vitro assays of CFTR function to iPSC-derived airway cells. In both a 3-D spheroid assay using forskolin-induced swelling as well as planar cultures composed of polarized mucociliary airway epithelial cells, we quantified CFTR baseline function and response to CFTR modulators and detected genotype-specific differences. Our results demonstrate the potential of the human iPSC platform as a research tool to study cystic fibrosis and in particular accelerate therapeutic development for CF caused by rare mutations.

category: developmental biology


title: A UDP-GlcNAc : βGal β1-6 GlcNAc transferase involved in bloodstream form N-glycan and procyclic form GPI anchor elaboration in Trypanosoma brucei.

abstract: Trypanosoma brucei has large carbohydrate extensions on its N-linked glycans and glycosylphosphatidylinositol (GPI) anchors in its bloodstream form (BSF) and procyclic form (PCF), respectively. The parasites glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are unknown. Here, we probe the function(s) of a putative {beta}3GT gene, TbGT10. The BSF null mutant is viable in vitro and in vivo and can differentiate into PCF, demonstrating non-essentiality. However, the absence of TbGT10 led to impaired elaboration of N-glycans and GPI anchor sidechains in BSF and PCF parasites, respectively. Glycosylation defects include reduced BSF glycoprotein binding to ricin and to monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope of lysosomal glycoprotein p67 that we show here, using synthetic glycans, consists of (-6Gal{beta}1-4GlcNAc{beta}1-)[≥] 4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase glycopeptides isolated from BSF wild-type and TbGT10 null parasites show a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. Together, these data suggest that TbGT10 encodes a UDP-GlcNAc : {beta}Gal {beta}1-6 GlcNAc-transferase active in both BSF and PCF life-cycle stages elaborating complex N-glycans and GPI sidechains, respectively. The {beta}1-6 specificity of this {beta}3GT gene product and its dual roles in N-glycan and GPI glycan elaboration are notable.


title: Mapping lignification dynamics with a combination of chemistry, data segmentation and ratiometric analysis

abstract: This article describes a new methodology for detailed mapping of the lignification capacity of plant cell walls that we have called "REPRISAL" for REPorter Ratiometrics Integrating Segmentation for Analyzing Lignification. REPRISAL consists of the combination of three separate approaches. In the first approach, H*, G* and S* monolignol chemical reporters, corresponding to p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, are used to label the growing lignin polymer in a fluorescent triple labelling strategy based on the sequential use of 3 main bioorthogonal chemical reactions. In the second step, an automatic parametric and/or artificial intelligence (AI) segmentation algorithm is developed that assigns fluorescent image pixels to 3 distinct cell wall zones corresponding to cell corners (CC), compound middle lamella (CML) and secondary cell walls (SCW). The last step corresponds to the exploitation of a ratiometric approach enabling statistical analyses of differences in monolignol reporter distribution (ratiometric method 1) and proportions (ratiometric method 2) within the different cell wall zones. In order to demonstrate the potential of REPRISAL for investigating lignin formation we firstly describe its use to map developmentally-related changes in the lignification capacity of WT Arabidopsis interfascicular fiber cells. We then show how it can be used to reveal subtle phenotypical differences in lignification by analyzing the Arabidopsis prx64 peroxidase mutant and provide further evidence for the implication of the AtPRX64 protein in floral stem lignification. Finally, we demonstrate the general applicability of REPRISAL by using it to map lignification capacity in poplar, flax and maize.

category: plant biology


title: EEG-Based Focus Estimation Using Neurable's Enten Headphones and Analytics Platform

abstract: We introduce Neurables research on focus using our recently developed Enten EEG headphones. First we quantify Entens performance on standard EEG protocols, including eyes-closed alpha rhythms, auditory evoked response and the P300 event-related potential paradigm. We show that Entens performance is on-par with established industry-standard hardware. We then introduce a series of experimental tasks designed to mimic how focus might be maintained or disrupted in a real-world office setting. We show that (A) these tasks induce behavioral changes that reflect underlying changes in focus levels and (B) our proprietary algorithm detects these changes across a large number of sessions without needing to adjust the model per participant or recording session. Through manipulation of our experimental protocol, we show that our algorithm is not dependent on gross EMG artifacts and it is driven by changes in EEG. Finally, we evaluated the models performance on the same subject across several days, and show that performance remained consistent over time. Our model correctly captured 80% {+/-} 4.1% of distractions present in our experiments with statistical significance. This indicates that our model generalizes across subjects, time points, and conditions. Our findings are based on EEG data collected from 132 participants across 337 sessions and 45 different experiments.


title: Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins

abstract: Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity and detect phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or {beta}2 glycoprotein I have been shown to be independent risk factors for vascular thrombosis and pregnancy morbidity in APS. aPL production is thought to be triggered by - among other factors - viral infections, though infection-associated aPL have mostly been considered non-pathogenic. Recently, the potential pathogenicity of infection-associated aPL has gained momentum since an increasing number of patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been described with coagulation abnormalities and hyperinflammation, together with the presence of aPL. Here, we present data from a multicentric, mixed-severity study including three cohorts of individuals who contracted SARS-CoV-2 as well as non-infected blood donors. We simultaneously measured 10 different criteria and non-criteria aPL (IgM and IgG) by using a line immunoassay. Further, IgG antibody response against three SARS-CoV-2 proteins was investigated using tripartite automated blood immunoassay technology. Our analyses revealed that selected non-criteria aPL were enriched concomitant to or after an infection with SARS-CoV-2. Linear mixed-effects models suggest an association of aPL to prothrombin (PT) with the strength of the antibody response against SARS-CoV-2 and that it is further influenced by SARS-CoV-2 disease severity and sex of the individuals. In conclusion, our study is the first to report an association between disease severity, anti-SARS-CoV-2 immunoreactivity and aPL against PT in patients with SARS-CoV-2.


title: Horizontal transfer and southern migration: the tale of Hydrophiinae's marine journey.

abstract: Transposable elements (TEs), also known as jumping genes, are sequences able to move or copy themselves within a genome. As TEs move throughout genomes they often act as a source of genetic novelty, hence understanding TE evolution within lineages may help in understanding environmental adaptation. Studies into the TE content of lineages of mammals such as bats have uncovered horizontal transposon transfer (HTT) into these lineages, with squamates often also containing the same TEs. Despite the repeated finding of HTT into squamates, little comparative research has examined the evolution of TEs within squamates. Here we examine a diverse family of Australo-Melanesian snakes (Hydrophiinae) to examine if the previously identified, order-wide pattern of variable TE content and activity holds true on a smaller scale. Hydrophiinae diverged from Asian elapids [~]30 Mya and have since rapidly diversified into six amphibious, [~]60 marine and [~]100 terrestrial species which fill a broad range of ecological niches. We find TE diversity and expansion differs between hydrophiines and their Asian relatives and identify multiple HTTs into Hydrophiinae, including three likely transferred into the ancestral hydrophiine from fish. These HTT events provide the first tangible evidence that Hydrophiinae reached Australia from Asia via a marine route.

category: evolutionary biology


title: Horizontal transposon transfer and its implications for the ancestral ecology of hydrophiine snakes


title: A cattle graph genome incorporating global breed diversity

abstract: Despite only 8% of cattle being found in Europe, European breeds dominate current genetic resources. This adversely impacts cattle research in other important global cattle breeds. To mitigate this issue, we have generated the first assemblies of African breeds, which have been integrated with genomic data for 294 diverse cattle into the first graph genome that incorporates global cattle diversity. We illustrate how this more representative reference assembly contains an extra 116.1Mb (4.2%) of sequence absent from the current Hereford sequence and consequently inaccessible to current studies. We further demonstrate how using this graph genome increases read mapping rates, reduces allelic biases and improves the agreement of structural variant calling with independent optical mapping data. Consequently, we present an improved, more representative, reference assembly that will improve global cattle research.


title: Single-molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA

abstract: Telomeres are essential chromosome end capping structures that safeguard the genome from dangerous DNA processing events. DNA strand invasion occurs during vital transactions at telomeres, including telomere length maintenance by the alternative lengthening of telomeres (ALT) pathway. During telomeric strand invasion, a single stranded guanine-rich (G-rich) DNA invades at a complimentary duplex telomere repeat sequence forming a displacement loop (D-loop) in which the displaced DNA consists of the same G-rich sequence as the invading single stranded DNA. Single stranded G-rich telomeric DNA readily folds into stable, compact, structures called G-quadruplexes (GQ) in vitro, and is anticipated to form within the context of a D-loop; however, evidence supporting this hypothesis is lacking. Here we report a magnetic tweezers assay that permits the controlled formation of telomeric D-loops (TDLs) within uninterrupted duplex human telomere DNA molecules of physiologically relevant lengths. Our results are consistent with a model wherein the displaced single stranded DNA of a TDL folds into a GQ. This study provides new insight into telomere structure and establishes a framework for development of novel therapeutics designed to target GQs at telomeres in cancer cells.


title: The Imaging and Molecular Annotation of Xenografts and Tumours (IMAXT) High Throughput Data and Analysis Infrastructure

abstract: With the aim of producing a 3D representation of tumours, IMAXT uses a large variety of modalities in order to acquire tumour samples and produce a map of every cell in the tumour and its host environment. With the large volume and variety of data produced in the project we develop automatic data workflows and analysis pipelines and introduce a research methodology where scientists connect to a cloud environment to perform analysis close to where data are located instead of bringing data to their local computers. Here we present the data and analysis infrastructure, discuss the unique computational challenges and describe the analysis chains developed and deployed to generate molecularly annotated tumour models. Registration is achieved by use of a novel technique involving spherical fiducial marks that are visible in all imaging modalities used within IMAXT.


title: Genome-Scale Metabolic Model Accurately Predicts Fermentation of Glucose by  Chromochloris zofingiensis

abstract: Algae have the potential to be sources of renewable fuels and chemicals. One particular strain, Chromochloris zofingiensis, is of interest due to the co-production of triacylglycerols (TAGs) and astaxanthin, a valuable nutraceutical. To aid in future engineering efforts, we have developed the first genome-scale metabolic model on C. zofingiensis, iChr1915. This model includes 1915 genes, 3413 metabolic reactions and 2652 metabolites. We performed detailed biomass composition analysis for three growth conditions: autotrophic, mixotrophic and heterotrophic and used this data to develop biomass formation equations for each growth condition. The completed model was then used to predict flux distributions for each growth condition; interestingly, for heterotrophic growth, the model predicts the excretion of fermentation products due to overflow metabolism. We confirmed this experimentally via metabolomics of spent medium and fermentation product assays. An in silico gene essentiality analysis was performed on this model, as well as a flux variability analysis to test the production capabilities of this organism. In this work, we present the first genome scale metabolic model of C. zofingiensis and demonstrate its use predicting metabolic activity in different growth conditions, setting up a foundation for future metabolic engineering studies in this organism.

category: systems biology


title: Hypothalamic effective connectivity at rest is associated with body weight and energy homeostasis

abstract: Hunger and satiety states drive eating behaviours via changes in brain function. The hypothalamus is a central component of the brain networks that regulate food intake. Animal research parsed the roles of the lateral hypothalamus (LH) and the medial hypothalamus (MH) in hunger and satiety respectively. Here, we examined how hunger and satiety change information flow between human LH and MH brain networks, and how these interactions are influenced by body mass index. Forty participants (15 overweight/obese) underwent two resting-state functional MRI scans: after overnight fasting (fasted state) and following a standardised meal (sated state). The direction and valence (excitatory/inhibitory influence) of information flow between the MH and LH was modelled using spectral dynamic causal modelling. Our results revealed two core networks interacting across homeostatic state and weight status: subcortical bidirectional connections between the LH, MH and the substantia nigra pars compacta (prSN), and cortical top-down inhibition from frontoparietal and temporal areas. During fasting relative to satiety, we found higher inhibition between the LH and prSN, whereas the prSN received greater top-down inhibition from across the cortex. Individuals with higher BMI showed that these network dynamics occur irrespective of fasted or satiety states. Our findings reveal fasting affects brain dynamics over a distributed hypothalamic-midbrain-cortical network. This network is less sensitive to state-related fluctuations among people with obesity.


title: A Multiparametric Activity Profiling Platform for Neuron Disease Phenotyping and Drug Screening

abstract: Patient stem cell-derived models enable imaging of complex disease phenotypes and the development of scalable drug discovery platforms. Current preclinical methods for assessing cellular activity do not, however, capture the full intricacies of disease-induced disturbances, and instead typically focus on a single parameter, which impairs both the understanding of disease and the discovery of effective therapeutics. Here, we describe a cloud-based image processing and analysis platform that captures the intricate activity profile revealed by GCaMP fluorescent recordings of intracellular calcium changes and enables discovery of molecules that correct 153 parameters that define the amyotrophic lateral sclerosis motor neuron disease phenotype. In a high-throughput screen we identified compounds that revert the multiparametric disease profile to that found in healthy cells, a novel and robust measure of therapeutic potential quite distinct from unidimensional screening. This platform can guide the development of therapeutics that counteract the multifaceted pathological features of diseased cellular activity.


title: Dopamine release in nucleus accumbens core during social behaviors in mice

abstract: Social behaviors are among the most important and rewarding motivational behaviors. How dopamine, a "reward" signal, releases in the nucleus accumbens (NAc) during social behaviors has become a topic of interest for decades. However, limitations in early recording methods, such as microdialysis, prevented a complete understanding of moment-to-moment dopamine responses during social behaviors. Here, we employ a genetically encoded dopamine sensor, GRABDA2h, to record dopamine activity in the NAc core in mice and find acute changes in extracellular dopamine levels during all three phases of social behaviors: approach, investigation and consummation. Dopamine release during approach phase correlates with animals motivation towards the conspecific whereas its release during consummatory phase signals the valence of the experience. Furthermore, dopamine release during sexual and aggressive behaviors shows sex differences that correlate with the potential value of those experiences. Overall, our results reveal rich and temporally precise motivation and value information encoded by NAc dopamine during social behaviors and beyond.


title: Ultrashort treatment with telacebec alone and with companion drugs in immunocompetent and immunosuppressed mouse footpad models of Buruli ulcer

abstract: The antimicrobial treatment of Mycobacterium ulcerans infection, or Buruli ulcer (BU), has a long duration and is therefore burdensome and linked to indirect costs for affected patients. The new antimycobacterial drug telacebec (Q203) has previously shown promising treatment-shortening potential in mouse models of BU. In the present study, we investigated the potential of Q203 to reduce the treatment duration further. The first experiment investigated the possibility of cure by one, three or five doses of Q203 (2 mg/kg) with or without a companion drug (bedaquiline, BDQ, clofazimine, CFZ, or clarithromycin, CLR) in immunocompetent BALB/c mice. The second experiment assessed the effect of five doses of Q203 with or without BDQ or CFZ on Mycobacterium ulcerans infection of immunocompromised SCID-beige mice with the aim to evaluate the contribution of host immunity to treatment efficacy. In BALB/c mice, a treatment duration as short as 3 days was sufficient to prevent relapse in nearly all footpads and a single dose of Q203 with or without BDQ or CFZ prevented relapse in approximately 50% of footpads. Unlike in BALB/c mice, a small percentage of SCID-beige mouse footpads were culture-positive after a treatment duration of five days, highlighting an important role of host immunity for M. ulcerans clearance. Our results confirm the marked potency and prolonged bactericidal and sterilizing effects of Q203, even in immunocompromised SCID-beige mice.


title: A novel Toxoplasma IMC sutures-associated protein regulates suture protein targeting and colocalizes with membrane trafficking machinery

abstract: The cytoskeleton of Toxoplasma gondii is composed of the inner membrane complex (IMC) and an array of underlying microtubules that provide support at the periphery of the parasite. Specific subregions of the IMC carry out distinct roles in replication, motility, and host cell invasion. Building on our previous in vivo biotinylation (BioID) experiments of the IMC, we identify here a novel protein that localizes to discrete punctae that are embedded in the parasites cytoskeleton along the IMC sutures. Gene knockout analysis shows that loss of the protein results in defects in cytoskeletal suture protein targeting, cytoskeletal integrity, parasite morphology, and host cell invasion. We then use deletion analyses to identify a domain in the N-terminus of the protein that is critical for both localization and function. Finally, we use the protein as bait for in vivo biotinylation which identifies several other proteins that colocalize in similar spot-like patterns. These putative interactors include several proteins that are implicated in membrane trafficking and are also associated with the cytoskeleton. Together, this data reveals an unexpected link between the IMC sutures and membrane trafficking elements of the parasite and suggests that the sutures punctae are likely a portal for trafficking cargo across the IMC.


title: Copy-out-paste-in transposition of a Tn6283-like integrative element assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis

abstract: The exchange of antimicrobial resistance (AMR) genes between aquaculture and terrestrial microbial populations has emerged as a serious public health concern. However, the nature of the mobile genetic elements in marine bacteria is poorly documented. To gain insight into the genetic mechanisms underlying AMR gene transfer from marine bacteria, we mated a multidrug-resistant Vibrio alfacsensis strain with an Escherichia coli strain, and then determined the complete genome sequences of the donor and transconjugant strains. Sequence analysis revealed a multidrug resistance conjugative plasmid in the donor strain, which was integrated into the chromosome of the recipient. The plasmid backbone in the transconjugant chromosome was flanked by two copies of a 7.1 kb unclassifiable integrative element harboring a {beta}-lactamase gene. The 7.1 kb element (named SE-6945) and previously reported Tn6283 share four coding sequences, two of which encode the catalytic R-H-R-Y motif of tyrosine recombinases. Polymerase chain reaction and sequencing experiments revealed that SE-6945 generates a circular copy of its one specific strand without generating an empty donor site, in contrast to the movement of integron gene cassette or ICE/IMEs discovered to date. Copy numbers and location of SE-6945 in the chromosome affected antibiotic resistance levels of the transconjugants. SE-6945/Tn6283-related integrative elements are collectively termed SEs (strand-biased circularizing integrative elements). SEs were identified in the genomes of other Vibrio species. Overall, these results suggest that in addition to conjugative plasmids, SEs are involved in the spread of AMR genes among marine bacteria.


title: Atypical integrative element with strand-biased circularization activity assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis


title: Identification of PAX6 and NFAT4 as the transcriptional regulators of lncRNA Mrhl in neuronal progenitors

abstract:
LncRNA Mrhl has been shown to be involved in regulating meiotic commitment of mouse spermatogonial progenitors and coordinating differentiation events in mouse embryonic stem cells. Here we have characterized the interplay of Mrhl with lineage-specific transcription factors during mouse neuronal lineage development. Our results demonstrate that Mrhl is predominantly expressed in the neuronal progenitor populations in mouse embryonic brains and in retinoic acid derived radial-glia like neuronal progenitor cells. Mrhl levels are significantly down regulated in postnatal brains and in maturing neurons. In neuronal progenitors, a master transcription factor, PAX6, acts to regulate the expression of Mrhl through direct physical binding at a major site in the distal promoter, located at 2.9kb usptream of the TSS of Mrhl. Furthermore, NFAT4 occupies the Mrhl proximal promoter at two sites, at 437bp and 143bp upstream of the TSS. ChIP studies reveal that PAX6 and NFAT4 interact with each other, suggesting co-regulation of lncRNA Mrhl expression in neuronal progenitors. Our studies herewith are crucial towards understanding how lncRNAs are regulated by major lineage-specific TFs towards defining specific development and differentiation events.

Summary statementTranscriptional regulation of lncRNA Mrhl by multiple lineage-specific transcription factors in neuronal progenitors highlights context-dependent regulation important for lineage specification.


title: Tissue-specific modification of cellular bioelectrical activities using the chemogenetic tool, DREADD, in zebrafish

abstract: Cellular electronic activity plays an essential role in neuronal communication. Manipulation and visualization of cellular membrane potential remain essential tasks in order to study electrical signaling in living organisms. Light-controlled optogenetic and designed chemical-controlled chemogenetic tools were developed to manipulate cellular electric activities for neuroscience research. One of the most common chemogenetic tools is DREADD (designer receptors exclusively activated by designer drugs). It has been extensively utilized due to its convenience and long-lasting effects in murine and primate models, but not in zebrafish, a leading model organism in various research fields. Here, we first establish multiple tissue-specific transgenic zebrafish lines that express two different DREADDs with a genetically encoded voltage indicator, ASAP2s. We observed voltage changes in zebrafish melanophores, epidermis, and neurons by hM4DGi or rM3DGs receptors measured by ASAP2s fluorescence intensity. Alteration to melanophore bioelectricity by DREADD generated dynamic electric signals and resulted in morphological alterations to pigment cells. We also tested a few agonists and found that the latest generation performs better than clozapine N-oxide (CNO). Collectively, our experiments demonstrate that DREADD can be utilized to manipulate cell-specific membrane potential in the zebrafish model. The availability of this tool in zebrafish will offer a new resource for a variety of bioelectricity research fields such as neuroscience, cardiology, and developmental biology.


title: ZFP541 is indispensable for pachytene progression by interacting with KCTD19 and activates meiotic gene expression in mouse spermatogenesis

abstract: Meiosis is essential for fertility in sexually reproducing species, extensive studies tried to delineate this sophisticated process. Notwithstanding, the molecules involved in meiosis have not been fully characterized. In this study, we investigate the role of zinc finger protein 541 (ZFP541) and its interacting protein potassium channel tetramerization domain containing 19 (KCTD19) in mice. We demonstrate that they are indispensable for male fertility by regulating proper pachytene progression. ZFP541 is expressed starting from leptotene to round spermatids, and KCTD19 is initially expressed in pachytene. Depletion of Zfp541 or Kctd19 leads to infertility in male mice, and exhibits retarded progression from early to mid/late pachynema. In addition, Zfp541-/- spermatocytes show abnormal programmed DNA double-strand breaks (DSBs) repair, impaired crossover formation/resolution, and asynapsis of the XY chromosomes. Immunoprecipitation-mass spectrometry (IP-MS) and in vitro Co-IP reveal that ZFP541 interacts with KCTD19, histone deacetylase 1/2 (HDAC1), HDAC2 and deoxynucleotidyltransferase terminal-interacting protein 1 (DNTTIP1). Furthermore, RNA-seq and CUT&Tag analyses demonstrate that ZFP541 binds to the promoter regions of genes involved in meiosis and post-meiosis including Kctd19, and activates their transcription. Taken together, our studies reveal a ZFP541-Kctd19 transcription regulatory axis and the crucial role of ZFP541 and KCTD19 for pachytene progression and fertility in male mice.


title: Trash-basket epiphytes as secondary foundation species: a review of their distribution and effects on biodiversity and ecosystem functions

abstract:
BackgroundSecondary foundation species (FS) are organisms that inhabit ecosystems structurally defined by a primary foundation species, providing additional structure to habitats and communities. Trash-basket epiphytes (TBE) are secondary FS that enhance arboreal soil accumulation, providing shelter to animals, and rooting sites for plants. While their importance may vary across biomes, TBE have been overlooked as drivers of biodiversity and ecosystem functions. Here, we discuss the prevalence of TBE across biomes, their effects on biodiversity and ecosystem functions, and future research directions.

MethodsWe performed a systematic literature review of articles, books and theses and collated and synthesised information about the taxonomic distribution of TBE, their effects on ecosystem functions, and reports of plant-animal and plant-plant interactions. Then, we analysed the global distribution of TBE using a generalized linear model and summarised two studies to assess their effects on soil invertebrates.

ResultsWe identified 120 publications describing 209 species of TBE. Most TBE belong to Araceae (43%), Polypodiaceae (23%), and Orchidaceae (14%) and occur in all tropical and southern temperate forests. TBE richness peaks in the South-American Pacific mangroves, Eastern Cordillera Real, and the Napo moist forests. TBE effects on ecosystem functions include arboreal soil accumulation, water retention and temperature regulation in the canopy, and nutrient leaching through stem-flow. TBE provide shelter to species in more than 97 animal families, including from invertebrates to mammals, while 72 vascular plants have been reported to root in arboreal soil of TBE.

ConclusionsTBE are a compelling group of model organisms that can be used to study ecological processes such as facilitation cascades, niche construction, extended phenotypes, or the effects of secondary FS on biodiversity and ecosystem functioning. TBE should also be included in forest management plans to enhance the availability of microhabitats in the canopy supporting its associated flora and fauna.

category: ecology


title: nzffdr: an R package to import, clean and update data from the New Zealand Freshwater Fish Database

abstract: The New Zealand Freshwater Fish Database (NZFFD) is a repository of more than 155,000 records of freshwater fish observations from around New Zealand, maintained by the National Institute of Water and Atmospheric Research (NIWA). Records from the NZFFD can be downloaded using a web interface. The statistical computing language R is now widely used for data wrangling, analysis, and visualisation. Here, we present nzffdr, an open source R software package that: i) allows users to query and download data from the New Zealand Freshwater Fish Database directly in R, ii) provides functions to clean imported data, iii) facilitates the addition of information such as species names and Department of Conservation threat classification status, and iv) a workflow for visualising information from the NZFFD. The nzffdr package aims to standardise, simplify, and speed up a workflow likely already used in an ad hoc manner by scientists across New Zealand and abroad.


title: SIA: Selection Inference Using the Ancestral Recombination Graph

abstract: Detecting signals of selection from genomic data is a central problem in population genetics. Coupling the rich information in the ancestral recombination graph (ARG) with a powerful and scalable deep learning framework, we developed a novel method to detect and quantify positive selection: Selection Inference using the Ancestral recombination graph (SIA). Built on a Long Short-Term Memory (LSTM) architecture, a particular type of a Recurrent Neural Network (RNN), SIA can be trained to explicitly infer a full range of selection coefficients, as well as the allele frequency trajectory and time of selection onset. We benchmarked SIA extensively on simulations under a European human demographic model, and found that it performs as well or better as some of the best available methods, including state-of-the-art machine-learning and ARG-based methods. In addition, we used SIA to estimate selection coefficients at several loci associated with human phenotypes of interest. SIA detected novel signals of selection particular to the European (CEU) population at the MC1R and ABCC11 loci. In addition, it recapitulated signals of selection at the LCT locus and several pigmentation-related genes. Finally, we reanalyzed polymorphism data of a collection of recently radiated southern capuchino seedeater taxa in the genus Sporophila to quantify the strength of selection and improved the power of our previous methods to detect partial soft sweeps. Overall, SIA uses deep learning to leverage the ARG and thereby provides new insight into how selective sweeps shape genomic diversity.