title: In vivo dissection of Rhoa function in vascular development using zebrafish

abstract:
RationaleThe small monomeric GTPase RHOA acts as a master regulator of signal transduction cascades by activating effectors of cellular signaling, including the Rho-associated protein kinases ROCK1/2. Previous in vitro cell culture studies suggest that RHOA can regulate many critical aspects of vascular endothelial cell (EC) biology, including focal adhesion, stress fiber formation, and angiogenesis. However, the specific in vivo roles of RHOA during vascular development and homeostasis are still not well understood.

ObjectiveIn this study we examine the in vivo functions of RHOA in regulating vascular development and integrity in zebrafish.

Methods and ResultsWe use zebrafish RHOA-ortholog (rhoaa) mutants, transgenic embryos expressing wild type, dominant-negative, or constitutively active forms of rhoaa in ECs, and a pharmacologic inhibitor of ROCK1/2 to study the in vivo consequences of RHOA gain- and loss-of-function in the vascular endothelium. Our findings document roles for RHOA in vascular integrity, developmental angiogenesis, and vascular morphogenesis.

ConclusionsOur results indicate that either too much or too little RHOA activity leads to vascular dysfunction in vivo.

category: developmental biology


title: Insights into in vivo adipocyte differentiation through cell-specific labeling in zebrafish

abstract:
White adipose tissue hyperplasia has been shown to be crucial for handling excess energy in healthy ways. Though adipogenesis mechanisms have been underscored in vitro, we lack information on how tissue and systemic factors influence the differentiation of new adipocytes. While this could be studied in zebrafish, adipocyte identification currently relies on neutral lipid labeling, thus precluding access to cells in early stages of differentiation. Here we report the generation and analysis of a zebrafish line with the transgene fabp4(-2.7):EGFPcaax. In vivo confocal microscopy of the pancreatic and abdominal visceral depots of transgenic larvae, revealed the presence of labeled mature adipocytes as well as immature cells in earlier stages of differentiation. Through co-labeling for blood vessels, we observed a close interaction of differentiating adipocytes with endothelial cells through cell protrusions. Finally, we implemented hyperspectral imaging and spectral phasor analysis in Nile Red labeled transgenic larvae and revealed the lipid metabolic transition towards neutral lipid accumulation of differentiating adipocytes. Altogether our work presents the characterization of a novel adipocyte-specific label in zebrafish and uncovers previously unknown aspects of in vivo adipogenesis.

Summary statementAnalysis of the differentiation of adipocytes in vivo through cell-specific labeling in zebrafish, revealed their early interaction with blood vessels as well as early lipid metabolic changes.


title: In vivo regulation of fluorescent fusion proteins by engineered kinases

abstract: Reversible protein phosphorylation by kinases in extensively used to control a plethora of processes essential for proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases for direct, robust and tissue-specific phosphorylation of target fluorescent protein fusions in vivo. We show that synthetic Rok kinases, based on the Drosophila ortholog of Rho-associated protein kinase (ROCK), are functional enzymes and can activate myosin II through phosphorylation of Sqh::GFP or Sqh::mCherry in different morphogenetic processes in a developing fly embryo. We next use the system to study the impact of actomyosin activation specifically in the developing tracheal branches and showed that ectopic activation of actomyosin with engineered Rok kinase did not prevent cell intercalation nor the formation of autocellular junctions. We assume that this approach can be adapted to other kinases and targets in various eukaryotic genetic systems.


title: Rapid Evolution of SARS-CoV-2 Challenges Human Defenses

abstract:
Evolutionary ecology theory provides an avenue to anticipate the future behavior of SARS-CoV-2. Here we quantify the accelerating evolution of SARS-CoV-2 by tracking the SARS-CoV-2 mutation globally, with a focus on the Receptor Binding Domain (RBD) of the spike protein believed to determine infectivity. We estimate that 384 million people were infected by March 1st, 2021, producing up to 1021 copies of the virus, with one new RBD variant appearing for every 600,000 human infections, resulting in approximately three new effective RBD variants produced daily. Doubling the number of RBD variants every 71.67 days followed by selection of the most infective variants challenges our defenses and calls for a shift to anticipatory, rather than reactive tactics.

One-Sentence SummaryAccelerating evolution of SARS-CoV-2 demands formulating universal vaccines and treatments based on big-data simulations of possible new variants.

category: evolutionary biology


title: Validating Pore Size Estimates in a Complex Microfibre Environment on a Human MRI System

abstract:
PurposeRecent advances in diffusion-weighted MRI provide  restricted diffusion signal fraction and restricting pore size estimates. Materials based on co-electrospun oriented hollow cylinders have been introduced to provide validation for such methods. This study extends this work, exploring accuracy and repeatability using an extended acquisition on a 300 mT/m gradient human MRI scanner, in substrates closely mimicking tissue, i.e., non-circular cross-sections, intra-voxel fibre crossing, intra-voxel distributions of pore-sizes and smaller pore-sizes overall.

MethodsIn a single-blind experiment, diffusion-weighted data were collected from a biomimetic phantom on a 3T Connectom system using multiple gradient directions/diffusion times. Repeated scans established short-term and long-term repeatability. The total scan time (54 minutes) matched similar protocols used in human studies. The number of distinct fibre populations was estimated using spherical deconvolution, and median pore size estimated through the combination of CHARMED and AxCaliber3D framework. Diffusion-based estimates were compared with measurements derived from scanning electron microscopy.

ResultsThe phantom contained substrates with different orientations, fibre configurations and pore size distributions. Irrespective of one or two populations within the voxel, the pore-size estimates (~5m) and orientation-estimates showed excellent agreement with the median values of pore-size derived from scanning electron microscope and phantom configuration. Measurement repeatability depended on substrate complexity, with lower values seen in samples containing crossing-fibres. Sample-level repeatability was found to be good.

ConclusionWhile no phantom mimics tissue completely, this study takes a step closer to validating diffusion microstructure measurements for use in vivo by demonstrating the ability to quantify microgeometry in relatively complex configurations.

category: biophysics


title: The nucleotide addition cycle of the SARS-CoV-2 polymerase

abstract: Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We have used a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide the first evidence that an RdRp uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enables the direct observation of coronavirus polymerase deep and long-lived backtrack that are strongly stimulated by secondary structure in the template. The framework presented here elucidates one of the most important structure-dynamics-function relationships in human health today, and will form the grounds for understanding the regulation of this complex.


title: Single-cell motion of magnetotactic bacteria in microfluidic confinement: interplay between surface interaction and magnetic torque

abstract: Swimming microorganisms often experience complex environments in their natural habitat. The same is true for microswimmers in envisioned biomedical applications. The simple aqueous conditions typically studied in the lab differ strongly from those found in these environments and often exclude the effects of small volume confinement or the influence that external fields have on their motion. In this work, we investigate magnetically steerable microswimmers, specifically magnetotactic bacteria, in strong spatial confinement and under the influence of an external magnetic field. We trap single cells in micrometer-sized microfluidic chambers and track and analyze their motion, which shows a variety of different trajectories, depending on the chamber size and the strength of the magnetic field. Combining these experimental observations with simulations using a variant of an active Brownian particle model, we explain the variety of trajectories by the interplay between the wall interactions and the magnetic torque. We also analyze the pronounced cell-to-cell heterogeneity, which makes single-cell tracking essential for an understanding of the motility patterns. In this way, our work establishes a basis for the analysis and prediction of microswimmer motility in more complex environments.


title: High Throughput Virtual Screening and Validation of a SARS-CoV-2 Main Protease Non-Covalent Inhibitor

abstract:
Despite the recent availability of vaccines against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the search for inhibitory therapeutic agents has assumed importance especially in the context of emerging new viral variants. In this paper, we describe the discovery of a novel non-covalent small-molecule inhibitor, MCULE-5948770040, that binds to and inhibits the SARS-Cov-2 main protease (Mpro) by employing a scalable high throughput virtual screening (HTVS) framework and a targeted compound library of over 6.5 million molecules that could be readily ordered and purchased. Our HTVS framework leverages the U.S. supercomputing infrastructure achieving nearly 91% resource utilization and nearly 126 million docking calculations per hour. Downstream biochemical assays validate this Mpro inhibitor with an inhibition constant (Ki) of 2.9 {micro}M [95% CI 2.2, 4.0]. Further, using room-temperature X-ray crystallography, we show that MCULE-5948770040 binds to a cleft in the primary binding site of Mpro forming stable hydrogen bond and hydrophobic interactions. We then used multiple {micro}s-timescale molecular dynamics (MD) simulations, and machine learning (ML) techniques to elucidate how the bound ligand alters the conformational states accessed by Mpro, involving motions both proximal and distal to the binding site. Together, our results demonstrate how MCULE-5948770040 inhibits Mpro and offers a springboard for further therapeutic design.

O_TEXTBOXSignificance StatementThe ongoing novel coronavirus pandemic (COVID-19) has prompted a global race towards finding effective therapeutics that can target the various viral proteins. Despite many virtual screening campaigns in development, the discovery of validated inhibitors for SARS-CoV-2 protein targets has been limited. We discover a novel inhibitor against the SARS-CoV-2 main protease. Our integrated platform applies downstream biochemical assays, X-ray crystallography, and atomistic simulations to obtain a comprehensive characterization of its inhibitory mechanism. Inhibiting Mpro can lead to significant biomedical advances in targeting SARS-CoV-2 treatment, as it plays a crucial role in viral replication.

C_TEXTBOX


title: Stress-driven tissue fluidization physically segments vertebrate somites

abstract: Shaping functional structures during embryonic development requires both genetic and physical control. During somitogenesis, cell-cell coordination sets up genetic traveling waves in the presomitic mesoderm (PSM) that orchestrate somite formation. While key molecular and genetic aspects of this process are known, the mechanical events required to physically segment somites from the PSM remain unclear. Combining direct mechanical measurements during somite formation, live imaging of cell and tissue structure, and computer simulations, here we show that somites are mechanically sectioned off from the PSM by a large, actomyosin-driven increase in anisotropic stress at the nascent somite-somite boundary. Our results show that this localized increase in stress drives the regional fluidization of the tissue adjacent to the forming somite border, enabling local tissue remodeling and the shaping of the somite. Moreover, we find that active tension fluctuations in the tissue are optimized to mechanically define sharp somite boundaries while minimizing somite morphological defects. Altogether, these results indicate that mechanical changes at the somite-somite border and optimal tension fluctuations in the tissue are essential physical aspects of somite formation.


title: Rbf/E2F1 control growth and endoreplication via steroid-independent Ecdysone Receptor signalling in Drosophila prostate-like secondary cells

abstract: Dysregulation of cell cycle components results in the development and progression of several cancer types. Unusually, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 have been linked to late-stage tumour progression in prostate cancer, rather than early-stage events. This change is associated with an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG) share several functional and signalling similarities with human prostate epithelial cells. Upon mating, SC growth regulation switches from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control that induces genome endoreplication. Here, we demonstrate that the Drosophila Rb homologue, Rbf, and E2F1, as well as cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key mediators of SC growth and endoreplication both in virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and associated growth in SCs after mating, mirroring changes in CRPC. We also demonstrate that excess Rbf activity reversibly suppresses binucleation in adult SCs. Overall, our work reveals mechanistic parallels between the physiological switch to hormone-independent EcR signalling in SCs, and the pathological switch seen in CRPC, and suggests that the latter may represent the dysregulation of a currently unidentified physiological process, which permits AR signalling when androgen levels are low.

category: cell biology


title: Signal requirement for cortical potential of transplantable human neuroepithelial stem cells

abstract: The cerebral cortex develops from dorsal forebrain neuroepithelial progenitor cells. Initial expansion of the progenitor cell pool is followed by the generation of neurons of all the cortical layers and later, astrocytes and oligodendrocytes. However, the regulatory pathways that control the expansion and maintenance of the neuroepithelial progenitor cell pool are currently unknown. Here we define six basic pathway components that regulate proliferation of cortically specified human neuroepithelial stem cells (cNESCs) in vitro without the loss of developmental potential. We show that activation of FGF and inhibition of BMP and ACTIVIN A signalling are required for long-term cNESC proliferation. We also demonstrate that cNESCs preserve dorsal telencephalon-specific potential when GSK3, AKT and nuclear CATENIN-{beta}1 activity are low. Remarkably, regulation of these six pathway components supports the clonal expansion of cNESCs. Moreover, cNESCs differentiate to lower and upper layer cortical neurons both in vitro and in vivo. Identifying the mechanisms that drive the self-renewal and fate of cNESCs decision of neuroepithelial stem cells is key to developing new stem cell-based therapeutic approaches to treat neurological conditions.


title: RNA polymerase II depletion from the inactive X chromosome territory is not mediated by physical compartmentalization.

abstract: Sub-nuclear compartmentalization has been proposed to play an important role in gene regulation by segregating active and inactive parts of the genome in distinct physical and biochemical environments, where transcription and epigenetic factors are either concentrated or depleted. The inactive X chromosome offers a paradigm for studying sub-nuclear compartmentalization. When the non-coding Xist RNA coats the X chromosome, it recruits repressors and chromatin factors that trigger gene silencing, and forms a dense body of heterochromatin from which the transcription machinery appears to be excluded. Phase separation has been proposed to be involved in X-chromosome inactivation (XCI) and might explain exclusion of the transcription machinery by preventing its diffusion into the Xist-coated territory. Here, using quantitative fluorescence microscopy and single particle tracking, we show that RNA polymerase II (RNAPII) freely accesses the Xist territory during initiation of XCI, and that its diffusion is not prevented by biophysical constraints. Instead, the apparent depletion of RNAPII is due to the loss of its chromatin bound fraction. These findings demonstrate that initial exclusion of RNA Pol2 from the inactive X is a consequence of its reduced binding rate at the chromatin and gene level, rather than the biophysical compartmentalization of the inactive X heterochromatin domain. The Xist silent compartment is thus a biochemical rather than a biophysical compartment, at least during initiation of XCI.


title: Robust, Fiducial-Free Drift Correction for Super-resolution Imaging

abstract: We describe a robust, fiducial-free method of drift correction for use in single molecule localization-based super-resolution methods. The method combines periodic 3D registration of the sample using brightfield images with a fast post-processing algorithm that corrects residual registration errors and drift between registration events. The method is robust to low numbers of collected localizations, requires no specialized hardware, and provides stability and drift correction for an indefinite time period.


title: Structural variant selection for high-altitude adaptation using single-molecule long-read sequencing

abstract: Structural variants (SVs) can be important drivers of human adaptation with strong effects, but previous studies have focused primarily on common variants with weak effects. Here, we used large-scale single-molecule long-read sequencing of 320 Tibetan and Han samples, to show that SVs are key drivers of selection under high-altitude adaptation. We expand the landscape of global SVs, apply robust models of selection and population differentiation combining SVs, SNPs and InDels, and use epigenomic analyses to predict driver enhancers, target genes, upstream regulators, and biological functions, which we validate using enhancer reporter and DNA pull-down assays. We reveal diverse Tibetan-specific SVs affecting the cis- and trans-regulatory circuitry of diverse biological functions, including hypoxia response, energy metabolism, lung function, etc. Our study greatly expands the global SV landscape, reveals the central role of gene-regulatory circuitry rewiring in human adaptation, and illustrates the diverse functional roles that SVs can play in human biology.

category: genomics


title: Learning brain dynamics for decoding and predicting individual differences

abstract:
Insights from functional Magnetic Resonance Imaging (fMRI), as well as recordings of large numbers of neurons, reveal that many cognitive, emotional, and motor functions depend on the multivariate interactions of brain signals. To decode brain dynamics, we propose an architecture based on recurrent neural networks to uncover distributed spatiotemporal signatures. We demonstrate the potential of the approach using human fMRI data during movie-watching data and a continuous experimental paradigm. The model was able to learn spatiotemporal patterns that supported 15-way movie-clip classification (~90%) at the level of brain regions, and binary classification of experimental conditions (~60%) at the level of voxels. The model was also able to learn individual differences in measures of fluid intelligence and verbal IQ at levels comparable or better than existing techniques. We propose a dimensionality reduction approach that uncovers low-dimensional trajectories and captures essential informational (i.e., classification related) properties of brain dynamics. Finally, saliency maps were employed to characterize brain-region/voxel importance, and uncovered how dynamic but consistent changes in fMRI activation influenced decoding performance. When applied at the level of voxels, our framework implements a dynamic version of multivariate pattern analysis. We believe our approach provides a powerful framework for visualizing, analyzing, and discovering dynamic spatially distributed brain representations during naturalistic conditions.1

Author summaryBrain signals are inherently dynamic and evolve in both space and time as a function of cognitive or emotional task condition or mental state. To characterize brain dynamics, we employed an architecture based on recurrent neural networks, and applied it to functional magnetic resonance imaging data from humans watching movies or during continuous experimental conditions. The model learned spatiotemporal patterns that allowed it to correctly classify which clip a participant was watching based entirely on data from other participants; the model also learned a binary classification of experimental conditions at the level of voxels. We developed a dimensionality reduction approach that uncovered low-dimensional "trajectories" and captured essential information properties of brain dynamics. When applied at the level of voxels, our framework implements a dynamic version of multivariate pattern analysis. We believe our approach provides a powerful framework for visualizing, analyzing, and discovering dynamic spatially distributed brain representations during naturalistic conditions.

category: neuroscience


title: Dopaminergic regulation of vestibulo-cerebellar circuits through unipolar brush cells

abstract: While multiple monoamines modulate cerebellar output, the mechanistic details of dopaminergic signaling in the cerebellum remain poorly understood. Here we show that Drd1 dopamine receptors are expressed in unipolar brush cells (UBCs) of the cerebellar vermis. Drd1 activation increases UBC firing rate and postsynaptic NMDA receptor-mediated currents. Using anatomical tracing and in situ hybridization, we tested three hypotheses about the source of cerebellar dopamine. We exclude midbrain dopaminergic nuclei and tyrosine hydroxylase-positive Purkinje cells as potential sources, supporting the possibility of dopaminergic co-release from locus coeruleus (LC) axons. Using an optical dopamine sensor GRABDA, electrical stimulation, and optogenetic activation of LC fibers in the acute slice, we find evidence for monoamine release onto Drd1-expressing UBCs. Altogether, we propose that the LC regulates cerebellar cortex activity by co-releasing dopamine onto UBCs to modulate their response to cerebellar inputs. Purkinje neurons directly inhibit these Drd1-positive UBCs, forming a dopamine-sensitive recurrent vestibulo-cerebellar circuit.


title: Modulation masking and fine structure shape neural envelope coding to predict speech intelligibility across diverse listening conditions

abstract: A fundamental question in the neuroscience of everyday communication is how scene acoustics shape the neural processing of attended speech sounds and in turn impact speech intelligibility. While it is well known that the temporal envelopes in target speech are important for intelligibility, how the neural encoding of target-speech envelopes is influenced by background sounds or other acoustic features of the scene is unknown. Here, we combine human electroencephalography with simultaneous intelligibility measurements to address this key gap. We find that the neural envelope-domain signal-to-noise ratio in target-speech encoding, which is shaped by masker modulations, predicts intelligibility over a range of strategically chosen realistic listening conditions unseen by the predictive model. This provides neurophysiological evidence for modulation masking. Moreover, using high-resolution vocoding to carefully control peripheral envelopes, we show that target-envelope coding fidelity in the brain depends not only on envelopes conveyed by the cochlea, but also on the temporal fine structure (TFS), which supports scene segregation. Our results are consistent with the notion that temporal coherence of sound elements across envelopes and/or TFS influences scene analysis and attentive selection of a target sound. Our findings also inform speech-intelligibility models and technologies attempting to improve real-world speech communication.


title: The Detection of Bergeyella zoohelcum in Young Children after Cat Bite

abstract: Bergeyella zoohelcum is an uncommon zoonotic pathogen typically associated with cat or dog bites. Previously, only 14 cases of B. zoohelcum infection have been reported. We isolated the bacteria from the face of a 2-year-old girl who was bitten by a cat. The organism was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequence, and phylogenetic analysise supported that this isolate was belong to B. zoohelcum. Due to the contradicts of culture characteristics of B. zoohelcum which described in reported literatures, we used different media to culture the bacteria. After 24 h incubation, Colombia blood agar (CBA), Mueller-Hinton agar plate with 5% sheep blood and chocolate agar (CA) grew well, but blood agar (BA) grew well until 72 h. The strain did not grow on McConkey agar (MAC), Mueller-Hinton agar (MHA) and chocolate agar (containing vancomycin). The low detection rate of this strain was related to its harsh growth conditions and the limitations of traditional identification techniques. With the popularization of MALDI-TOF MS and 16S rRNA gene sequence and our further understanding of the fastidious bacteria, we will quickly and accurately identify the B. zoohelcum, meanwhile the detection rate of this bacterium will also be significantly improved. So that clinicians can achieve precise anti-infective treatment according to antimicrobial susceptibility testing.

category: microbiology


title: Chromatic bacteria version 2 - A Himar1 transposon based delivery vector to extend the host range of a toolbox to fluorescently tag bacteria

abstract: A recent publication described the construction and utility of a comprehensive "Chromatic Bacteria" toolbox containing a set of genetic tools that allows for fluorescently tagging a variety of Proteobacteria. In an effort to expand the range of bacteria taggable with the Chromatic Bacteria toolbox, a Himar1 transposon vector was constructed to mediate insertion of fluorescent protein and abiotic resistant genes. The Himar1 transposon was chosen as it is known to function in a wide range of bacteria. To test the use of the Himar1 derivative of the Chromatic Bacteria plasmid series for fluorescently tagging bacteria, recently isolated non-model organisms were conjugated with the Himar1 transposon plasmids. We were unsuccessful in delivering the plasmids into Gram positive bacterial isolates but were able to successfully integrate the transposon in isolates that we were previously unable to modify such as Sphingomonas sp. Leaf357 and Acidovorax sp. Leaf84. This manuscript reports on the currently available plasmids and transposition success in different bacteria. The manuscript will be updated regularly until a full plasmid series is available and a large range of recipient strains was tested.


title: Rapid evolutionary turnover of mobile genetic elements drives microbial resistance to viruses

abstract: Although it is generally accepted that viruses (phages) drive bacterial evolution, how these dynamics play out in the wild remains poorly understood. Here we show that the arms race between phages and their hosts is mediated by large and highly diverse mobile genetic elements. These phage-defense elements display exceedingly fast evolutionary turnover, resulting in differential phage susceptibility among clonal bacterial strains while phage receptors remain invariant. Protection afforded by multiple elements is cumulative, and a single bacterial genome can harbor as many as 18 putative phage-defense elements. Overall, elements account for 90% of the flexible genome amongst closely related strains. The rapid turnover of these elements demonstrates that phage resistance is unlinked from other genomic features and that resistance to phage therapy might be as easily acquired as antibiotic resistance.


title: Th17 cell master transcription factor RORC2 regulates HIV-1 gene expression and viral outgrowth

abstract:
Among CD4+ T-cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier resulting in microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long-term in the gastro-intestinal lymphatic tract, where low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence, however it is unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host-cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T-cells. Depletion of RORC2 inhibited HIV-1 infection, whereas RORC2 overexpression enhanced it. RORC2 was found to promote HIV-1 gene expression. Chromatin immune precipitation revealed that RORC2 binds to the nuclear receptor responsive element (NRRE) in the HIV-1 LTR. In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2- cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T-cells from antiretroviral-treated patients. Altogether, these results provide a new explanation as to why Th17 cells are highly susceptible to HIV-1 infection and point to RORC2 as a cell-specific target for HIV-1 therapy.

Significance statementHIV-1 infects CD4 T cells and among these, Th17 cells are known to be particularly permissive for virus replication. Infection of Th17 cells is critical for AIDS pathogenesis and viral persistence, however it is not clear why these cells are highly permissive to HIV-1. We found that Th17 cell permissiveness depends on expression of the hormone receptor RORC2, which is the master transcriptional regulator of Th17 cell differentiation. We identify RORC2 as a new, cell-specific host-dependency factor that can be targeted by small molecules. Our results point to RORC2 as a cell-specific target for HIV-1 therapy, an entirely new concept in the field, and suggest HIV-1 might have evolved to exploit RORC2 to promote its own persistence.


title: Host genetic control of succession in the switchgrass leaf fungal microbiome

abstract: Leaf fungal microbiomes can be fundamental drivers of host plant success, as they contain pathogens that devastate crop plants and taxa that enhance nutrient uptake, discourage herbivory, and antagonize pathogens. We measured leaf fungal diversity with amplicon sequencing across an entire growing season in a diversity panel of switchgrass (Panicum virgatum). We also sampled a replicated subset of genotypes across three additional sites to compare the importance of time, space, ecology, and genetics. We found a strong successional pattern in the microbiome shaped both by host genetics and environmental factors. Further, we used genome-wide association mapping and RNA-sequencing to show that three cysteine-rich receptor-like kinases were linked to a genetic locus associated with microbiome structure. These genes were more highly expressed in genotypes susceptible to fungal pathogens, which were central to microbial covariance networks, suggesting that host immune genes are a principal means of controlling the entire leaf microbiome.

category: ecology


title: Transcriptome-based phylogeny and whole-genome duplication in Theaceae

abstract: Theaceae, with three tribes and nine genera, is a family of great economic and ecological importance. Recent phylogenetic analyses based on plastid genome resolved the relationship among three tribes and the intergeneric relationships within Gordonieae and Stewartieae. However, generic level relationships within the largest tribe Theeae were not fully resolved and potential hybridization among genera within Theeae revealed previously also remains to be tested further. Here we conducted a comprehensive phylogenomic study of Theaceae based on transcriptomes and low-depth whole-genome sequencing of 57 species as well as additional plastome sequence data from previous work. Phylogenetic analyses suggested that Stewartieae was the first-diverging clade in Theaceae, consistent with previous study using plastomic data. Within Theeae, the highly supported Apterosperma-Laplacea clade grouped with Pyrenaria with maximum support based on the partitioned and unpartitioned concatenation analyses using the 610 low-copy nuclear genes, leaving Camellia and Polyspora as another sister genera in the tribe. PhyloNet analyses suggested one reticulation event within Camellia and Pyrenaria respectively, but no intergeneric reticulations were detected in Theeae. Another introgression was found between Gordonia lasianthus and the common ancestor of Gordonieae during the Late Oligocene. The existing land bridges (e.g. Bering land bridge) might have facilitated this ancient introgression. Further researches need to be conducted to uncover the interspecific introgression pattern within Camellia. Ks distribution analyses supported the tea family shared one whole-genome duplication (WGD) event Ad-{beta}, which was recently mapped to the clade containing core Ericales, Primuloids, Polemonioids and Lecythidaceae.


title: A Synthesis of Game Theory and Quantitative Genetic Models of Social Evolution

abstract: Two popular approaches for modeling social evolution, evolutionary game theory and quantitative genetics, ask complementary questions but are rarely integrated. Game theory focuses on evolutionary outcomes, with models solving for evolutionarily stable equilibria, whereas quantitative genetics provides insight into evolutionary processes, with models predicting short-term responses to selection. Here we draw parallels between evolutionary game theory and interacting phenotypes theory, which is a quantitative genetic framework for understanding social evolution. First, we show how any evolutionary game may be translated into two quantitative genetic selection gradients, nonsocial and social selection, which may be used to predict evolutionary change from a single round of the game. We show that synergistic fitness effects may alter predicted selection gradients, causing changes in magnitude and sign as the population mean evolves. Second, we show how evolutionary games involving plastic behavioral responses to partners can be modeled using indirect genetic effects, which describe how trait expression changes in response to genes in the social environment. We demonstrate that repeated social interactions in models of reciprocity generate indirect effects and conversely, that estimates of parameters from indirect genetic effect models may be used to predict the evolution of reciprocity. We argue that a pluralistic view incorporating both theoretical approaches will benefit empiricists and theorists studying social evolution. We advocate the measurement of social selection and indirect genetic effects in natural populations to test the predictions from game theory, and in turn, the use of game theory models to aid in the interpretation of quantitative genetic estimates.


title: Manifestations of genetic risk for Alzheimer's Disease in the blood: a cross-sectional multi-omic analysis in healthy adults aged 18-90+

abstract: Deeply phenotyped cohort data can elucidate differences associated with genetic risk for common complex diseases across an age spectrum. Previous work has identified genetic variants associated with Alzheimers disease (AD) risk from large-scale genome-wide association study meta-analyses. To explore effects of known AD-risk variants, we performed a phenome-wide association study on ~2000 clinical, proteomic, and metabolic blood-based analytes obtained from 2,831 cognitively normal adult clients of a consumer-based scientific wellness company. Results uncovered statistically significant SNP-analyte associations for five genetic variants after correction for multiple testing (for SNPs in or near NYAP1, ABCA7, INPP5D, and APOE). These effects were detectable from early adulthood. Sex modified the effects of four genetic variants, with multiple interrelated immune-modulating effects associated with the PICALM variant. Sex-stratified GWAS results from an independent AD case-control meta-analysis supported sexspecific disease effects of the PICALM variant, highlighting the importance of sex as a biological variable. These analyses support evidence from previous functional genomics studies in the identification of a causal variant within the PILRA gene. Taken together, this study highlights clues to the earliest effects of AD genetic risk variants in individuals where disease symptoms have not (yet) arisen.

category: genetics


title: The formation of the bacterial RNA polymerase-promoter open complex involves a branched pathway

abstract: The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)-{sigma}70 holoenzyme to the promoter DNA. This initial RNAP-promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; these changes lead to the catalytically active open complex (RPO) state. Recent cryo-electron microscopy studies have provided detailed structural insight on the RPO and putative intermediates on its formation pathway. Here, we employ single-molecule fluorescence microscopy to interrogate the conformational dynamics and reaction kinetics during real-time RPO formation. We find that the RPO pathway is branched, generating RPO complexes with different stabilities. The RNAP cleft loops, and especially the {beta} rudder, stabilise the transcription bubble. The RNAP interactions with the promoter upstream sequence (beyond -35) stimulate transcription bubble nucleation and tune the reaction path towards stable forms of the RPO. The mechanistic heterogeneity of the RPO pathway may be a prerequisite for its regulation since such heterogeneity allows the amplification of small promoter sequence or transcription-factor-dependent changes in the free energy profile of the RPO pathway to large differences in transcription efficiency.

category: biochemistry


title: A second specificity-determining loop in Class A sortases: Biochemical characterization of natural sequence variation in chimeric SrtA enzymes

abstract: Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice for SML reactions. However, the stringent specificity of saSrtA for the sequence motif LPXTG limits its uses. Here, we use principal component analysis to identify a structurally conserved loop with a high degree of variability in all classes of sortases. We investigate the contribution of this {beta}7-{beta}8 loop, located between the catalytic cysteine and arginine residues and immediately adjacent to the target binding cleft, by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of Class A sortases from 8 species engineered into the SrtA sequence from Streptococcus pneumoniae (spSrtA). While some of our chimeric enzymes mimic the activity and selectivity of the wild-type protein from which the loop sequence is derived (e.g., that of saSrtA), others result in chimeric spSrtA enzymes able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural, and sequence analyses, we identify three interactions facilitated by {beta}7-{beta}8 loop residues that appear to be broadly characteristic of Class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.


title: Thermoplasmonic nano-rupture of cells reveals Annexin V function in plasma membrane repair

abstract: Maintaining the integrity of the cell plasma membrane (PM) is critical for the survival of cells. While an efficient PM repair machinery can aid survival of healthy cells by preventing influx of extracellular calcium, it can also constitute an obstacle in drug delivery and photothermal therapy. We show how nanoscopic holes can be applied to the cell surface thus allowing identification of molecular components with a pivotal role in PM repair. Cells are punctured by locally heating gold nanostructures at the cell surface which causes nano-ruptures in cellular PMs. Recruitment of annexin V near the hole is found to locally reshape the ruptured plasma membrane. Experiments using model membranes, containing recombinant annexin V, provide further biophysical insight into the ability of annexin V to reshape edges surrounding a membrane hole. The thermoplasmonic method provides a general strategy to monitor the response to nanoscopic injuries to the cell surface.


title: Regulatory FOXP3+ T cells in uterine sarcomas are associated with favorable prognosis, low extracellular matrix expression and reduced YAP activation

abstract:
PurposeUterine sarcomas are rare but deadly malignancies without effective treatment. The goal of this study was to characterize and identify potential mechanisms underlying observed variations in the immune microenvironment of different sarcoma subtypes, using integrated clinicopathological and molecular methods.

Experimental designFifty-eight cases of uterine sarcoma with full clinicopathological annotation were analyzed for their immune landscape in the tumor microenvironment, gene, and protein expression. Cases included leiomyosarcoma (LMS; n=13), low-grade endometrial stromal sarcoma (ESS; n=16), undifferentiated uterine sarcoma (UUS; n=26), and YWHAE-FAM22 translocation-bearing ESS (YFAM; n=3). Image analysis was used to quantify immune cells and immune regulatory proteins. Gene ontology and network enrichment analysis of matching transcriptomic data was used to relate over- and under expressed genes to pathways and further to the immune phenotype and clinicopathological findings.

ResultsImmune cell characterization revealed overall prevalence of regulatory T cells and the pro-tumor M2-like macrophages. Cytotoxic T cells were only found in ESS and UUS tumors. Expression of immune regulatory proteins was heterogeneous, with PD-L1 being undetectable. Hierarchical clustering of patients showed four immune signatures independent of tumor type, where infiltration of non-exhausted FOXP3+ cells and M1-like macrophages were associated with greater overall survival. High CD8+/FOXP3+ ratio in UUS and ESS was associated with poor survival and upregulation of extracellular matrix (ECM)-related genes and proteins and YAP nuclear localization.

ConclusionsUterine sarcomas present distinct immune signatures with prognostic value, independent of tumor type. This study suggests that the ECM is a potential regulator of the immune microenvironment in uterine sarcomas.

category: cancer biology


title: Single-nuclei transcriptomes from human adrenal gland reveals distinct cellular identities of low and high-risk neuroblastoma tumors.

abstract: Childhood neuroblastoma has a remarkable variability in outcome. Age at diagnosis is one of the most important prognostic factors, with children less than 1 year old having favorable outcomes. We studied single-cell and single-nuclei transcriptomes of neuroblastoma with different clinical risk groups and stages, including healthy adrenal gland. We compared tumor cell populations with embryonic mouse sympatho-adrenal derivatives, and post-natal human adrenal gland. We provide evidence that low and high-risk neuroblastoma have different cell identities, representing two disease entities. Low-risk neuroblastoma presents a transcriptome that resembles sympatho- and chromaffin cells, whereas malignant cells enriched in high-risk neuroblastoma resembles an unknown subtype of TRKB+ cholinergic progenitor population identified in human post-natal gland. Analyses of these populations revealed different gene expression programs for worst and better survival in correlation with age at diagnosis. Our findings reveal two cellular identities and a composition of human neuroblastoma tumors reflecting clinical heterogeneity and outcome.


title: Tribbles 2 confers enzalutamide resistance in prostate cancer by promoting lineage plasticity.

abstract: Second-generation anti-androgen, such as enzalutamide (Xtandi), is commonly prescribed for prostate cancer therapy, but enzalutamide-resistant, lethally incurable disease invariably develops. To understand the molecular basis of enzalutamide resistance, we comprehensively analyzed prostate tumors and clinically relevant models. These studies revealed that enzalutamide resistant prostate cancer cells overexpress Tribbles 2 (Trib2), a pseudokinase. Expression of Trib2 is negatively regulated by androgen receptor signaling. Overexpression of Trib2 makes prostate cancer cells completely resistant to clinically relevant doses of enzalutamide. Trib2 downregulates expression of luminal markers but upregulates the neuronal transcription factor, BRN2, and the stemness factor, SOX2, to induce neuroendocrine differentiation. Our findings indicate that Trib2 confers resistance to enzalutamide therapy via a mechanism involving increased cellular plasticity and lineage switching.


title: Evaluation of the effect of hydrocortisone in 2D and 3D HEp-2 cell culture

abstract: Cancer is one of the diseases with the highest incidence globally and that associated with the patients emotional state, can act positively or negatively in the treatment. Cortisol is a principal primary stress hormone in the human body. The corticoids can increase cell proliferation and reactive oxygen species that contribute to DNA damage. Prolonged exposure to stress can contribute to tissues becoming insensitive to cortisol, the primary human stress hormone. This study explores cortisols influence on tumor cell development, particularly in human cells of carcinoma of the human laryngeal (HEp-2). HEp-2 cells were exposed to increasing cortisol (hydrocortisone) concentrations for 24 or 48 hours, and cytotoxicity (MTT assay) proliferation assay (crystal violet assay), and immunolabeled 3D culture for fibronectin and FAK were analyzed. The group treated with hydrocortisone showed a significant increase in mitochondrial activity, as for the evaluation by the violet crystal, the treated group showed similar behavior to the control. The 3D culture showed dispersed cells within 24 hours with reduced FAK labeling; however, no changes were observed within 48 hours. Although some cases favored corticosteroid use in cancer patients, a more detailed analysis is necessary before prescribing them.


title: Exploring functional diversity of Nicotiana tabacum Aquaporins

abstract:
Aquaporins (AQPs) are involved in a variety of vital plant physiological processes, including water relations, development, stress responses and photosynthesis. Nicotiana tabacum (tobacco) has 84 AQP genes due to its recent hybridisation and allotetraploid genome. We functionally characterised a diverse subset of tobacco AQPs spanning the 3 largest AQP subfamilies, selecting nine isoforms from the PIPs, TIPs, and NIPs. Using high-throughput yeast-based functional assays, we determined AQP permeability to water, hydrogen peroxide (H2O2), boric acid (BA) and urea. For each AQP, subcellular localisations in planta were established using GFP translational fusions. From 3D protein homology modelling, we found that the monomeric pore shape/size, selectivity filter region and NPA motifs is insufficient to comprehensively predict their transport capabilities. PIPs had the narrowest pore diameter and were permeable to water, H2O2 and BA. The pore in TIPs was wider and more cylindrical in shape than for the PIPs. TIP1;1s was permeable to all four substrates tested, and is highly expressed in leaves and flowers, suggesting it functions in multiple roles. By contrast, NIP5;1t, with a larger pore size than the NtTIPs, is only expressed in young flowers and enhanced permeability only to BA. Its homolog in Arabidopsis (AtNIP5;1) has the same substrate specificity and functions as a boron channel.

Sentence SummaryDiverse tobacco PIP, TIP and NIP aquaporin isoforms were functionally characterised using high-throughput yeast-based assays, assessing their transport capabilities for key plant solutes: water, H2O2, boric acid and urea.

category: plant biology


title: Verticillium dahliae effector VDAL protects MYB6 from degradation by interacting with PUB25/26 E3 ligases for enhancing Verticillium wilt resistance in Arabidopsis

abstract:
Verticillium wilt is a severe plant disease that causes massive losses in multiple crops. Increasing the plant resistance to Verticillium wilt is a critical challenge worldwide. Here, we report that the hemibiotrophic Verticillium dahliae (V. dahliae)-secreted Asp f2-like protein VDAL causes leaf wilting when applied to cotton leaves in vitro, but enhances the resistance to V. dahliae when overexpressed in Arabidopsis or cotton without affecting the plant growth and development. VDAL protein interacts with Arabidopsis E3 ligases PUB25 and PUB26 (PUBs) and is ubiquitinated by PUBs in vitro. However, VDAL is not degraded by PUB25 or PUB26 in planta. Besides, the pub25 pub26 double mutant shows higher resistance to V. dahliae than the wild type. PUBs interact with the transcription factor MYB6 in a yeast two-hybrid screen. MYB6 promotes plant resistance to Verticillium wilt while PUBs ubiquitinate MYB6 and mediate its degradation. VDAL competes with MYB6 for binding to PUBs, and the role of VDAL in increasing Verticillium wilt resistance depends on MYB6. Taken together, these results suggest that plants evolute a strategy to utilize the invaded effector protein VDAL to resist the V. dahliae infection without causing a hypersensitive response (HR), Alternatively, in order to take nutrients from host cells, hemibiotrophic pathogens may use some effectors to keep plant cells alive during its infection in order to take nutrients from host cells. This study provides the molecular mechanism for plants increasing disease resistance when overexpressing some effector proteins without inducing HR, and may promote searching for more genes from pathogenic fungi or bacteria to engineer plant disease resistance.

One-sentence summaryEctopically expressed VDAL in Arabidopsis alleviates the degradation of a positive disease response factor MYB6 through its interaction with PUB25/26 E3 ligases.


title: Histone H3 dopaminylation in ventral tegmental area underlies heroin-induced maladaptive plasticity

abstract:
Persistent transcriptional eventsamples were incubated on ice for 30 min and then centrifugeds in ventral tegmental area (VTA) and other reward relevant brain regions contribute to enduring behavioral adaptations that characterize substance use disorder (SUD). Recent data from our laboratory indicate that aberrant accumulation of the newly discovered histone post-translational modification (PTM), H3 dopaminylation at glutamine 5 (H3Q5dop), contributes significantly to cocaine-seeking behavior following prolonged periods of abstinence. It remained unclear, however, whether this modification is important for relapse vulnerability in the context of other drugs of abuse, such as opioids. Here, we showed that H3Q5dop plays a critical role in heroin-mediated transcriptional plasticity in midbrain. In rats undergoing abstinence from heroin self-administration (SA), we found acute and persistent accumulation of H3Q5dop in VTA. By attenuating H3Q5dop during abstinence, we both altered gene expression programs associated with heroin withdrawal and reduced heroin-primed reinstatement behavior. These findings thus establish an essential role for H3Q5dop, and its downstream transcriptional consequences, in opioid-induced plasticity in VTA.

SIGNIFICANCE STATEMENTOpioid addiction is a chronically relapsing disorder defined by pathological drug-seeking behavior. Persistent relapse vulnerability is hypothesized to reflect a functional "rewiring" of reward-processing circuitry in the brain. This phenomenon is precipitated by drug-induced alterations in transcriptional plasticity of midbrain dopamine neurons, indicating an important role for epigenetic regulatory mechanisms in opioid dependence. We found that the newly discovered histone modification, H3 dopaminylation at glutamine 5 (H3Q5dop), was increased in ventral tegmental area (VTA) during periods of abstinence. H3Q5dop accumulation was found to regulate transcriptional programs associated with chronic heroin exposure. Using viral-mediated gene therapy to reduce H3Q5dop accumulation during abstinence resulted in attenuated heroin-primed seeking behaviors. Together, our results indicate that H3Q5dop plays an abstinence-specific role in regulating heroin-induced behavioral plasticity during prolonged abstinence.


title: Parental-fetal interplay of immune genes leads to intrauterine growth restriction

abstract: Intrauterine growth restriction (IUGR) of fetuses affects 5-10% of pregnancies and is associated with perinatal morbidity, mortality and long-term health issues. Understanding genetic predisposition to IUGR is challenging, owing to extensive gene polymorphisms, linkage disequilibrium, and maternal and paternal influence. Here, we demonstrate that the inhibitory receptor, KIR2DL1, expressed on maternal uterine natural killer (uNK) cells, in interaction with the paternally-inherited HLA-C*05, an HLA-C group 2 allotype, expressed on fetal trophoblast cells, causes IUGR in a humanised mouse model. Micro-CT imaging of the uteroplacental vasculature revealed reduced uterine spiral artery diameter and increased segment length, increasing fetal blood flow resistance. Single cell RNA-Seq from the maternal-fetal interface highlighted expression programs activated by KIR2DL1-induced IUGR in several placental cell types, including degradation of extracellular matrix components, angiogenesis, and uNK cell communication, suggesting a complex response underlying IUGR. As current IUGR treatments are insufficient, our findings provide important insight for drug development.

category: immunology


title: Kenyan Traditional Medicine: Exploring Old Solutions to the Modern Antibacterial Crises Through Natural Products Chemistry

abstract: Infectious diseases are a major cause of morbidity and mortality around the world, accounting for approximately 50% of all deaths in tropical countries. Despite remarkable progress in the field of microbiology, inability to control or mitigate, epidemics caused by drug-resistant microorganisms pose a serious health hazard to the global population. New therapeutic strategies must be developed as a global initiative for the prevention and control of infectious diseases. This study focuses on Kenyan medicinal plants and their activity against bacteria. Plant extracts obtained from seven Kenyan plants used in traditional medicine, were screened for their antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Mycobacterium smegmatis. Extracts from all these plants showed antibacterial activity against at least one of the tested organisms at a concentration of 2 mg/mL. Chemical screening showed the presence of different classes of phytochemicals such as alkaloids, terpenes, tannins, in some active extracts.


title: Human herpesvirus 8 molecular mimicry of ephrin ligands facilitates cell entry and triggers EphA2 signaling

abstract: Human herpesvirus 8 (HHV-8) is an oncogenic virus that enters cells by fusion of the viral and endosomal cellular membranes in a process mediated by viral surface glycoproteins. One of the cellular receptors hijacked by HHV-8 to gain access to cells is the EphA2 tyrosine kinase receptor, and the mechanistic basis of EphA2-mediated viral entry remains unclear. Using X-ray structure analysis, targeted mutagenesis and binding studies, we here show that the HHV-8 envelope glycoprotein complex gH/gL binds with sub-nanomolar affinity to EphA2 via molecular mimicry of the receptors cellular ligands, ephrins, revealing a pivotal role for the conserved gH residue E52 and the amino-terminal peptide of gL. Using FSI-FRET and cell contraction assays, we further demonstrate that the gH/gL complex also functionally mimics ephrin ligand by inducing EphA2 receptor association via its dimerization interface, thus triggering receptor signaling for cytoskeleton remodeling. These results now provide novel insight into the entry mechanism of HHV-8, opening avenues for the search of therapeutic agents that could interfere with HHV-8 related diseases.


title: Machine-learning prediction of resistance to sub-inhibitory antimicrobial concentrations from Escherichia coli genomes

abstract:
Escherichia coli is an important cause of bacterial infections worldwide, with multidrug resistant strains incurring substantial costs on human lives. Besides therapeutic concentrations of antimicrobials in healthcare settings, the presence of sub-inhibitory antimicrobial residues in the environment and in the clinics selects for antimicrobial resistance (AMR), but the underlying genetic repertoire is less well understood. We used machine-learning to predict the population doubling time and growth yield of 1,432 genetically diverse E. coli expanding under exposure to three sub-inhibitory concentrations of six classes of antimicrobials from single nucleotide genetic variants, accessory gene variation and the presence of known AMR genes. We could predict cell yields in the held-out test data with an average correlation (Spearmans {rho}) of 0.63 (0.32 - 0.90 across concentrations) and cell doubling time with an average correlation of 0.47 (0.32 - 0.74 across concentrations), with moderate increases in sample size unlikely to improve predictions further. This points to the remaining missing heritability of growth under antimicrobials exposure being explained by effects that are too rare or weak to be captured unless sample size is dramatically increased, or by effects other than those conferred by the presence of individual SNPs and genes. Predictions based on whole genome information were generally superior to those based only on known AMR genes, and also accurate for AMR resistance at therapeutic concentrations. We also pinpointed genes and SNPs determining the predicted growth and thereby recapitulated the known AMR determinants. Finally, we estimated the effect sizes of resistance genes across the entire collection of strains, disclosing growth effects for known resistance genes for each strain. Our results underscore the potential of predictive modelling of growth patterns from genomic data under sub-inhibitory concentrations of antimicrobials, although the remaining missing heritability poses an issue for achieving the accuracy and precision required for clinical use.

ImportancePredicting bacterial growth from genome sequences is important not only for a rapid characterization of strains in clinical diagnostic applications but for the identification of novel targets for drug discovery. Previous studies examined the relationship between bacterial growth and genotype in mutant libraries for laboratory strains, yet no study has so far examined the prediction power of genome sequences for bacterial growth in natural strains. In this study, we used a high throughput phenotypic assay to measure bacterial growth of a systematic collection of natural Escherichia coli strains and then employed machine learning models to predict bacterial growth from genomic data under non-therapeutic sub-inhibitory concentrations of antimicrobials that are common in nonclinical settings. Our results revealed a moderate to strong correlation between predicted and actual values for different antimicrobials concentrations. Furthermore, the quantified effect of resistance genes on bacterial growth indicate these genes are still effective at sublethal antimicrobial concentrations.


title: Draft Genome of the Korean smelt (Hypomesus nipponensis) and its transcriptomic responses to heat stress in the liver and muscle

abstract: Pond smelt (Hypomesus nipponensis) is a cold-freshwater fish species as a winter economic resource of aquaculture in South Korea. Due to its high susceptibility to abnormal water temperature from global warming, a large number of smelt die in hot summer. Here, we present the first draft genome of H. nipponensis and transcriptomic changes in molecular mechanisms or intracellular responses under heat stress. We combined Illumina and PacBio sequencing technologies to generate the draft genome of H. nipponensis. Based on the reference genome, we conducted transcriptome analysis of liver and muscle tissues under normal (NT, 5{degrees}C) versus warm (HT, 23{degrees}C) conditions, to identify heat stress-induced genes and gene categories. We observed a total of 1,987 contigs, with N50 of 0.46 Mbp with a largest contig (3.03 Mbp) in the assembled genome. A total number of 20,644 protein coding genes were predicted, and 19,224 genes were functionally annotated: 15,955 genes for Gene Ontology (GO) terms; and 11,560 genes for KEGG Orthology (KO). We conducted the lost and gained genes analysis compared with three species that human, zebrafish and salmon. In the lost genes analysis, we detected smelt lost 4,461 (22.16%), 2,825 (10.62%), and 1,499 (3.09%) genes compare with above three species, respectively. In the gained genes analysis, we observed smelt gain 1,133 (5.49%), 1,670 (8.09%), and 229 (1.11%) genes compare with above species, respectively. From transcriptome analysis, a total of 297 and 331 differentially expressed genes (DEGs) with False discovery rate (FDR) < 0.05 were identified in the liver and muscle tissues, respectively. Gene enrichment analysis of DEGs indicates that up-regulated genes were significantly enriched for lipid biosynthetic process (GO:0008610, P < 0.001) and regulation of apoptotic process (GO:0042981, P < 0.01), and down-regulated genes by immune responses such as myeloid cell differentiation (GO:0030099, P < 0.001) in the liver under heat stress. In muscle tissue, up-regulated genes were enriched for hypoxia (GO:0001666, P < 0.05), transcription regulator activity (GO:0140110, P < 0.001) and calcium-release channel activity (GO:0015278, P < 0.01), and down-regulated genes for nicotinamide nucleotide biosynthetic process (GO:0019359, P < 0.01). The results of KEGG pathway analysis were similar to that of gene enrichment analysis. The draft genome and transcriptomic of H. nipponensis will be used as a useful genetic resource for functional and evolutionary studies. Our findings will improve understanding of the molecular mechanisms and heat responses and will be useful for predicting survival of the smelt and its closely related species under global warming.


title: Genomic analysis of response to neoadjuvant chemotherapy in esophageal adenocarcinoma

abstract: Neoadjuvant therapy followed by surgery is the standard of care for locally advanced esophageal adenocarcinoma (EAC). Unfortunately, response to neoadjuvant chemotherapy (NAC) is poor (<20%), as is the overall survival benefit at 5 years (5%). The EAC genome is complex and heterogeneous between patients, and it is not yet understood whether specific mutational patterns may result in chemotherapy sensitivity or resistance. To identify associations between genomic events and response to NAC in EAC, a comparative genomic analysis was performed in 65 patients with extensive clinical and pathological annotation using whole-genome sequencing (WGS). We defined response using Mandard Tumor Regression Grade (TRG), with responders classified as TRG1-2 (n=27) and non-responders classified as TRG4-5 (n=38). We report a higher non-synonymous mutation burden in responders (median 2.08/Mb vs 1.70/Mb, P=0.036) and elevated copy number variation in non-responders (282 vs 136/patient, P<0.001). We identified copy number variants unique to each group in our cohort, with cell cycle (CDKN2A, CCND1), c-Myc (MYC), RTK/PIK3 (KRAS, EGFR) and gastrointestinal differentiation (GATA6) pathway genes being specifically altered in non-responders. Of note, NAV3 mutations were exclusively present in the non-responder group with a frequency of 22%. Thus, lower mutation burden, higher chromosomal instability and specific copy number alterations are associated with resistance to NAC.


title: Origins of cancer genome complexity revealed by haplotype-resolved genomic analysis of evolution of Barretts esophagus to esophageal adenocarcinoma

abstract: Complex chromosomal alterations are a hallmark of advanced cancers but rarely seen in normal tissue. The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations that can drive their transformation through focal oncogenic amplifications. However, the etiology and evolution dynamics of these alterations are poorly understood. Here we study chromosomal copy-number evolution in the progression of Barretts esophagus (BE) to esophageal adenocarcinoma (EAC) by multi-regional whole-genome sequencing analysis of BE samples with dysplasia and microscopic EAC foci. Through haplotype-specific copy-number analysis of BE genome evolution, we identified distinct patterns of episodic copy-number evolution consistent with the outcomes of abnormal mitosis and dicentric chromosome breakage. While abnormal mitosis, including whole-genome duplication, accounts for most chromosome or arm-level copy-number changes, segmental copy-number alterations display signatures of multi-generational evolution of unstable dicentric chromosomes. Continuous evolution of dicentric chromosomes through breakage-fusion-bridge cycles and chromothripsis rapidly increases genomic complexity and diversity among BE cells, culminating in the generation of distinct focal amplifications. These mutational processes enable multiple subclones within small dysplastic areas to undergo parallel transformation to cancer following acquisition of distinct oncogenic amplifications. Our results demonstrate how chromosomal instability drives clonal diversification in precancer evolution and promotes tumorigenesis in primary human samples.


title: Quantification of the evolutionary constraint on intronic branch point sequences in the bovine and human genome

abstract: The branch point sequence is a cis-acting intronic motif required for mRNA splicing. Despite their functional importance, branch point sequences are not routinely annotated. Here we predict branch point sequences in 179,476 bovine introns and investigate their variability using a catalogue of 29.4 million variants detected in 266 cattle genomes. We localize the bovine branch point within a degenerate heptamer "nnyTrAy". An adenine residue at position 6, that acts as branch point, and a thymine residue at position 4 of the heptamer are more strongly depleted for mutations than coding sequences suggesting extreme purifying selection. We provide evidence that mutations affecting these evolutionarily constrained residues lead to alternative splicing. We confirm evolutionary constraints on branch point sequences using a catalogue of 115 million SNPs established from 3,942 human genomes of the gnomAD database.


title: Identification and prediction of developmental enhancers in sea urchin embryos

abstract:
BackgroundThe transcription of developmental regulatory genes is often controlled by multiple cis-regulatory elements. The identification and functional characterization of distal regulatory elements remains challenging, even in tractable model organisms like sea urchins.

ResultsWe evaluate the use of chromatin accessibility, transcription and RNA Polymerase II for their ability to predict enhancer activity of genomic regions in sea urchin embryos. ATAC-seq, PRO-seq, and Pol II ChIP-seq from early and late blastula embryos are manually contrasted with experimental cis-regulatory analyses available in sea urchin embryos, with particular attention to common developmental regulatory elements known to have enhancer and silencer functions differentially deployed among embryonic territories. Using the three functional genomic data types, machine learning models are trained and tested to classify and quantitatively predict the enhancer activity of several hundred genomic regions previously validated with reporter constructs in vivo.

ConclusionsOverall, chromatin accessibility and transcription have substantial power for predicting enhancer activity. For promoter-overlapping cis-regulatory elements in particular, the distribution of Pol II is the best predictor of enhancer activity in blastula embryos. Furthermore, ATAC- and PRO-seq predictive value is stage dependent for the promoter-overlapping subset. This suggests that the sequence of regulatory mechanisms leading to transcriptional activation have distinct relevance at different levels of the developmental gene regulatory hierarchy deployed during embryogenesis.


title: Appraisal of Cell Cycle Delay and Cytotoxicity Inducing Potential of 3-epicaryoptin in Allium Cepa L.

abstract: Diterpenoid 3-epicaryoptin (C26H36O9) is abundant in leaves of Clerodendrum inerme, a traditionally used medicinal plant, having insect antifeedant activities. Here, we aim to explore its cell cycle delay and cytotoxic effects in Allium cepa root apical meristem cells. Allium cepa roots were treated with 3-epicaryoptin (100, 150 & 200 {micro}g mL-1 concentration) and colchicine (200 {micro}g mL-1 concentration) for 2, 4, 4+16 h (4 h treatment followed by 16 h recovery) and the induced cell cycle delay and cytotoxic effects were analyzed. The highest metaphase cells frequency for 3-epicaryoptin (150 {micro}g mL-1) and colchicine (200 {micro}g mL-1) treated samples were found to be 66.2{+/-}0.58 % and 82.35{+/-}0.74 % respectively at 4 h treatment. Treatment of 3-epicaryoptin and colchicine increased the aberrant cells %, CA, MN, c-metaphase, and PP cells. Therefore, indiscriminate use of C. inerme in traditional medicine should be restricted and the cell cycle delay and cytotoxicity inducing effects of 3-epicaryoptin needs further exploration for its cancer chemotherapeutic applications.

category: pharmacology and toxicology


title: Paenibacillus sp. strain UY79, isolated from a root nodule of Arachis villosa, displays a broad spectrum of antifungal activity

abstract:
A nodule-inhabiting Paenibacillus sp. strain (UY79) isolated from wild peanut (Arachis villosa) was screened for its antagonistic activity against diverse fungi and oomycetes (Botrytis cinerea, Fusarium verticillioides, Fusarium oxysporum, Fusarium graminearum, Fusarium semitectum, Macrophomina phaseolina, Phomopsis longicolla, Pythium ultimum, Phytophthora sojae, Rhizoctonia solani, Sclerotium rolfsii and Trichoderma atroviride). Results obtained show that Paenibacillus sp. UY79 was able to antagonize these fungi/oomycetes and that agar-diffusible metabolites and enzymes, as well as volatile compounds (different from HCN), participate in the antagonism exerted. We found that Paenibacillus sp. strain UY79 did not affect symbiotic association or growth promotion of alfalfa plants when co-inoculated with rhizobia. By whole genome sequence analysis, we determined that strain UY79 is a new species of Paenibacillus within the Paenibacillus polymyxa complex. Diverse genes putatively involved in biocontrol activity were identified in the UY79 genome. Moreover, according to genome mining and antibiosis assays, strain UY79 would have the capability to modulate the growth of bacteria commonly found in soil/plant communities.

IMPORTANCEPhytopathogenic fungi and oomycetes are responsible for causing devastating losses in agricultural crops. Therefore, there is an enormous interest in the development of effective and complementary strategies that allow the control of the phytopathogens, reducing the input of agrochemicals in croplands. Discovery of new strains with expanded antifungal activities and with a broad spectrum of action is challenging and of great future impact. Diverse strains belonging to the P. polymyxa complex have been reported to be effective biocontrol agents. Results presented here show that the novel discovered strain of Paenibacillus sp. presents diverse traits involved in antagonistic activity against a broad spectrum of pathogens and would be a potential and valuable strain to be further assessed for the development of biofungicides.


title: Regenerating vascular mural cells in zebrafish fin blood vessels are not derived from pre-existing ones and differentially require pdgfrb signaling for their development

abstract: Vascular networks are comprised of endothelial cells and mural cells, which include pericytes and smooth muscle cells. It is well established that new endothelial cells are derived from pre-existing ones during the angiogenic phase of blood vessel growth. By contrast, mural cell ontogeny is less clear with an ongoing debate whether mural cells possess mesenchymal stem cell properties. To elucidate the mechanisms controlling mural cell recruitment during development and tissue regeneration, we studied the formation of zebrafish caudal fin arteries. Mural cells showed morphological heterogeneity: cells colonizing arteries proximal to the body wrapped around them, while those in more distal regions extended protrusions along the proximo-distal vascular axis. Despite these differences, both cell populations expressed platelet-derived growth factor receptor beta (Pdgfrb) and the smooth muscle cell marker myosin heavy chain 11a (Myh11a). Loss of Pdgfrb signalling during development or tissue regeneration resulted in a substantial decrease in mural cells at the vascular front, while those proximal to the body were less affected. Using lineage tracing, we demonstrate that precursor cells located in periarterial regions of the caudal fin and expressing Pgdfrb can give rise to mural cells, while in regeneration newly formed mural cells were not derived from pre-existing ones. Together, our findings reveal conserved roles for pdgfrb signalling in development and regeneration, while at the same time illustrating a limited capacity of mural cells to self-renew or contribute to other cell types during tissue regeneration.


title: Single cell trajectory modeling identifies a primitive trophoblast state defined by BCAM enrichment

abstract:
In early placental development, progenitor cytotrophoblasts (CTBs) differentiate along one of two cellular trajectories: the villous or extravillous pathways. CTBs committed to the villous pathway fuse with neighboring CTBs to form the outer multinucleated syncytiotrophoblast (SCT), while CTBs committed to the extravillous pathway differentiate into invasive extravillous trophoblasts (EVT). Unfortunately, little is known about the processes controlling human CTB progenitor maintenance and differentiation. To address this, we established a single cell RNA sequencing (scRNA-seq) dataset from first trimester placentas to identify cell states important in trophoblast progenitor establishment, renewal, and differentiation. Multiple distinct trophoblast states were identified, representing progenitor CTBs, column CTBs, SCT precursors, and EVT. Lineage trajectory analysis identified a progenitor origin that was reproduced in human trophoblast stem cell organoids. Heightened expression of basal cell adhesion molecule (BCAM) defined this primitive state, where BCAM enrichment or gene silencing resulted in enhanced or diminished organoid growth. Together, this work describes at high-resolution trophoblast heterogeneity within the first trimester, resolves gene networks within human CTB progenitors, and identifies BCAM as a primitive progenitor marker and possible regulator.

Summary StatementLineage trajectory modeling identifies multiple human progenitor trophoblast states and defines trophoblast differentiation kinetics, where BCAM-expressing progenitors demonstrate enhanced regenerative ability.


title: Cell trajectory modeling identifies a primitive trophoblast state defined by BCAM enrichment


title: High content 3D imaging method for quantitative characterization of organoid development and phenotype.

abstract: Quantitative analysis on a large number of organoids can provide meaningful information from the morphological variability observed in 3D organotypic cultures, called organoids. Yet, gathering statistics of growing organoids is currently limited by existing imaging methods and subsequent image analysis workflows that are either restricted to 2D, limited in resolution, or with a low throughput. Here, we present an automated high content imaging platform synergizing high density organoid cultures with 3D live light-sheet imaging. The platform is an add-on to a standard inverted microscope. We demonstrate our capacity to collect libraries of 3D images at a rate of 300 organoids per hour, enabling training of artificial intelligence-based algorithms to quantify the organoid morphogenetic organization at multiple scales with subcellular resolution. We validate our approach on different organotypic cell cultures (stem, primary, and cancer), and quantify the development of hundreds of neuroectoderm organoids (from human Embryonic Stem Cells) at cellular, multicellular and whole organoid scales.


title: Consensus tandem tetratricopeptide repeat proteins are complex superhelical nanosprings

abstract:
Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs) -- superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between folded and unfolded conformations. We rationalise the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the centre towards both termini simultaneously. Strikingly, we also directly observe the proteins superhelical tertiary structure in the force signal. Using protein engineering, crystallography and single-molecule experiments, we show how the superhelical geometry can be altered by carefully placed amino-acid substitutions and examine how these sequence changes affect intrinsic repeat stability and inter-repeat coupling. Our findings provide the means to dissect and modulate repeat-protein stability and dynamics, which will be essential for researchers to understand the function of natural repeat proteins and to exploit artificial repeats proteins in nanotechnology and biomedical applications.

Significance statementRepetition of biological building blocks is crucial to modulating and diversifying structure and function of biomolecules across all organisms. In tandem-repeat proteins, the linear arrangement of small structural motifs leads to the formation of striking supramolecular shapes. Using a combination of single-molecule biophysical techniques and modelling approaches, we dissect the spring-like nature of a designed repeat protein and demonstrate how its shape and mechanics can be manipulated by design. These novel insights into the biomechanical and biochemical characteristics of this protein class give us a methodological basis from which to understand the biological functions of repeat proteins and to exploit them in nanotechnology and biomedicine.


title: Dynamic phase separation of the androgen receptor and its coactivators to regulate gene expression.

abstract: Numerous cancers, including prostate cancer (PCa), are addicted to transcription programs driven by superenhancers (SEs). The transcription of genes at SEs is enabled by the formation of phase-separated condensates by transcription factors and co-activators with intrinsically disordered regions. The androgen receptor (AR), main oncogenic driver in PCa, contains large disordered regions and is co-recruited with the co-activator MED1 to SEs to promote oncogenic programs. In this work, we show that dynamic AR-rich, liquid-like foci form in PCa models upon androgen stimulation and correlate with AR transcriptional activity. The co-activator MED1 plays an essential role in the formation of AR foci while AR antagonists hinder their formation. These results suggest that enhanced compartmentalization of AR and co-activators at SEs may play an important role in the activation of oncogenic transcription programs in PCa. A better understanding of the assembly and the regulation of these AR-rich compartments may provide novel therapeutic options.


title: Canagliflozin increases adenoma burden in female APCMin/+ mice

abstract: The diabetes drug canagliflozin acts primarily by inhibiting glucose reuptake by the sodium glucose transporter 2 (SGLT2) in the kidney proximal tubule, thereby lowering serum glucose levels. Canagliflozin also acts on SGLT1, a related transporter responsible for glucose uptake in the small intestine and more distal kidney tubules. Several cancers overexpress SGLT1 and SGLT2, where these transporters fuel tumor metabolism. A recent study by NIAs Interventions Testing Program (ITP) showed that canagliflozin treatment extends lifespan in male mice. Since cancer is the major cause of death in most mouse strains, including the UM-HET3 strain used by the ITP, this observation suggests that canagliflozin might exert anti-cancer effects in this context. Here, we treated a commonly-used mouse neoplasia model -- the intestinal adenoma-prone APCMin/+ strain -- with canagliflozin, to test the effects of drug treatment on tumor burden. Surprisingly, canagliflozin increased the total area of intestine involved by adenomas, an effect that was most marked in the distal intestine and in female mice. Immunohistochemical analysis suggested that canagliflozin may not influence adenoma growth via direct SGLT1/2 inhibition in neoplastic cells themselves. Instead, our results are most consistent with a model whereby canagliflozin aggravates adenoma development by altering the anatomic distribution of intestinal glucose absorption, as evidenced by increases in postprandial GLP-1 levels consistent with delayed glucose absorption. Our results suggest that canagliflozin exacerbates adenomatosis in the APCMin/+ model via complex, cell-non-autonomous mechanisms, and hint that sex differences in incretin responses may underlie differential effects of this drug on lifespan.


title: Decoding regulatory specificity of human ribosomal proteins on global gene expression

abstract: Human ribosomes, made of around 80 ribosomal proteins (RPs) and four ribosomal RNAs, have long been thought as uniform passive protein-making factories with little regulatory function. Recently, accumulating evidence showed heterogeneity of RP composition in ribosomes responsible for regulating gene expression in development and tumorigenesis. However, a comprehensive understanding of regulatory spectrum of RPs is still lacking. In this study, we conducted a systematic survey of regulatory specificity of human RPs on global gene expression. We quantified translational and transcriptional changes of gene expression upon deficiency of 75 RPs, including 44 from the large subunit (60S) and 31 from the small subunit (40S), by ribosomal profiling and RNA sequencing analysis. We showed that RP deficiency induced diverse expression changes, particularly at the translational level. RPs were subjected to co-translational regulation under ribosomal stress where deficiency of the 60S and the 40S RPs had opposite effects on the two subunits. RP deficiency perturbed expression of genes related to cell cycle, cellular metabolism, signal transduction and development. Deficiency of RPs from the 60S led to a greater repression effect on cell growth than that from the 40S by perturbing P53 signaling and cell cycle pathways. To demonstrate functional specificity of RPs, we showed that RPS8 deficiency stimulated cellular apoptosis and RPL13 or RPL18 deficiency promoted cellular senescence. We also showed that RPL11 and RPL15 played important roles in retina development and angiogenesis, respectively. Overall, our study demonstrated a widespread regulatory role of RPs in controlling cellular activity, providing an important resource which can offer novel insights into ribosome regulation in human diseases and cancer.


title: Decoding regulatory specificity of human ribosomal proteins


title: Single Cell, Single Nucleus and Spatial RNA Sequencing of the Human Liver Identifies Hepatic Stellate Cell and Cholangiocyte Heterogeneity

abstract: The critical functions of the human liver are coordinated through the interactions of hepatic parenchymal and non-parenchymal cells. Recent advances in single cell transcriptional approaches have enabled an examination of the human liver with unprecedented resolution. However, dissociation related cell perturbation can limit the ability to fully capture the human livers parenchymal cell fraction, which limits the ability to comprehensively profile this organ. Here, we report the transcriptional landscape of 73,295 cells from the human liver using matched single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq). The addition of snRNA-seq enabled the characterization of interzonal hepatocytes at single-cell resolution, revealed the presence of rare subtypes of hepatic stellate cells previously only seen in disease, and detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and NK cells were only distinguishable using scRNA-seq, highlighting the importance of applying both technologies to obtain a complete map of tissue-resident cell-types. We validated the distinct spatial distribution of the hepatocyte, cholangiocyte and stellate cell populations by an independent spatial transcriptomics dataset and immunohistochemistry. Our study provides a systematic comparison of the transcriptomes captured by scRNA-seq and snRNA-seq and delivers a high-resolution map of the parenchymal cell populations in the healthy human liver.


title: Accessory genomic epidemiology of co-circulating Acinetobacter baumannii clones

abstract: A. baumannii has become one of the most important multidrug resistant nosocomial pathogens all over the world. Nonetheless, very little is known about the diversity of A. baumannii lineages co-existing in hospital settings. Here, using whole-genome sequencing, epidemiological data and antimicrobial susceptibility tests, we uncover the transmission dynamics of extensive and multidrug resistant A. baumannii in a tertiary hospital for a decade. Our core genome phylogeny of almost 300 genomes suggests that there were several introductions of lineages from international clone 2 into the hospital. The molecular dating analysis shows that these introductions happened between 2004 and 2015. Furthermore, using the accessory genome, we show that these lineages were extensively disseminated across many wards in the hospital. Our results demonstrate that accessory genome variation can be a very powerful tool for conducting genomic epidemiology. We anticipate future studies employing the accessory genome as a phylogenomic marker over very short microevolutionary scales.


title: Gene structure, differential exon usage, and expression of the testis long intergenic non-protein coding RNA 1016 in humans reveals isoform-specific roles in controlling biological processes

abstract:
Long noncoding RNAs (lncRNAs) have emerged as critical regulators of biological processes. The constant expansion of newly-identified lncRNA genes requires that each one be comprehensively annotated to understand its molecular functions. Here, we describe a detailed characterization of the gene which encodes long intergenic non-protein coding RNA 01016 (LINC01016, a.k.a., LncRNA1195) with a focus on its structure, exon usage, and expression in human and macaque tissues. In this study, we show that it is exclusively conserved among non-human primates, suggesting its recent evolution and is expressed and processed into 12 distinct RNAs in testis, cervix, and uterus tissues. Further, we integrate de novo annotation of expressed LINC01016 transcripts and isoform-dependent gene expression analyses to show that human LINC01016 is a multi-exon gene, processed through differential exon usage with isoform-specific functions. Furthermore, in gynecological cancers, such as cervical squamous cell carcinoma and uterine corpus endometrial carcinoma, LINC01016 is downregulated; however, its higher expression is predictive of relapse-free survival in these cancers. Collectively, these analyses reveal that, unlike coding RNAs, lncRNA isoforms are differentially regulated and precisely processed in specific tissues to perform distinct biological roles.

One sentence summaryThe distinct molecular role of LINC01016 isoforms reveals intricate biology associated with lncRNA transcription and processing.


title: Swimming kinematics and performance of spinal transected lampreys with different levels of axon regeneration

abstract: Neural and functional recovery in lampreys from spinal cord transection has been well documented. However, the extent of axon regeneration is highly variable and it is not known whether it is related to the level of behavioral recovery. To address this, we examined how swimming kinematics were related to axon regeneration by quantifying the relationship between swimming performance and percent axon regeneration of transected lampreys after 11 weeks of recovery. We found that swimming speed was not related to percent axon regeneration but it was closely related to body wave frequency and speed. However, wave frequency and speed varied greatly within individuals which resulted in swimming speed also varying within individuals. In fact, most recovered individuals, regardless of percent axon regeneration, could swim at fast and slow speeds. However, none of the transected individuals were able to generate body waves as large as the control lampreys. In order to swim faster, transected lampreys increased their wave frequencies and, as a result, transected lampreys had much higher frequencies than control lamprey at comparable swimming velocities. These data suggest that the control lampreys swam more efficiently than transected lampreys. In conclusion, there appears to be a minimal recovery threshold in terms of percent axon regeneration required for lampreys to be capable of swimming, however, there also seems to be a limit to how much they can behaviorally recover.

category: animal behavior and cognition


title: Edge strand of Escherichia coli BepA interacts with immature LptD on the β-barrel assembly machine to direct it to on- and off-pathways

abstract: The outer membrane (OM) of gram-negative bacteria functions as a selective permeability barrier. Escherichia coli periplasmic Zn-metallopeptidase BepA contributes to the maintenance of OM integrity through its involvement in the biogenesis and degradation of LptD, a {beta}-barrel protein component of the lipopolysaccharide translocon. BepA either promotes the maturation of LptD when it is on the normal assembly pathway (on-pathway) or degrades it when its assembly is compromised (off-pathway). BepA performs these functions probably on the {beta}-barrel assembly machinery (BAM) complex. However, how BepA recognizes and directs an immature LptD to different pathways remains unclear. Here, we explored the interactions among BepA, LptD, and the BAM complex. We found that the interaction of the BepA edge-strand located adjacent to the active site with LptD was crucial not only for proteolysis but also, unexpectedly, for assembly promotion of LptD. Site-directed crosslinking analyses indicated that the unstructured N-terminal half of the {beta}-barrel-forming domain of an immature LptD contacts with the BepA edge-strand. Furthermore, the C-terminal region of the {beta}-barrel-forming domain of the BepA-bound LptD intermediate interacted with a "seam" strand of BamA, suggesting that BepA recognized LptD assembling on the BAM complex. Our findings provide important insights into the functional mechanism of BepA.


title: Decoding Object Weight from Electromyography during Human Grasping

abstract: Human urges, desires, and intentions manifest themselves in voluntary action. The final stages of such voluntary action are the muscle contractions that bring it about. Electromyography (EMG) signals measure such muscle contractions. Decoding action contents from EMG require advanced methods for detection, decomposition, processing, and classification and remains a challenge in neuroscience. This study presents a new, time-domain method of classifying EMG for grasping different types of objects. Our proposed method can classify objects with different weights with an accuracy of up to 90%. This progress in neuroscience affects other fields like physiology, brain-computer interfaces, robotics, and so on.

category: bioengineering


title: Silk-based antimicrobial peptide mixed with recombinant spidroin creates functionalized spider silk

abstract: 1.Surgical site infection (SSI) from sutures is a global health emergency because of the antibiotic crisis. Methicillin-resistant S. aureus and other emerging strains are difficult to treat with antibiotics, so drug-free sutures with antimicrobial properties are a solution. Functionalized spider silk protein (spidroin) is a candidate for its extraordinary strength because it has a large repetitive region (150Rep) that forms crosslinked beta-sheets. The antimicrobial peptide HNP-1 can be connected to recombinant spidroin to create antimicrobial silk. Ni-NTA purified 2Rep-HNP1 fusion protein was mixed with recombinant NT2RepCT spidroin at 1:25, 1:20, 1:10 ratios, and spun into silk fibers by syringe-pumping protein into a 100% isopropanol bath. Beta-sheet crosslinking of the identical 2Rep regions tagged the 2Rep-HNP1 permanently onto the resultant silk. Silk showed no sign of degradation in an autoclave, PBS, or EtOH. The tagged 2Rep-HNP1 retained broad-spectrum antimicrobial activity >90% against S. aureus and E. coli as measured by log reduction and radial diffusion assay. Furthermore, a modified expression protocol increased protein yield of NT2RepCT 2.8-fold, and variable testing of the spinning process demonstrated the industrial viability of silk production. We present a promising suture alternative in antimicrobial recombinant spider silk.


title: TPST-dependent and -independent regulation of root development and signaling by PSK LRR receptor kinases in Arabidopsis

abstract:
Tyrosine-sulfated peptides are key regulators of plant growth and development. The disulfated pentapeptide phytosulfokine (PSK) mediates growth via leucine-rich repeat receptor-like kinases, PSKR1 and PSKR2. PSKRs are part of a response module at the plasma membrane that mediates short-term growth responses, but downstream signaling of transcriptional regulation remains unexplored. In Arabidopsis, tyrosine sulfation is catalyzed by a single-copy gene (TPST). We performed a microarray-based transcriptome analysis in the tpst-1 mutant background that lacks sulfated peptides to identify PSK-regulated genes and genes that are regulated by other sulfated peptides. Of the 160 PSK-regulated genes, several had functions in root growth and development in agreement with shorter roots and a higher lateral root density in tpst-1. Further, tpst-1 roots developed higher numbers of root hairs and PSK induced expression of WEREWOLF (WER), its paralog MYB DOMAIN PROTEIN 23 (MYB23) and At1g66800 that maintain non-hair cell fate. The tpst-1 pskr1-3 pskr2-1 mutant showed even shorter roots, and higher lateral root and root hair density than tpst-1 revealing unexpected synergistic effects of ligand and PSK receptor deficiencies. While residual activities may exist, overexpression of PSKR1 in the tpst-1 background induced root growth suggesting that PSKR1 may be active in the absence of sulfated ligands.

HighlightPhytosulfokine (PSK) receptor signaling promotes root elongation, determines lateral root density and maintains non-hair cell fate partially independent of TPST responsible for the activating sulfation of PSK.