title: Analysis of chemical and mechanical behaviors in living cells by continuum mechanics-based FRAP

abstract: Fluorescence recovery after photobleaching (FRAP) is a common technique to analyze the turnover of molecules in living cells. Numerous physicochemical models have been developed to quantitatively evaluate the rate of turnover driven by chemical reaction and diffusion that occurs in a few seconds to minutes. On the other hand, they have limitations in interpreting long-term FRAP responses where intracellular active movement inevitably provides target molecular architectures with additional effects other than chemical reaction and diffusion, namely directed transport and structural deformation. To overcome the limitations, we develop a continuum mechanics-based model that allows for decoupling FRAP response into the intrinsic turnover rate and subcellular mechanical characteristics such as displacement vector and strain tensor. Our approach was validated using fluorescently-labeled beta-actin in an actomyosin-mediated contractile apparatus called stress fibers, revealing spatially distinct patterns of the multi-physicochemical events, in which the turnover rate of beta-actin was significantly higher at the center of the cell. We also found that the turnover rate is negatively correlated with the strain rate along stress fibers but, interestingly, not with the absolute strain magnitude. Moreover, stress fibers are subjected to centripetal flow as well as both contractile and tensile strains along them. Taken together, this novel framework for long-term FRAP analysis allows for unveiling the contribution of overlooked microscopic mechanics to molecular turnover in living cells.


title: Dynamic cholesterol redistribution favors membrane fusion pore constriction

abstract: Previous experiments have shown that cholesterol strongly prefers concave leaflets (which have negative curvature and are typically thin), but cholesterol also orders and thickens bilayers (promoting liquid-ordered phases with positive curvature). Our all-atom molecular dynamics simulations resolve this discrepancy for highly curved fusion pores, similar to those found in the nascent fusion and terminal fission steps of endo-/exocytosis. We find that cholesterol is strongly excluded by bilayer thinning in the fusion pore neck, which is caused by the necks net negative Gaussian (saddle) curvature. Consistent with experiment and our fusion pore simulations, analysis of liquid-disordered planar bilayers indicates that cholesterol prefers overall thicker bilayers, but negative leaflet curvature. The exclusion of cholesterol from the neck because of saddle Gaussian curvature implies that it helps drive fusion pore closure, consistent with literature evidence that membrane reshaping is connected to lateral phase separation.

category: biophysics


title: Structural and Dynamic Effects of PTEN C-terminal Tail Phosphorylation

abstract: The phosphatase and tensin homolog deleted on chromosome ten (PTEN) tumor suppressor gene encodes a tightly regulated dual-specificity phosphatase that serves as the master regulator of PI3K/AKT/mTOR signaling. The carboxy-terminal tail (CTT) is key to regulation and harbors multiple phosphorylation sites (Ser/Thr residues 380-385). CTT phosphorylation suppresses the phosphatase activity by inducing a stable, closed conformation. However, little is known about the mechanisms of phosphorylation-induced CTT-deactivation dynamics. Using explicit solvent microsecond molecular dynamics simulations, we show that CTT phosphorylation leads to a partially collapsed conformation, which alters the secondary structure of PTEN and induces long-range conformational rearrangements that encompass the active site. The active site rearrangements prevent localization of PTEN to the membrane, precluding lipid phosphatase activity. Notably, we have identified phosphorylation-induced allosteric coupling between the interdomain region and a hydrophobic site neighboring the active site in the phosphatase domain. Collectively, the results provide a mechanistic understanding of CTT phosphorylation dynamics and reveal potential druggable allosteric sites in a previously believed clinically undruggable protein.


title: Cell-free Protein Crystallization for Nanocrystal Structure Determination

abstract: In-cell protein crystallization (ICPC) has attracted attention as a next-generation structural biology tool because it does not require multistep purification processes and large-scale crystallization screenings. However, significant issues remain to be solved in context of obtaining various protein crystals in sufficient amounts and quality for structure determination by ICPC. Here, we report the development of cell-free protein crystallization (CFPC), a direct protein crystallization technique which uses cell-free protein synthesis. The most crucial advantages of CFPC are that the reaction scale and time can be minimized and that various reagents can be added during the reaction. We obtained high-quality nano-sized polyhedra crystals, which are produced in insect cells by infection with cytoplasmic polyhedrosis virus, at a 200 L reaction scale within 6 h. We applied this technology to structure determination of crystalline inclusion protein A (CipA) by suppressing twin crystal formation with addition of an inhibitor to the reaction solution. We succeeded in determining a 2.11 [A] resolution structure from the nanocrystals of CipA. This technology, which integrates in-cell and in vitro crystallizations significantly expands the tools available for high throughput protein structure determination, particularly in context of unstable, low-yield, or substrate-binding proteins, which are difficult to analyze by conventional methods.


title: Chronically radiation-exposed survivor glioblastoma cells display poor response to Chk1 inhibition

abstract: Glioblastoma is the most common type of primary brain tumor with an aggressive clinical course, and one of the cornerstones in its treatment regimen is radiotherapy. However, tumor cells surviving after radiation is an indicator of treatment failure; therefore, better understanding of the molecular mechanisms regulating radiotherapy response is of utmost importance. In this study, we generated multiple clinically relevant irradiation exposed models, where we applied fractionated radiotherapy over a long period of time and selected irradiation-survivor (IR-Surv) glioblastoma cell populations. In these cells, we examined the transcriptomic alterations, cell cycle and growth rate changes as well as responses to secondary radiotherapy and DNA damage response (DDR) modulators. Accordingly, IR-Surv cells exhibited slower growth and partly retained their ability to resist secondary irradiation. Transcriptomic analysis revealed that IR-Surv cells upregulated the expression of DDR-related genes, such as CHK1, ATM, ATR, MGMT, and had better DNA repair capacity as an adaptive mechanism. Separately, we report IR-Surv cells to display downregulation of hypoxic signature and the lower induction of hypoxia target genes and not exhibiting the same level of hypoxia-induced changes with naive glioblastoma cells, as gauged by exposing cells to different hypoxia conditions. We also showed that Chk1 inhibition alone or in combination with irradiation significantly reduces cell viability in both naive and IR-Surv cells. However, IR-Surv cells were markedly less sensitive to Chk1 inhibition under hypoxic conditions. In conclusion, consistent with previous reports, we demonstrate the utility of combining DDR inhibitors and irradiation as a successful approach for both naive and IR-Surv glioblastoma cells as long as cells are refrained from hypoxic conditions. Thus, our findings with clinically relevant radiation survivor models will have future translational implications and benefit the optimization of combination therapies for glioblastoma patients.

category: cancer biology


title: Quantitative MHC class-I/-II expression levels in CDK12 mutated prostate cancer patients reveal intratumorally T cell adaptive immune response in tumors

abstract: Treatment of advanced prostate cancer (PCa) remains a therapeutic challenge. The promising improvements provided by new immune-checkpoint blockade (ICB) therapies have shown only modest benefits in a minority of PCa patients. CDK12 defective-PCa is a recently described immune active class of advanced PCa with an elevated mutational burden, high inflammatory levels, and increased immune cell infiltrate. CDK12 has been proposed as a pre-dictive biomarker of treatment response to ICB since partial response treatment has been found among CDK12 PCa patients treated with ICB. This study was designed to investigate potential molecular causes of alterations to immune response among patient tumors with defects of CDK12 in primary (TCGA-PRAD, n = 48) and metastatic (mCRPC-SU2C, n = 10) PCa. We hypothesize that the expression of the MHC-I and -II genes would differ in the CDK12 defective tumors, which would explain the lack of response in some patients treated with ICB. Using RNA-seq data, we were able to distinguish two groups of tumors with CDK12 defective tumors based on differential MHC expression. Tumors with increased MHC levels showed the activation of several pathways associated with the immune system and elevated PD-L1, IDO1, and TIM3 expression. There was also increased composition of CD8+ T cells, B cells, {gamma}{delta} T cells, and M1 Macrophages in tumors with elevated MHC levels. These data suggest that MHC expression and associated immune response pathways may provide new biomarkers for ICB therapeutic response in CDK12-defective PCa.


title: C/EBPα confers dependence to fatty acid anabolic pathways and vulnerability to lipid oxidative stress in FLT3-mutant leukemia

abstract:
While transcription factor C/AAT-enhancer binding protein  (C/EBP) is critical for normal and leukemic differentiation, its role on cell and metabolic homeostasis is largely unknown in cancer. Here, multi-omics analyses uncovered a coordinated activation of C/EBP and Fms-like tyrosine kinase 3 (FLT3) that increased lipid anabolism in vivo and in patients with FLT3-mutant acute myeloid leukemia (AML). Mechanistically, C/EBP regulated FASN-SCD axis to promote fatty acid (FA) biosynthesis and desaturation. We further demonstrated that FLT3 or C/EBP inactivation decreased mono-unsaturated FAs incorporation to membrane phospholipids through SCD downregulation. Consequently, SCD inhibition enhanced susceptibility to lipid redox stress. Moreover, this C/EBP-dependent adaptation of FA homeostasis was exploited by combining FLT3 and glutathione peroxidase 4 (GPX4) inhibition to trigger lipid oxidative stress, enhancing ferroptotic death of FLT3-mutant AML cells. Altogether, our study reveals a C/EBP function in lipid homeostasis and adaptation to redox stress, and a previously unreported vulnerability of FLT3-mutant AML with promising therapeutic application.

SIGNIFICANCEThe transcription factor C/EBP is as a master regulator of normal and leukemic myeloid differentiation. Here, we discovered that C/EBP regulates fatty acid biosynthesis and metabolic adaptation to redox imbalance in leukemic cells. This confers a vulnerability to lipid oxidative stress to FLT3-mutant cells and supports novel therapeutic opportunities for patients.


title: Prostaglandin EP3 receptor-expressing preoptic neurons bidirectionally control body temperature via tonic GABAergic signaling

abstract: The circuit mechanism of the thermoregulatory center in the preoptic area (POA) is unknown. Using rats, here we show prostaglandin EP3 receptor-expressing POA neurons (POAEP3R neurons) as a pivotal bidirectional controller in the central thermoregulatory mechanism. POAEP3R neurons are activated in response to elevated ambient temperature, but inhibited by prostaglandin E2, a pyrogenic mediator. Chemogenetic stimulation of POAEP3R neurons at room temperature reduces body temperature by enhancing heat dissipation, whereas inhibition of them elicits hyperthermia involving brown fat thermogenesis, mimicking fever. POAEP3R neurons innervate sympathoexcitatory neurons in the dorsomedial hypothalamus (DMH) via tonic inhibitory signaling. Although many POAEP3R neuronal cell bodies express a glutamatergic mRNA marker, paradoxically, their axons in the DMH predominantly contain terminals with GABAergic presynaptic proteins, which are increased by chronic heat exposure. These findings indicate that tonic GABAergic inhibitory signaling from POAEP3R neurons is a fundamental determinant of body temperature for thermal homeostasis and fever.

category: physiology


title: Evaluation of Nav1.8 as a therapeutic target for Pitt Hopkins Syndrome

abstract: Pitt Hopkins Syndrome (PTHS) is a rare syndromic form of autism spectrum disorder (ASD) caused by autosomal dominant mutations in the Transcription Factor 4 (TCF4) gene. TCF4 is a basic helix-loop-helix transcription factor that is critical for neurodevelopment and brain function through its binding to cis-regulatory elements of target genes. One potential therapeutic strategy for PTHS is to identify dysregulated target genes and normalize their dysfunction. Here, we propose that SCN10A is an important target gene of TCF4 that is an applicable therapeutic approach for PTHS. Scn10a encodes the voltage-gated sodium channel Nav1.8 and is consistently shown to be upregulated in PTHS mouse models. In this perspective, we review prior literature and present novel data that suggests inhibiting Nav1.8 in PTHS mouse models is effective at normalizing neuron function, brain circuit activity and behavioral abnormalities and posit this therapeutic approach as a treatment for PTHS.

category: neuroscience


title: Quantitative landscapes reveal trajectories of cell-state transitions associated with drug resistance in melanoma

abstract: Drug resistance and tumor relapse in melanoma patients is attributed to a combination of genetic and non-genetic mechanisms. Non-genetic mechanisms of drug resistance commonly involve reversible changes in the cell-state or phenotype, i.e., alterations in molecular profiles that can help cells escape being killed by targeted therapeutics. In melanoma, one of the most common mechanisms of non-genetic resistance is dedifferentiation, which is characterized by loss of melanocytic markers. While various molecular attributes of de-differentiation have been identified, the transition dynamics remains poorly understood. Here, we construct cell-state transition landscapes, to quantify the stochastic dynamics driving phenotypic switch in melanoma based on its underlying regulatory network. These landscapes reveal the existence of multiple alternative paths to resistance - de-differentiation and transition to a hyper-pigmented phenotype. Finally, by visualizing the changes in the landscape during in silico molecular perturbations, we identify combinatorial strategies that can lead to the most optimal outcome - a landscape with the minimal occupancy of the two drug-resistant states. Therefore, we present these landscapes as platforms to screen possible therapeutic interventions in terms of their ability to lead to most favourable patient outcomes.

category: systems biology


title: Induction of a CD8 T cell intrinsic DNA damage and repair response is associated with clinical response to PD-1 blockade in uterine cancer.

abstract:
Despite the success of immune checkpoint blockade (ICB), many patients fail to achieve durable clinical benefit, and the underlying immunological mechanisms remain poorly understood. Here, we investigated immune reinvigoration by ICB in advanced or recurrent hypermutated or microsatellite instability-high, mismatch repair deficient (MSI-H/MMRd) uterine cancer patients treated with anti-PD-1 (nivolumab). CD8 T cells underwent rapid pharmacodynamic proliferation 2-4 weeks after initiating PD-1 blockade. This immunological response, however, did not correlate with clinical response. We hypothesized that the T cell-intrinsic response to proliferative and genotoxic stress might contribute to the disparity between immunological and clinical response. We developed a high-dimensional single cell cytometric platform to simultaneously analyze T cell differentiation with changes in DNA damage and repair (DDR) pathways. This DDR-Immune platform revealed T cell subset-specific patterns of DDR, and distinct DDR pathways induced by different types of DNA damage. Applying this platform to MSI-H/MMRd or hypermutated uterine cancer patients revealed a signature of DDR exemplified by rapid increase in phosphorylated-ATM (pATM) intrinsic to CD8 T cells proliferating in response to PD-1 blockade that distinguished clinical responders and non-responders. ATM regulated transcriptional circuits in T cells were associated with better clinical response to PD-1 blockade. These findings highlight a previously unrecognized role for CD8 T cell-intrinsic DDR as a potential determinant of immune fitness and clinical outcome of PD-1 blockade therapy.

STATEMENT OF SIGNIFICANCEUsing high-dimensional immune and DNA damage response (DDR) profiling in T cell subsets we identified distinct signatures in anti-PD-1 treated MSI-H/hypermutated uterine cancer patients associated with clinical response versus non-response. Thus, T cell-intrinsic DDR is a potential determinant of immune responsiveness and clinical outcome to PD-1 blockade therapy in cancer.

category: immunology


title: Global shark species richness is more constrained by energy than evolutionary history

abstract:
AimTo examine the support of two ecological diversity theories- The Ecological Limits Hypothesis (ELH) and the Niche Conservatism Hypothesis (NCH) - in explaining patterns of global shark diversity.

LocationGlobal scale and two ecological realms: the Tropical Atlantic and the Central Indo-Pacific.

Time PeriodPast 100 years

Major Taxa StudiedWe examined 534 species of sharks and chimaeras, and we performed two subclade analyses on 272 species of ground sharks and 15 species of mackerel sharks.

MethodsWe compared the species richness, mean root distance (MRD), and tree imbalance patterns to those simulated under the ELH and NCH with temperate and tropical centers of origin. We used sea temperature as a proxy for energy availability. We examined the importance of biogeographic history by comparing the model fits between two taxonomic groups, ground and mackerel sharks, and two geographic regions, the Tropical Atlantic realm and Central Indo-Pacific realm.

ResultsThe ELH, temperate-origin model had the best fit to the global dataset and the sub-analyses on ground sharks, mackerel sharks, and the Tropical Atlantic. The NCH temperate-origin model provided the best fit for the Central Indo-Pacific. The {beta} metric of tree symmetry showed the best potential for differentiating between the ELH and NCH models, and the correlation coefficient for temperature vs MRD performed the best at differentiating between temperate and tropical origin of ancestors.

Main ConclusionsThe global and subclade analyses indicate the ELH provides the best explanation for global scale shark diversity gradients even in clades with varying ecology. However, at the realm scale, biogeographic history has an impact on richness patterns. Comparing multiple metrics in relation to a simulation model provides a more rigorous comparison of these models than simple regression fits.

category: ecology


title: Data availability of open T-cell receptor repertoire data, a systematic assessment

abstract: The improvement of next-generation sequencing technologies has promoted the field of immunogenetics and produced numerous immunogenomics data. Modern data-driven research has the power to promote novel biomedical discoveries through secondary analysis of such data. Therefore, it is important to ensure data-driven research with great reproducibility and robustness for promoting a precise and accurate secondary analysis of the immunogenomics data. In scientific research, rigorous conduct in designing and conducting experiments is needed, specifically in scientific and articulate writing, reporting and interpreting results. It is also crucial to make raw data available, discoverable, and well described or annotated in order to promote future re-analysis of the data. In order to assess the data availability of published T cell receptor (TCR) repertoire data, we examined 11,918 TCR-Seq samples corresponding to 134 TCR-Seq studies ranging from 2006 to 2022. Among the 134 studies, only 38.1% had publicly available raw TCR-Seq data shared in public repositories. We also found a statistically significant association between the presence of data availability statements and the increase in raw data availability (p=0.014). Yet, 46.8% of studies with data availability statements failed to share the raw TCR-Seq data. There is a pressing need for the biomedical community to increase awareness of the importance of promoting raw data availability in scientific research and take immediate action to improve its raw data availability enabling cost-effective secondary analysis of existing immunogenomics data by the larger scientific community.

category: scientific communication and education


title: Dynamic mode structure of active turbulence

abstract: Dense suspensions of swimming bacteria exhibit chaotic flow patterns that promote the mixing and transport of resources and signalling chemicals within cell colonies. While the importance of active turbulence is widely recognized, the structure and dynamics of the resulting collective flows are the subject of intense investigation. Here, we combine microfluidic experiments with proper orthogonal decomposition (POD) analysis to quantify the dynamical flow structure of this model active matter system under a variety of conditions. In isotropic bulk turbulence, the modal representation shows that the most energetic flow structures dictate the spatio-temporal dynamics across a range of suspension activity levels. In confined geometries, POD analysis illustrates the role of boundary interactions for the transition to bacterial turbulence, and it quantifies the evolution of coherent active structures in externally applied flows. Beyond establishing the physical flow structures under-pinning the complex dynamics of bacterial turbulence, the low-dimensional representation afforded by this modal analysis offers a potential path toward data-driven modelling of active turbulence.


title: Vibrio tarriae sp. nov., a novel member of the Cholerae clade isolated from across the United States

abstract:
A number of bacteria with close resemblance to Vibrio cholerae has been isolated over the years by the Centers for Disease Control and Prevention (CDC), which could not be assigned a proper taxonomic designation based on preliminary identification methods. Nine such isolates have been found to share 16S rRNA gene identity exceeding 99% with V. cholerae, yet DNA-DNA hybridization (60.4-62.1%) and average nucleotide identity values (94.4-95.1%) were below the species cut-off, indicating a potentially novel species. Phylogenetic analysis of core genomes places this group of isolates in a monophyletic clade, within the "Cholerae clade," but distinct from any other species. Extensive phenotypic characterization reveals unique biochemical properties that distinguish this novel species from V. cholerae. Comparative genomic analysis reveals a unique set of siderophore genes, suggesting that iron acquisition strategies could be vital for the divergence of the novel species from a common ancestor with V. cholerae. Based on genetic, phylogenetic, and phenotypic differences observed, we propose these isolates represent a novel species of the genus Vibrio, for which the name Vibrio tarriae sp. nov. is proposed. Strain 2521-89 (= DSM 112461 = CCUG 75318), isolated from lake water, is the type strain.

Author NotesThe GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 2521-89 is MW773202.1. The genome sequences (genome assemblies) of strains 2521-89, 2523-88, 2015V-1076, 2016V-1018, 2016V-1062, 2017V-1038, 2017V-1070 and 2017V-1124 are deposited under the accession number NZ_CP022353.1, QKKG01000001.1, QKKH00000000.1, QKKI00000000.1, QKKJ00000000.1, QKKK00000000.1, QKKM00000000.1, and QKKN00000000.1, respectively. All the whole genome sequences are deposited under bioproject ID: PRJNA391152.

category: microbiology


title: Meta-analysis using new methods for three-stressor combinations reveal substantial higher-order interactions and emergent properties

abstract: Although natural populations are typically subjected to multiple stressors, most past research has focused on single stressors and two-stressor interactions, with little attention paid to higher-order interactions among three or more stressors. However, higher-order interactions increasingly appear to be widespread. Consequently, we used a recently introduced and improved framework to re-analyze higher-order ecological interactions. We conducted a literature review of the last 100 years (1920-2020) and reanalyzed 151 ecological three-stressor interactions from 45 published papers. We found that 89% (n=134) of the three-stressor combinations resulted in new or different interactions than previously reported. We also found substantial levels of emergent properties-- interactions that are only revealed when all three stressors are present. Antagonism was the most prevalent net interaction whereas synergy was the most prevalent emergent interaction. Understanding multiple stressor interactions is crucial for fundamental questions in ecology and also has implications for conservation biology and population management.


title: ModCRE: a structure homology-modeling approach to predict TF binding in cis-regulatory elements

abstract: Transcription factor (TF) binding is a key component of genomic regulation. There are numerous high-throughput experimental methods to characterize TF-DNA binding specificities. Their application, however, is both laborious and expensive, which makes profiling all TFs challenging. For instance, the binding preferences of [~]25% human TFs remain unknown; they neither have been determined experimentally nor inferred computationally. Here, we introduce ModCRE, a web server implementing a structure homology-modelling approach to predict TF motifs and automatically model higher-order TF regulatory complexes. Starting from a TF sequence or structure, ModCRE predicts a set of motifs for that TF. The predicted motifs are then used to scan the DNA for occurrences of each of them, and the best matches are either profiled with a binding score or collected for their subsequent modeling into a higher-order regulatory complex with DNA, as well as other TFs and co-factors. Moreover, we demonstrate that incorporating high-throughput TF binding data, such as from protein binding microarrays, addresses the protein-DNA structure scarcity problem for deriving statistical potentials. In turn, these statistical potentials are proven to be capable predictors of TF motifs. We also show the conditional advantage of using ModCRE over a nearest-neighbor approach for predicting TF binding sites as well as an improvement in prediction accuracy when using a rank-enrichment selection system. Finally, as case examples, we apply ModCRE to model the interferon beta enhanceosome and the complex of SOX2 and 11 with a nucleosome.

category: bioinformatics


title: Wide-Field Multicontrast Nonlinear Microscopy for Histopathology

abstract: A multicontrast polarimetric wide-field second harmonic generation (SHG) and multiphoton excitation fluorescence (MPF) microscope is optimized for large area imaging of hematoxylin and eosin (H&E) stained and unstained histology slides. The bleaching kinetics of MPF and SHG are examined with various laser intensities at different pulse repetition rates to determine the optimal wide-filed imaging conditions for H&E stained histology slides. Several polarimetric parameters are used to investigate the organization of extracellular matrix collagen in the histology samples.


title: Accelerated mass transfer from frozen thin films during thin-film freeze-drying

abstract: Freeze-drying, or lyophilization, is widely used to produce pharmaceutical solids from temperature-sensitive materials but the process is time and energy inefficient. Herein, using E. coli as a model live organism, whose viability in dry powders is highly sensitive to the water content in the powders, we demonstrated that the drying rate of thin-film freeze-drying (TFFD) is significantly higher than that of the conventional shelf freeze-drying, likely because the large total surface area from the loosely stacked frozen thin films and the low thickness of the thin-films enable faster and more efficient mass transfer during freeze-drying. The highly porous nature and high specific surface area of the thin-film freeze-dried powders may have contributed to the faster mass transfer as well. Moreover, we demonstrated that TFFD can be applied to produce dry powders of E. coli and L. acidophilus with minimum bacterial viability loss (i.e., within one log reduction), and the L. acidophilus dry powder is suitable for intranasal delivery. It is concluded that TFFD technology is promising in addressing the time-and cost-inefficient issue of conventional shelf freeze-drying.

category: pharmacology and toxicology


title: Improved metabolic syndrome and timing of weight loss is crucial for influenza vaccine-induced immunity in obese mice

abstract: Persons with obesity are at higher risk for developing severe complications upon influenza virus infection making vaccination a priority. Yet, studies demonstrate vaccine responses are less effective in obese hosts. In these studies, we examined how the timing of weight loss influenced influenza vaccine efficacy in male and female diet- induced obese mice. Here, we show weight loss post-vaccination is insufficient to rescue poor vaccine efficacy; however, weight loss occurring pre-vaccination successfully improves outcomes at viral challenge. Pre-vaccination weight loss improved vaccine immunogenicity and restored a functional recall response at challenge. Through tracking sera metabolic biomarkers, we propose the metabolic state at the time of vaccination is predictive of vaccine immunogenicity. Altogether, these findings highlight how timing of host-directed interventions is vital when seeking to improve influenza vaccine immunogenicity in obese hosts.


title: A fijivirus major viroplasm protein shows RNA-stimulated ATPase activity by adopting pentameric and hexameric assemblies of dimers

abstract: The fijivirus Mal de Rio Cuarto virus (MRCV) causes a devastating maize disease. Its non-structural protein P9-1, which shows ATPase and RNA binding activities, is the major component of the intracellular viroplasm where virus replication takes place. Here, we established that the 24 C-terminal residues (C-arm) of P9-1 are required for the formation of viroplasm-like structures (VLS) in vivo and for the protein multimerization in vitro. Employing an integrative structural approach, we found that the C-arm is dispensable for P9-1 dimer assembly, but essential for the formation of doughnut-shaped pentamers and hexamers of dimers (decamers and dodecamers). Both assemblies, larger than those reported for other reoviruses, contain disordered loops oriented towards the inner pore of the structures, where RNA binding sites and conditional proteasome-mediated degradation signals (PEST) were predicted. In vitro assays demonstrated that ssRNA binding is favored towards P9-1 (do)decamers over the dimeric {Delta}C-arm version. In addition, although both P9-1 and P9-1{Delta}C-arm catalyze the hydrolysis of ATP with similar activity values, an RNA-stimulated ATPase activity was only observed in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. Computational studies revealed a stronger preference of phosphate moieties to the decamer in the pore and the C-arm regions, suggesting that the allosteric communication between the ATP and RNA binding sites is favored with this protein arrangement. Overall, our work reveals the structural versatility of a major viroplasm protein providing unprecedented insights into fijivirus viroplasm assembly and function and establishes the structural basis for the development of antiviral strategies against the Mal de Rio Cuarto crop disease.

category: biochemistry


title: Structural dynamics of Zika Virus NS1 via a reductionist approach reveal the disordered nature of its beta roll domain in isolation

abstract:
Flavivirus Non-structural 1 (NS1) protein performs multiple functions such as host immune evasion, interaction with complement system factors, membrane rearrangement, etc. Therefore, it is highly plausible that significant structural and folding dynamics of NS1 might play a role in its multifunctionality. The dimeric structures of NS1 of multiple flaviviruses, including Zika virus (ZIKV), are available. However, its domain-wise dynamics perspective has not been explored so far. Therefore, it is of utmost importance to understand the structural conformations of NS1 and its domains in isolation, possibly highlighting the implications on the overall NS1 protein dynamics. Here, we have employed extensively long molecular dynamic (MD) simulations to understand the role of monomer, dimer, and a reductionist approach in understanding the dynamics of the three structural domains (i.e., {beta}- roll, wing, and {beta}-ladder) in isolation. Further, we experimentally validated our findings using CD spectroscopy and confirmed the intrinsically disordered behavior of NS1 {beta}-roll in isolation and lipid mimetic environments. We also found that the {beta}-ladder domain is highly flexible during long simulations. Therefore, we believe this study may have implications for significant dynamics played by NS1 protein, specifically during oligomerization of NS1.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/488568v1_ufig1.gif" ALT="Figure 1">
View larger version (37K):
org.highwire.dtl.DTLVardef@1b2928org.highwire.dtl.DTLVardef@12b0b68org.highwire.dtl.DTLVardef@f9da77org.highwire.dtl.DTLVardef@175fbc1_HPS_FORMAT_FIGEXP  M_FIG C_FIG Schematic representation of the ZIKV NS1 protein and the models that we have used in this study.


title: Protein kinase D promotes prostate cancer cell bone metastasis by positively regulating Runx2 in a MEK/ERK1/2-dependent manner

abstract: Most cancer patients die because of tumor metastasis not due to tumors at the primary site. Prostate tumors in advance stages frequently metastasize to the bone, which is the main cause of death for the disease. The family of protein kinase D (PKD) has been implicated in prostate cancer development, however, its role in prostate cancer metastasis has not been investigated. In this study, we examined the contribution of PKD to the metastatic potential of prostate cancer cells and the impact of PKD inhibition on prostate cancer bone metastasis in vivo. Our data showed that depletion of PKDs by siRNA or inhibition of PKD by an inhibitor CRT0066101 in a highly invasive prostate cancer PC3-ML cells potently inhibited colony formation and cell migration. Furthermore, depletion or inhibition of PKD significantly blocked invasion of PC3-ML cells and suppressed expression of genes related to bone metastasis. The reduced invasive activity resulted from PKD depletion was in part mediated through the transcription factor Runx2 as its silencing decreased PKD-mediated metastatic gene expression. Mechanistically, our data indicate that PKD modulated Runx2 target gene expression through the MEK/ERK1/2 signaling axis. Additionally, we examined whether PKD inhibitor CRT0066101 could prevent prostate cancer bone metastasis in a mouse model of metastasis, where intracardiac injection of PC3-ML cells led to metastasis of cells to the bone. We found that CRT0066101 potently decreased the frequency of micrometastases in mouse bone. These results indicate that PKDs play an important role in bone metastasis of prostate cancer cells and its inhibition may be beneficial for treatment of advanced stages of cancer.


title: Plant-on-Chip: core morphogenesis processes in the tiny plant Wolffia australiana

abstract:
A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the tiniest known angiosperm to overcome this challenge and provide an ideal model system for plant morphogenesis. We present a detailed morphological description of the monocot Wolffia australiana, as well as high-quality genome information. Further, we developed the Plant-on-Chip culture system and demonstrate the application of advanced technologies such as snRNA-seq, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate how W. australiana can open a new horizon for deciphering the core regulatory mechanisms of plant morphogenesis.

SignificanceWhat is the core morphogenetic process in angiosperms, a plant like a tree indeterminately growing, or a bud sequentially generating limited types of organs? Wolffia australiana, one of the smallest angiosperms in the world may help to make a distinction. Wolffia plantlet constitutes of only three organs that are indispensable to complete life cycle: one leaf, one stamen and one gynoecium. Before the growth tip is induced to flower, it keeps branching from the leaf axil and the branches separate from the main plantlet. Here we present a high-quality genome of W. australiana, detailed morphological description, a Plant-on-Chip cultural system, and some principle-proof experiments, demonstrating that W. australiana is a promising model system for deciphering core developmental program in angiosperms.

category: plant biology


title: Dual orexin antagonist DORA-22 suppressed posttraumatic seizures and enhances GABAergic inhibition in dentate granule cells

abstract:
BackgroundTraumatic brain injury (TBI) can result in posttraumatic epilepsy (PTE) and sleep disturbances. We hypothesized that treatment with sleep aids after TBI can ameliorate PTE.

MethodsCD-1 mice underwent controlled cortical impact (CCI), sham craniotomy or no craniotomy. Sham and CCI groups underwent a month-long treatment with sleep aids including a dual orexin antagonist (DORA-22) or THIP (gaboxadol). We performed week-long EEG recordings during week-1 of treatment and again at months 1, 2 and 3. Seizure analysis occurred at all-time points and sleep analysis occurred in week-1 and month-1 recordings in all groups. Subsets of animals in sleep aid-treated and untreated CCI, and sham groups were subjected to voltage clamp experiments.

ResultsDORA-22 treated group had seizures at week-1 but none at months 1-3. TBI reduced amplitude and frequency of miniature inhibitory synaptic currents (mIPSCs) in dentate granule cells and these changes were rescued by DORA-22 treatment. Sleep analysis showed that DORA-22 increased non-rapid eye movement sleep (NREM) in the first 4hours of lights-off whereas THIP increased REM sleep in the first 4-hours of lights-on in week-1, At month-1 both treatments reduced time in NREM during lights-off. TBI increased NREM delta power (N{Delta}) along with loss of the homeostatic overnight decline of N{Delta} in week-1 regardless of treatment. DORA-22 and THIP treatment restored N{Delta} to levels similar to no craniotomy animals at month-1.

ConclusionsDORA-22 treatment suppressed posttraumatic seizures possibly due to enhanced GABAergic inhibition in dentate granule cells. DORA-22 may have therapeutic potential in suppressing PTE.

Summary for Social MediaTraumatic brain injury (TBI) can result is posttraumatic epilepsy and sleep disturbances. There are no treatments to prevent these complications. We tested whether treatment with sleep aids after TBI can mitigate seizures and sleep disturbances. We found that a sleep aid DORA-22 but not THIP suppressed post traumatic seizures possibly be enhancing GABAergic inhibition in the hippocampus. Sleep analysis showed that TBI disrupts the sleep homeostatic drive and DORA treatment restored it. Findings may lead to potential disease modifying therapy for posttraumatic epilepsy.


title: Asymmetric activation of microglia in the hippocampus drives anxiodepressive consequences of trigeminal neuralgia

abstract: Patients suffering from trigeminal neuralgia (TN) are often accompanied by anxiety and depression. Whether and how microglia are involved in TN-induced anxiodepressive remains unclear. Here, we unconventionally report that TN activates ipsilateral but not contralateral hippocampal microglia, upregulates ipsilateral hippocampal ATP and interleukin1{beta} (IL-1{beta}) levels, impairs ipsilateral hippocampal long-term potentiation (LTP), and induces anxiodepressive-like behaviors in a time-dependent manner in rodents. Specifically, activation of ipsilateral hippocampal microglia is necessary for TN-induced anxiodepressive-like behaviors; and unilateral activating hippocampal microglia is sufficient to elicit an anxiodepressive state and impair LTP. Knockdown of ipsilateral hippocampal P2X7 receptor prevented TN-induced microglial activation and anxiodepressive-like behaviors. Furthermore, we demonstrate that microglia-derived IL-1{beta} mediates microglial activation-induced anxiodepressive-like behaviors and LTP impairment. Together, these findings suggest that priming of microglia with ATP/P2X7R in the ipsilateral hippocampus drives pain-related anxiodepressive-like behaviors via IL-1{beta}. Our results also reveal an asymmetric role of the bilateral hippocampus in TN-induced anxiety and depression.


title: mGlu2 and mGlu3 receptor negative allosteric modulators affect the discriminative stimulus effects of alcohol

abstract: All drugs with the potential for addiction liability, including alcohol, produce distinct interoceptive stimulus effects that influence drug intake. Our lab has shown involvement of Group II metabotropic receptors (mGlu2/3) in the expression of alcohol interoceptive effects using a nonspecific mGlu2/3 agonist and antagonist. Furthermore, the anterior insular cortex (aIC) is a hub for interoceptive processing, including the interoceptive effects of alcohol. Therefore, the goal of the present work was to assess the independent functional contribution of mGlu2 and mGlu3, and the role of mGlu3 in the aIC, in the interoceptive effects of alcohol. As such, male, Long-Evans rats were trained to discriminate the stimulus effects of alcohol (2 g/kg, i.g.) from water (i.g.) using an operant, 2-lever drug discrimination procedure. To assess involvement of mGlu2 and mGlu3 in the discriminative stimulus effects of alcohol, rats were administered the mGlu2 negative allosteric modulator (NAM, VU6001966; 0, 3, 6 mg/kg, i.p.) and the mGlu3-NAM (VU6010572; 0, 3, 6, 12 mg/kg, i.p.) before alcohol (2 g/kg, i.g.). mGlu2-NAM (6 mg/kg) and mGlu3-NAM (12 mg/kg) decreased alcohol-appropriate lever responses without affecting response rate, reflecting diminished interoceptive alcohol sensitivity. mGlu3-NAM microinjection (3 g) into the aIC before alcohol decreased response rate to such an extent that alcohol-appropriate lever responding could not be determined. As such, mGlu3-NAM was tested in the aIC before water administration to determine if VU6010572 substitutes for alcohol. mGlu3-NAM VU6010572 (3 g) in the aIC produced partial alcohol-like stimulus effects without affecting response rate. These data indicate that the newly developed mGlu2 and mGlu3 NAMs can modulate interoceptive effects of alcohol. The opposing effects of systemic vs. microinjected (aIC) mGlu3-NAM on alcohol interoceptive effects may reflect differences in receptor binding, a functionally relevant metabolite, and/or brain region differences in receptor function.


title: Peptidergic signaling controls the dynamics of sickness behavior in Caenorhabditis elegans

abstract: Pathogenic infection elicits sickness behaviors that promote recovery and survival of the host. For example, following infection with the pathogenic bacterium Pseudomonas aeruginosa PA14, the nematode Caenorhabditis elegans modifies its sensory preferences to avoid the pathogen. Here we identify antagonistic neuromodulatory circuits that shape this sickness behavior. Using an unbiased cell-directed neuropeptide screen, we show that AVK neurons upregulate and release FMRFamide-like FLP-1 neuropeptides during infection to drive pathogen avoidance. Manipulations that increase or decrease AVK signaling accelerate or delay pathogen avoidance, respectively, implicating AVK in the dynamics of sickness behavior. FLP-1 neuropeptides act via the G-protein-coupled receptor DMSR-7 in RIM/RIC neurons to reduce tyraminergic/octopaminergic signaling that opposes pathogen avoidance. RIM/RIC neurons relay parallel signals from neuropeptides and the cytokine TGF-{beta} that represent internal and external regulators of pathogen avoidance. Our results demonstrate that antagonism between neuromodulatory systems results in slow, graded transitions between alternative behavioral states.


title: CD200R1 promotes IL-17 production by ILC3s, by enhancing STAT3 activation

abstract:
Psoriasis is a common chronic inflammatory skin disease with no cure. It is driven by the IL-23/IL-17A axis and TH17 cells but, recently group 3 innate lymphoid cells (ILC3s) have also been implicated. However, the development, and factors regulating the activity of ILC3s remain incompletely understood.

Immune regulatory pathways are particularly important at barrier sites such as the skin, gut and lung, which are exposed to environmental substances and microbes. CD200R1 is an immune regulatory cell surface receptor which inhibits proinflammatory cytokine production in myeloid cells. CD200R1 is also highly expressed on ILCs, where its function remains largely unexplored. We previously observed reduced CD200R1 signalling in psoriasis skin, suggesting that dysregulation may promote disease. Here we show that contrary to this, psoriasis models are less severe in CD200R1-deficient mice due to reduced IL-17 production. Here we uncover a key cell-intrinsic role for CD200R1 in promoting IL-23-driven IL-17A production by ILC3s, by promoting STAT3 activation. CD200R1 is expressed on ILC precursors and is particularly high on neonatal ILC3s, suggesting CD200R1 may function during ILC development. Therefore, CD200R1 is required on ILC3s, potentially during their development, to promote IL-23-stimulated STAT3 activation triggering optimal IL-17 production.


title: Characterization of enterovirus D68 infection in four nonhuman primate species

abstract: Human enterovirus D68 (EV-D68) is a globally reemerging respiratory pathogen that is associated with the development of acute flaccid myelitis (AFM) in children. Currently, there are no approved vaccines or treatments for EV-D68 infection, and there is a paucity of data related to the virus and host specific factors that predict disease severity and progression to the neurologic syndrome. Published animal models of EV-D68 infection to date have been limited to mice, cotton rat and ferrets, and investigation of the susceptibility of nonhuman primate (NHP) species to contemporary EV-D68 isolates has not yet been reported. In this study, we challenged juvenile NHPs - cynomolgus macaques, rhesus macaques, pigtailed macaques, and African green monkeys - with one of five different 2014 or 2018 EV-D68 isolates by the respiratory route. Animals were monitored for clinical respiratory and neurologic signs, and serially collected nasal swabs, bronchoalveolar lavage fluid (BALF) and cerebrospinal fluid (CSF) were evaluated for EV-D68 RNA and infectious virus. Infection with 2014 and 2018 EV-D68 isolates resulted in mild respiratory and gastrointestinal disease in some animals, but no evidence of neurological disease. Neither EV-D68 RNA nor infectious virus could be detected from any sample collected from animals challenged with 2014 EV-D68 isolates. Limited viral shedding - based on viral RNA quantified from nasal swabs and BALF - was detected from some animals infected with 2018 EV-D68 isolates. No virus was detectable in CSF. The rate of seroconversion was 100% for cynomolgus macaques infected with the 2018 EV-D68 isolates, but averaged between 0-50% for the 2014 isolates. Based on the results of this study, there is some evidence that infection with 2018 EV-D68 isolates may be more reliable at establishing limited infection than 2014 EV-D68 isolates. Regardless of virus isolate, however, EV-D68 infection of juvenile NHP species resulted in mild and nonspecific clinical disease and limited viral shedding. These data suggest that further refinements to the NHP model system (e.g., immunosuppression and/or direct viral inoculation) may be required to reproduce EV-D68 infection of the central nervous system and the associated AFM phenotype.


title: Gene editing is suitable to treat GM1 Gangliosidosis: a proof-of-concept study

abstract: Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced {beta}-galactosidase ({beta}-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. Here, we show that 41% of GLB1 pathogenic SNVs might be cured by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring a therapeutic level of {beta}-gal activity. Unbiased off-target DNA analysis did not detect off-target editing activity in treated patients cells except a bystander edit without consequences on {beta}-gal activity. Altogether our results suggest that gene editing is an alternative strategy to cure GM1 gangliosidosis, by correcting the root cause of disease and avoiding repetitive adeno-associated virus injections.

category: genetics


title: Development of a high-density 665 K SNP array for rainbow trout genome-wide genotyping

abstract: Single nucleotide polymorphism (SNP) arrays, also named << SNP chips >>, enable very large numbers of individuals to be genotyped at a targeted set of thousands of genome-wide identified markers. We used preexisting variant datasets from USDA, a French commercial line and 30X-coverage whole genome sequencing of INRAE isogenic lines to develop an Affymetrix 665 K SNP array (HD chip) for rainbow trout. In total, we identified 32,372,492 SNPs that were polymorphic in the USDA or INRAE databases. A subset of identified SNPs were selected for inclusion on the chip, prioritizing SNPs whose flanking sequence uniquely aligned to the Swanson reference genome, with homogenous repartition over the genome and the highest Minimum Allele Frequency in both USDA and French databases. Of the 664,531 SNPs which passed the Affymetrix quality filters and were manufactured on the HD chip, 65.3% and 60.9% passed filtering metrics and were polymorphic in two other distinct French commercial populations in which, respectively, 288 and 175 sampled fish were genotyped. Only 576,118 SNPs mapped uniquely on both Swanson and Arlee reference genomes, and 12,071 SNPs did not map at all on the Arlee reference genome. Among those 576,118 SNPs, 38,948 SNPs were kept from the commercially available medium-density 57K SNP chip. We demonstrate the utility of the HD chip by describing the high rates of linkage disequilibrium at 2 kb to 10 kb in the rainbow trout genome in comparison to the linkage disequilibrium observed at 50 kb to 100 kb which are usual distances between markers of the medium-density chip.

category: genomics


title: An atlas of genetic scores to predict multi-omic traits

abstract: Genetically predicted levels of multi-omic traits can uncover the molecular underpinnings of common phenotypes in a highly efficient manner. Here, we utilised a large cohort (INTERVAL; N=50,000 participants) with extensive multi-omic data for plasma proteomics (SomaScan, N=3,175; Olink, N=4,822), plasma metabolomics (Metabolon HD4, N=8,153), serum metabolomics (Nightingale, N=37,359), and whole blood Illumina RNA sequencing (N=4,136). We used machine learning to train genetic scores for 17,227 molecular traits, including 10,521 which reached Bonferroni-adjusted significance. We evaluated genetic score performances in external validation across European, Asian and African American ancestries, and assessed their longitudinal stability within diverse individuals. We demonstrated the utility of these multi-omic genetic scores by quantifying the genetic control of biological pathways and by generating a synthetic multi-omic dataset of UK Biobank to identify disease associations using a phenome-wide scan. Finally, we developed a portal (OmicsPred.org) to facilitate public access to all genetic scores and validation results as well as to serve as a platform for future extensions and enhancements of multi-omic genetic scores.


title: Calculating ATP production rates from oxidative phosphorylation and glycolysis during cell activation

abstract: The balance between oxidative phosphorylation and glycolysis is shifted in a variety of cell types and tissues upon activation, proliferation, and differentiation. However, description of these metabolic switches has been mostly qualitative, particularly upon acute activation signals. We therefore applied a method to quantify rates of ATP production from oxygen consumption and extracellular acidification data obtained from the Seahorse XF Analyzer to study cell growth and activation. The method detects substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. Furthermore, quantifying ATP utilization during pro-inflammatory macrophage activation reveals the hallmark increase in glycolysis is not merely a compensatory response to decreased oxidative phosphorylation upon LPS treatment. Rather, these changes occur independently, and the profound increases in glycolysis and total ATP utilization upon activation is largely driven by MyD88-linked signaling and independent of oxidative phosphorylation.


title: Subchondral bone and synovial fluid metabolomic profiles are altered in injured and contralateral limbs 7 days after non-invasive joint injury in skeletally-mature C57BL/6 mice

abstract:
ObjectivePost-traumatic osteoarthritis (PTOA) is a common long-term outcome following ACL injury. However, early changes to bone and synovial fluid after ACL injury are not sufficiently understood. The objectives of this study were to (1) evaluate whether acute bone loss one week after ACL injury is accompanied by altered subchondral bone plate modulus, (2) determine if bone changes are localized to the injured limb or extend to the contralateral-to-injured limb compared with sham-loaded controls, and (3) identify shifts in synovial fluid metabolism unique to injured limbs.

DesignFemale C57Bl\6N mice (19 weeks at injury) were subjected to either a single tibial compression overload to simulate ACL injury (n=8) or a small pre-load (n=8). Mice were euthanized 7 days after injury, and synovial fluid was immediately harvested for metabolomic profiling. Bone microarchitecture, bone formation, and subchondral bone modulus at the proximal tibia were studied using microCT, histomorphometry, and nanoindentation, respectively. Osteoclast number density was assessed at the distal femur. For each bone measure a mixed model ANOVA was generated to determine the effects of injury and loaded side.

ResultsEpiphyseal and subchondral bone microarchitecture decreased while subchondral bone tissue modulus was unchanged after ACL injuries. Bone resorption increased but bone formation was not changed. Loss of bone microarchitecture also occurred for the contralateral-to-injured limb, demonstrating that the early response to ACL injury extended beyond the injured joint. While the metabolomic profiles of the injured and contralateral-to-injured limbs had many similarities, there were also distinct metabolic shifts present in only the injured limbs. The most prominent of the pathways was cysteine and methionine metabolism, which is associated with osteoclast activity.

ConclusionThese results add to the understanding of early bone changes following ACL injury. Confirming prior reports, we observe a decline in epiphyseal and subchondral bone microarchitecture. We add the finding that subchondral bone modulus remains unchanged at one week after ACL injury. A potential biomarker of this initial bone catabolic response may be synovial fluid cysteine and methionine metabolism, which was only dysregulated in injured knees. Our results implicate a rapidly changing biological and mechanical environment within both the injured and contralateral joints that has the potential for influencing the progression to PTOA.

category: bioengineering


title: Inferring functions of coding and non-coding genes using epigenomic patterns and deciphering the effect of combinatorics of transcription factors binding at promoters

abstract: The number of annotated genes in the human genome has increased tremendously, and understanding their biological role is challenging through experimental methods alone. There is a need for a computational approach to infer the function of genes, particularly for non-coding RNAs, with reliable explainability. We have utilized genomic features that are present across both coding and non-coding genes like transcription factor (TF) binding pattern, histone modifications, and DNase hypersensitivity profiles to predict ontology-based functions of genes. Our approach for gene function prediction (GFPred) made reliable predictions (>90% balanced accuracy) for 486 gene-sets. Further analysis revealed that predictability using only TF-binding patterns at promoters is also high, and it paved the way for studying the effect of their combinatorics. The predicted associations between functions and genes were validated for their reliability using PubMed abstract mining. Clustering functions based on shared top predictive TFs revealed many latent groups of gene-sets involved in common major biological processes. Available CRISPR screens also supported the inferred association of genes with the major biological processes of latent groups of gene-sets. For the explainability of our approach, we also made more insights into the effect of combinatorics of TF binding (especially TF-pairs) on association with biological functions.


title: HiCHub: A Network-Based Approach to Identify Domains of Differential Interactions from 3D Genome Data

abstract: Chromatin architecture is important for gene regulation. Existing algorithms for the identification of interactions changes focus on loops between focal loci. Here we develop a network-based algorithm HiCHub to detect chromatin interaction changes at larger scales. It identifies clusters of genomic elements in physical proximity in one state that exhibit concurrent decreases in interaction among them in the opposite state. The hubs exhibit concordant changes in chromatin state and expression changes, supporting their biological significance. HiCHub works well with data of limited sequencing coverage and facilitates the integration of the one-dimensional epigenetic landscape onto the chromatin architecture. HiCHub provides an approach for finding extended architectural changes and contributes to the connection with transcriptional output. HiCHub is freely available at https://github.com/WeiqunPengLab/HiCHub.


title: SOAR: a spatial transcriptomics analysis resource to model spatial variability and cell type interactions

abstract: Spatial transcriptomics is an emerging technology to profile spatially resolved gene expression. Its preserved capture locations allow researchers to investigate transcriptomes in the tissue context. Large volumes of spatial transcriptomics data under different study designs have been generated, but the lack of a public database with systematically collected and processed data makes data reuse challenging. We present Spatial transcriptOmics Analysis Resource (SOAR), a database with analysis capability and an extensive collection of spatial transcriptomics data. We systematically curated, reviewed, annotated, and pre-processed 132 datasets containing 1,633 samples across 22 tissue types from 6 species. SOAR provides interactive web interfaces for users to visualize spatial gene expression, evaluate gene spatial variability across cell types, and assess cell-cell interactions. Besides data access and download, SOAR can aid researchers in investigating whether a specific gene expression is associated with distinct spatial patterns or cell-cell communications. SOAR is publicly available at https://soar.fsm.northwestern.edu/.


title: MetaPhage: an automated pipeline for analyzing, annotating, and classifying bacteriophages in metagenomics sequencing data.

abstract: In the last decades, a great interest has emerged in the study and characterisation of the microbiota, especially the human gut microbiota, demonstrating that commensal microorganisms play a pivotal role in normal anatomical development and physiological function of the human body. To better understand the complex bacterial dynamics that characterize different environments, bacteriophage predation and gene transfer need to be considered as well, as they are important factors that may contribute to controlling the density, diversity, and network interactions among bacterial communities. To date, a variety of bacteriophage identification tools have been developed, differing on phage mining strategies, input files requested and results produced; however, new users approaching the bacteriophage analysis might struggle in untangling the variety of methods and comparing the different results produced. Here we present MetaPhage, a comprehensive reads-to-report pipeline that streamlines the use of multiple miners and generates an exhaustive report to both summarize and visualize the key findings and to enable further exploration of specific results with interactive filterable tables. The pipeline is implemented in Nextflow, a widely adopted workflow manager, that enables an optimized parallelization of the tasks on different premises, from local server to the cloud, and ensures reproducible results using containerized packages. MetaPhage is designed to allow scalability, reproducibility and to be easily expanded with new miners and methods, in a field that is constantly expanding. MetaPhage is freely available under a GPL-3.0 license at https://github.com/MattiaPandolfoVR/MetaPhage.


title: The bacterial lectin LecA from P. aeruginosa alters membrane organization by dispersing ordered domains

abstract: The assembly and dynamic reorganization of plasma membrane nanodomains (also known as "lipid rafts") play key roles in host cell infection by human pathogens (e.g. viruses and bacteria). Viruses and bacteria can trigger the reorganization of lipid rafts which leads to membrane invaginations and downstream signaling that promote infection. Such reorganizations can be induced by interactions of bacterial or viral carbohydrate proteins (so-called lectins) with lipid raft glycosphingolipids (GSLs). Here, we studied the GSL globotriaosylceramide (Gb3) which is a key receptor involved in the cellular uptake of the gram-negative bacterium P. aeruginosa. The bacterial surface lectin LecA targets Gb3 and promotes bacterial invasion via the "lipid zipper" mechanism. However, the impact of LecA on the organization of membrane nanodomains is unknown yet. We mimicked of the plasma membrane using supported lipid bilayers (SLBs) that contained liquid-ordered (Lo, "raft-like", enriched in sphingolipids and GSLs) and liquid-disordered (Ld, "non-raft-like" enriched in DOPC) lipid domains. Upon interaction with LecA, the Lo domains in the SLBs reshaped and dispersed. Moreover, deformation of SLBs was observed as LecA formed membrane multilayers on SLBs surface. We further dissected this process to reveal the impact of Gb3 structure, bilayer composition and LecA valence on the Lo reorganization.


title: Newly discovered alleles of the tomato antiflorigen gene SELF PRUNING provide a range of plant compactness and yield

abstract: In tomato cultivation, a rare natural mutation in the flowering repressor antiflorigen gene SELF-PRUNING (sp-classic) induces precocious shoot termination and is the foundation in determinate tomato breeding for open field production. Heterozygosity for induced mutations in the florigen gene SINGLE FLOWER TRUSS in the background of sp-classic provides a heterosis-like effect by delaying shoot termination, suggesting subtle suppression of determinacy by genetic modification of florigen-antiflorigen balance could improve yield. Here, we isolated three new sp alleles from the tomato germplasm that show modified determinate growth compared to sp-classic, including one allele that mimics the effect of sft heterozygosity. Two deletion alleles eliminate functional transcripts and showed similar shoot termination, determinate growth, and yields as sp-classic. In contrast, amino acid substitution allele sp-5732 showed semi-determinate growth with more leaves and sympodial shoots on all shoots. This translated to greater yield compared to the other stronger alleles by up to 42%. Transcriptome profiling of axillary (sympodial) shoot meristems (SYM) from sp-classic and wild type plants revealed six mis-regulated genes related to the floral transition, which were used as biomarkers to show that the maturation of SYMs in the weaker sp-5732 genotype is delayed compared to sp-classic, consistent with delayed shoot termination and semi-determinate growth. Assessing sp allele frequencies from over 500 accessions indicated that one of the strong sp alleles (sp-2798) arose in early breeding cultivars but was not selected. The newly discovered sp alleles are potentially valuable resources to quantitatively manipulate shoot growth and yield in determinate breeding programs, with sp-5732 providing an opportunity to develop semi-determinate field varieties with higher yields.


title: Uncovering the "ZIP code" for bZIP dimers reveals novel motifs, regulatory rules and one billion years of cis-element evolution

abstract: Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. For example, dimerization properties of the basic leucine zipper (bZIP) family play a critical role in regulating the unique biological functions in all eukaryotes. However, the molecular mechanism underlying the binding sequence and functional specificity of homo- versus heterodimers remains elusive. To fill this gap, we developed a double DNA Affinity Purification sequencing (dDAP-seq) technique that maps heterodimer DNA binding sites in an endogenous genome context. Our genome-wide binding profiles of twenty pairs of C/S1 bZIP heterodimers and S1 homodimers in Arabidopsis revealed that heterodimerization significantly expands the DNA binding preferences of bZIP TFs. Analysis of the heterodimer target genes in stress response and development suggest heterodimerization gives rise to regulatory responses that are distinct from the homodimers. In addition to the classic ACGT elements recognized by plant bZIPs, we found that the C/S1 heterodimers bound to motifs that might share an origin with the GCN4 cis-elements in yeast that diverged from plants more than one billion years ago. Importantly, heterodimer binding specificities can be distinguished by their relative preference for ACGT motifs versus GCN4-related motifs. More broadly, our study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation.

category: molecular biology


title: A cobB like protein in Oryza sativa indica regulates the mitochondrial machinery under stress conditions

abstract:
Sirtuins are ubiquitous in nature and are known to play an important role as metabolic regulators. In plants, these NAD+ dependent deacetylases are not well characterized. In this study, we are reporting a new member of sirtuin in Oryza sativa indica. It shares approx. 89% sequence identity with bacterial sirtuin, a class III sirtuin member. This protein is mostly present in mitochondria with trace amounts in the nucleus. It can physically interact with histones H3 and H4 and can specifically deacetylate histone H3 at K9 and K18 positions. In mitochondria, acetyl coA synthetase (AcS) and isocitrate dehydrogenase (IDH2) are the targets for its deacetylation. This removal of acetyl group is the mode of regulation under certain stress conditions. Thus, this is the first mitochondrial cobB targeting important plant machinery under changing growth environment. The OscobB deacetylase activity is not majorly affected by its products, NAM & ADP ribose but are sensitive to certain metal ions like Fe2+ and Mg2+. In contrast to some class III members, it does not have any ADP ribosyl transferase activity. In response to abiotic stress conditions like dehydration and low temperature, this enzyme can also mobilize to the nucleus to regulate the plant metabolism.

HighlightsO_LIA new class III member of the sirtuin family found in Oryza sativa indica.
C_LIO_LIUnder normal conditions, this protein is localized mostly in mitochondria, with trace amounts in the nucleus.
C_LIO_LIThis enzyme is capable of using both NAD+ and NADP+ as a substrate for catalysis.
C_LIO_LIIt specifically deacetylates the nuclear histone H3 at K9 and K18 positions.
C_LIO_LIMitochondrial proteins acetyl coA synthetase (AcS) and isocitrate dehydrogenase (IDH2) are the regulatory targets of OscobB.
C_LIO_LIUnder certain stress conditions in plants like dehydration, pathogenesis and low temperature, there is localisation of OscobB from mitochondria to nucleus.
C_LI


title: HOPX governs a molecular and physiological switch between cardiomyocyte progenitor and maturation gene programs

abstract: This study establishes the homeodomain only protein, HOPX, as a determinant controlling the molecular switch between cardiomyocyte progenitor and maturation gene programs. Time-course single-cell gene expression with genome-wide footprinting reveal that HOPX interacts with and controls core cardiac networks by regulating the activity of mutually exclusive developmental gene programs. Upstream hypertrophy and proliferation pathways compete to regulate HOPX transcription. Mitogenic signals override hypertrophic growth signals to suppress HOPX and maintain cardiomyocyte progenitor gene programs. Physiological studies show HOPX directly governs genetic control of cardiomyocyte cell stress responses, electro-mechanical coupling, proliferation, and contractility. We use human genome-wide association studies (GWAS) to show that genetic variation in the HOPX-regulome is significantly associated with complex traits affecting cardiac structure and function. Collectively, this study provides a mechanistic link situating HOPX between competing upstream pathways where HOPX acts as a molecular switch controlling gene regulatory programs underpinning metabolic, signaling, and functional maturation of cardiomyocytes.

category: developmental biology


title: Mycn regulates intestinal development through ribosomal biogenesis in a zebrafish model of Feingold syndrome 1

abstract: Feingold syndrome type 1, caused by loss-of-function of MYCN, is characterized by varied phenotypes including esophageal and duodenal atresia. However, no adequate model exists for studying the syndromes pathological or molecular mechanisms, nor is there a treatment strategy. Here, we developed a zebrafish Feingold syndrome type 1 model with nonfunctional mycn, which had severe intestinal atresia. Single-cell RNA-seq identified a subcluster of intestinal cells was highly sensitive to Mycn, and impaired cell proliferation decreased the overall number of intestinal cells in the mycn mutant fish. Bulk RNA-seq and metabolomic analysis showed that expression of ribosomal genes was downregulated and amino acid metabolism was abnormal. Ribosomal profiling analysis showed decreases in free 40S, 60S, and 80S ribosome particles, which led to impaired translation in the mutant. Further, both L-leucine and Rheb, which can elevate translation via TOR pathway, rescued the intestinal phenotype of mycn mutant. In summary, by this zebrafish Feingold syndrome type 1 model, we found that disturbance of ribosomal biogenesis and blockage of protein synthesis during development are primary causes of the intestinal defect in Feingold syndrome type 1. Importantly, our work suggests that leucine supplementation may be a feasible and easy treatment option for this disease.


title: In vitro and in vivo antioxidant therapeutic evaluation of Dodonaea viscosa Jacq.

abstract:
ContextNatural antioxidants are vital to promote health and treat critical diseased conditions in the modern healthcare system.

ObjectiveThis work adds to the index of natural medicines by exploring the antioxidant potential of Dodonaea viscosa Jacq. (Plant-DV).

Material and methodsThe aqueous extract of leaves and seeds from plant-DV in freshly prepared phosphate buffer is evaluated for antioxidant potential. In vitro antioxidant potential of the nascent and oxidatively stressed extracts was analyzed through glutathione (GSH) assay, hydrogen peroxide (H2O2) scavenging effect, glutathione-S-transferase (GST) assay, and catalase (CAT) activity. In vivo therapeutic assessment is performed in Wistar Albino rats using vitamin C as a positive control. The livers and kidneys of individual animals are probed for glutathione, glutathione-S-transferase, and catalase activities.

ResultsSeeds have GSH contents (59.61 {micro}M) and leaves (32.87 {micro}M) in the fresh aqueous extracts. The hydrogen peroxide scavenging effect of leaves is superior to seeds with 17.25% and 14.18% respectively after 30 min incubation. However, oxidatively stressed extracts with AgI and HgII show declining GSH and GST levels. The plant extracts are non-toxic in rats at 5000 mg/Kg body weight. Liver and kidneys homogenate reveal an increase in GSH, GST, and CAT levels after treatment with 150 {+/-} 2 mg/kg and 300 {+/-} 2 mg/kg body weight plant extract compared with normal saline-treated negative and vitamin C treated positive control.

Discussion and ConclusionThe crude aqueous extracts of leaves and seeds of plant DV show promising antioxidant potential both in in vitro and in vivo evaluation.


title: Public exams provide opportunities for deeper thought with less anxiety

abstract: Assessment methods across post-secondary education are traditionally constrained by logistics, built on prior practice instead of evidence, and contribute to the inequities in education outcomes. As part of attempts to improve and diversify the methods used in assessment, the authors have developed a flexible and low-tech style known as  public exams based in best practices. Public exams attempt to bring students authentically into the process of assessment through the use of pre-released portions of the exam. Through mixed-methods research at a closely-matched pair of an R1 and a community college classroom, we observe significant signals of positive impact from the public exam on student experiences. Public exams appear to result in deeper thought, more efficiently direct students to the core concepts in the discipline, and decrease anxiety in and around the exams. The public exam experience does not show evidence for exacerbating gaps in exam outcomes for students from minoritized backgrounds. This evidence suggests that public exams are an evidence-based, useful assessment style for instructors looking to improve their assessment design and implementation.


title: Application and Evaluation of Flipped Teaching Based on Video Conference in Standardized Training for Internal Medicine Residents during Vacation

abstract:
BackgroundThe training in the department of infectious diseases is a compulsory part of the standardized training for internal medicine residents, and the specialized hospitals for infectious diseases provide specialized training for residents in hospitals without the training standards for the department of infectious diseases in the region. However, due to holidays, coordination of training dates among hospitals, and vacation of other reasons, the actual training time of the department of infectious diseases may be insufficient.

ObjectiveFlipped teaching with "video conference" as the carrier was explored during vacation, to make up for the lack of actual training time of the department of infectious diseases for internal medicine residents caused by vacation, and to ensure the training quality of the department of infectious diseases.

MethodsVertical management mode was adopted, management team and lecturer team were established, training program and teaching implementation were formulated. Flipped teaching was carried out on the carrier of "video conference" for internal medicine residents of dispatched hospitals who participated in the training of infectious diseases department in April. Qualitative and quantitative analysis was made on the design and implementation of flipped teaching.

ResultsThe flipped teaching was organized and vertically managed by the contact person of the project of "Rotation Training for Infectious Diseases Department of Internal Medicine Residents". 19-member internal medicine residents participated in this teaching, and a 6-member management team was formed, and a 12-member lecturer team was composed of 12 internal medicine residents who had been trained in the department of infectious diseases from March to April. 12 training contents were selected to carry out teaching according to the requirements of training diseases in the department of infectious diseases, and the implementation rate of teaching plan was over 90%. A total of 197 feedback questionnaires were collected. The feedback that the teaching quality was "good" and "very good" accounted for more than 96%, and the attendance rate of the whole teaching process reached more than 94%. About this teaching, 6 internal medicine residents put forward 18 suggestions for improvement, accounting for 9.1%; and 11 internal medicine residents gave 110 commendation suggestions, accounting for 55.8%.

ConclusionFlipped teach based on video conference was generally effective in carrying out for internal medicine residents participating in the training of department of infectious diseases, and it could be used as a supplementary training method for standardized training of internal medicine residents to make up for the shortage of actual training period in a certain stage.


title: Insulated expression of periplasmic uricase in E. coli Nissle 1917 for the treatment of hyperuricemia

abstract: Hyperuricemia is a prevalent disease worldwide that is characterized by elevated urate levels in the blood owing to purine metabolic disorders, which can result in gout and comorbidities. As approximately one-third of urate is excreted by the small intestine and cleared by intestinal microorganisms, modulating the gut microbiota could be an attractive approach for hyperuricemia and gout treatment. In this study, we engineered a probiotic E. coli Nissle 1917 (EcN) strain, EcN C6, which expresses periplasmic uricase at an "insulated site", for urate degeneration. Oral administration of EcN C6 successfully alleviated hyperuricemia, related symptom and gut microbiota in a purine-rich food-induced hyperuricemia rat model and a uox-knockout mouse model. Importantly, the expression of periplasmic uricase in the insulated site did not influence the probiotic properties or global gene transcription of EcN, suggesting that EcN C6 is a safe, effective and low cost therapeutic candidate for hyperuricemia treatment.

category: synthetic biology


title: Chlamydia trachomatis effector Dre1 interacts with dynactin to reposition host organelles during infection

abstract: Chlamydia trachomatis is an obligate intracellular pathogen that replicates within a specialized membrane-bound compartment, called the inclusion. Chlamydia species express a unique class of effectors, Incs, which are translocated from the bacteria by a Type III secretion system and are inserted into the inclusion membrane where they modulate the host-bacterium interface. C. trachomatis repositions specific host organelles during infection to acquire nutrients and evade host cell surveillance, however the bacterial and host proteins controlling these processes are largely unknown. Here, we identify an interaction between the host dynactin complex and the C. trachomatis Inc CT192 (CTL0444), hereafter named Dre1 for Dynactin Recruiting Effector 1. We show that dynactin is recruited to the inclusion in a Dre1-dependent manner and that loss of Dre1 diminishes the recruitment of specific host organelles, including the centrosome, mitotic spindle, and Golgi apparatus to the inclusion. Inactivation of Dre1 results in decreased C. trachomatis fitness in cell-based assays and in a mouse model of infection. By targeting particular functions of the versatile host dynactin complex, Dre1 facilitates re-arrangement of certain organelles around the growing inclusion. Our work highlights how C. trachomatis employs a single effector to evoke specific, large-scale changes in host cell organization that establish an intracellular replicative niche without globally inhibiting host cellular function.


title: The Effects of Vitamin E Analogues α-Tocopherol and γ-Tocotrienol on the Human Osteocyte Response to Ultra-high Molecular Weight Polyethylene Wear Particles

abstract:
BackgroundPolyethylene (PE) liners are a common bearing surface of orthopaedic prostheses. In some products, vitamin E is added to PE as a preventive measure against oxidative degradation. Ultra-high molecular weight PE (UHMWPE) wear particles contribute to periprosthetic osteolysis, a major cause of aseptic loosening. Osteocytes are an important cell type for controlling both bone mineralisation and bone resorption, and respond to UHMWPE particles by inducing both osteocytic osteolysis and support of osteoclastogenesis. Here, we examined the effects of the vitamin E analogues -tocopherol and {gamma}-tocotrienol alone or in the context of wear particles on human osteocyte gene expression and mineralisation behaviour.

MethodsHuman primary osteoblasts isolated from patients undergoing hip arthroplasty were differentiated for 28d to an osteocyte-like stage. Cultures were exposed to UHMWPE wear particles in the presence or absence of either -Tocopherol or {gamma}-Tocotrienol. Gene expression related to the oxidative response, osteocytic osteolysis and osteoclastogenesis was measured by real-time RT-PCR. The effect on calcium release from mineralised osteocyte-like cultures was also measured.

ResultsBoth -Tocopherol and {gamma}-Tocotrienol upregulated expression of several antioxidant-related genes. UHMWPE particles also upregulated antioxidant gene expression, suggesting the involvement of oxidative stress pathways. UHMWPE particles upregulated the pro-osteoclastogenic RANKL:OPG mRNA ratio, however, this was reduced in the presence of vitamin E analogues. UHMWPE particles also upregulated mRNA expression of osteocytic-osteolysis related genes. However, treatment with either analogue alone and in combination with UHMWPE particles also resulted in significant upregulation of these genes. Consistent with this, both vitamin E analogues promoted calcium release from mineralised cultures of osteocyte-like cells.

DiscussionOur findings suggest that vitamin E analogues may suppress osteocyte support of osteoclastogenesis in response to UHMWPE particle exposure. Vitamin E analogues exert an antioxidant effect on osteocytes, however, this appears to involve the induction of osteocytic osteolysis, which could promote periprosthetic osteolysis. It will be important to further elucidate the effects of vitamin E on the cells of the prosthetic joint in order to understand the potential effects of their inclusion in prosthetic materials.

category: cell biology


title: Evolution of self-sustained circadian rhythms requires seasonal change of daylight

abstract: AbstractsSelf-sustained oscillation is a fundamental property of circadian rhythms and has been repeatedly tested since the early days of circadian research, resulting in the discovery of almost all organisms possessing self-sustained circadian oscillations. However, the evolutionary advantage of self-sustainability has been only speculatively discussed. In this theoretical study, we sought the environmental constraints and selection pressure that drive the acquisition or degeneration of self-sustainability through the process of evolution. We considered a random gene regulatory network dynamics under light cycles and optimized the network structure using an evolutionary algorithm. By designing the fitness function in the evolutionary algorithm, we investigated the environmental conditions that led to the evolution of the self-sustained oscillators. Then, we found that (i) networks showing self-sustained oscillation under constant light conditions are much rarer than those showing damped oscillation and hourglass-type behaviour, and (ii) among several types of fitness-based optimization, networks with self-sustainability property have a markedly high fitness score, especially when we assume that a network has to generate a constantly periodic expression profile regardless of day length. This study was the first to show that seasonality facilitated the evolution of the self-sustained circadian clock, which was consistent with empirical records.

category: evolutionary biology


title: Diversity chromosome evolution of Ty1-copia Retrotransposons in Pennisetum purpureum Revealed by FISH

abstract: Pennisetum purpureum is a potential species for biofuel production. Characterization and chromosomal distribution of retrotransposons could enhance the comprehension of the role and dynamics of the repetitive elements in plants. In this study, a phylogenetic tree was constructed according to the conserved reverse transcriptase sequences and revealed that these Ty1-copia retrotransposons had typical structure. Analysis showed that total Ty1-copia retrotransposons had a significant component, as high as 5.12 x 103 copy numbers in P. purpureum. Then, the chromosomal pattern of four known lineages were also analyzed with Pennisetum glaucum genome, which suggested that the Sire/Maximus lineage had the highest copy number and followed by Tork/Angela, Tork/TAR, Retrofit/Ale. Additionally, the chromosomal distribution of total Ty1-copia retrotransposons was detected by fluorescence in situ hybridization (FISH) to be a dispersed pattern with weak clustering, mostly near the centromeric regions of P. purpureum chromosomes; interestingly, there were four obvious signals in the subterminal chromosomes. These results suggested that there occurred differential dynamic evolution directions of Ty1-copia retrotransposons within P. purpureum. Furthermore, co-localization of Ty1-copia, 5S rDNA, and 35S rDNA indicated that two chromosome 2 and four chromosome 4 were identified. Concurrently, subterminal signals of Ty1-copia-type retrotransposons were located on four other homologous chromosomes. Altogether, these results shed light on the diversification of Ty1-copia retrotransposons and have the significance for generation of valid chromosomal markers in retrotransposon families.


title: Multi-marker DNA metabarcoding detects suites of environmental gradients from an urban harbour

abstract: There is increasing need for biodiversity monitoring, especially in places where potential anthropogenic disturbance may significantly impact ecosystem health. We employed a combination of traditional morphological and bulk macroinvertebrate metabarcoding analyses to benthic samples collected from Toronto Harbour (Ontario, Canada) to compare taxonomic and functional diversity of macroinvertebrates and their responses to environmental gradients. At the species rank, sites assessed using COI metabarcoding showed more variation than sites assessed using morphological methods. Depending on the assessment method, we detected gradients in magnesium (morphological taxa), ammonia (morphological taxa, COI sequence variants), pH (18S sequence variants) as well as gradients in contaminants such as metals (COI & 18S sequence variants) and organochlorines (COI sequence variants). Observed responses to contaminants such as aromatic hydrocarbons and metals align with known patchy distributions in harbour sediments. We determined that the morphological approach may limit the detection of macroinvertebrate responses to lake environmental conditions due to the effort needed to obtain fine level taxonomic assignments necessary to investigate responses. DNA metabarcoding, however, need not be limited to macroinvertebrates, can be automated, and taxonomic assignments are associated with a certain level of accuracy from sequence variants to named taxonomic groups. The capacity to detect change using a scalable approach such as metabarcoding is critical for addressing challenges associated with biodiversity monitoring and ecological investigations.


title: WFA-GPU: Gap-affine pairwise alignment using GPUs

abstract:
MotivationAdvances in genomics and sequencing technologies demand faster and scalable analysis methods that can process longer sequences with higher accuracy. However, classical pairwise alignment methods, based on dynamic programming (DP), impose impractical computational requirements to align long and noisy sequences like those produced by PacBio, and Nanopore technologies. The recently proposed WFA algorithm paves the way for more efficient alignment tools, improving time and memory complexity over previous methods. Notwithstanding, modern high performance computing (HPC) platforms rely on accelerator-based architectures that exploit parallel computing resources to improve over classical computing CPUs.

ResultsThis paper presents the WFA-GPU, a GPU (Graphics Processing Unit)-accelerated tool to compute exact gap-affine alignments based on the WFA algorithm. We present the algorithmic adaptations and performance optimizations that allow exploiting the massive parallel capabilities of modern GPU devices to accelerate the alignment computations. In particular, we propose a CPU-GPU co-design capable of performing inter and intra-sequence parallel alignment of multiple sequences, combining a succinct WFA-data representation with an efficient GPU implementation. As a result, we demonstrate that our implementation outperforms the original WFA implementation between 1.5-7.7x times, and up to 12x when using heuristic methods on long and noisy sequences. Compared to other state-of-the-art tools and libraries, the WFA-GPU is up to 175x faster than other GPU implementations and up to four orders of magnitude faster than other CPU implementations.

AvailabilityWFA-GPU code and documentation is publicly available at https://github.com/quim0/WFA-GPU

Contactquim.aguado.p@gmail.com


title: The field of protein function prediction as viewed by different domain scientists

abstract: Experimental biologists, biocurators, and computational biologists all play a role in characterizing a proteins function. The discovery of protein function in the laboratory by experimental scientists is the foundation of our knowledge about proteins. Experimental findings are compiled in knowledge-bases by biocurators to provide standardized, readily accessible, and computationally amenable information. Computational biologists train their methods using these data to predict protein function and guide subsequent experiments. To understand the state of affairs in this ecosystem, centered here around protein function prediction, we surveyed scientists from these three constituent communities. Our objective was to understand their views on this research area, including the importance of the problem, the usefulness of the methods, the bottlenecks in the field, and the level of interaction between the communities. We show that the three core communities have common but also idiosyncratic perspectives on the field. Most strikingly, experimentalists rarely use modern prediction software, but when presented with predictions, report many to be surprising and useful. Ontologies appear to be highly valued by biocurators, less so by experimentalists and computational biologists, yet controlled vocabularies bridge the communities and simplify the prediction task. Additionally, many software tools are not readily accessible and the predictions presented to the users can be broad and uninformative. To meet both the social and technical challenges in the field, a more productive and meaningful interaction between members of the core communities is necessary.


title: TITAN: A Toolbox for Information-Theoretic Analysis of Molecular Networks

abstract:
BackgroundReconstruction of molecular networks allows structural and functional characterization of cells, tissues, and organs. A network analytical approach to statistical correlations in molecular data can provide new insight into the underlying system, but currently there is not any analytical approach that is generalizable across different types of data. Here we present TITAN, an open-source toolbox in MATLAB and Octave for the reconstruction and graph analysis of molecular networks. Using an information-theoretical approach TITAN reconstructs networks from transcriptional data, revealing the topological structure of correlations in biological systems.

FindingsThe toolbox includes a data processing pipeline to construct gene co-expression networks from transcriptional data, mutual information (MI) and variation of information (VI) to quantify connectivity and graph-bas ed analysis for molecular network analysis. We show that the toolbox can accurately reconstruct network structure from synthetic data with predetermined network topology. We use the toolbox to construct the gene co-expression network of S. cerevisiae and show that the resulting network is indistinguishable from gene co-expression networks previously identified experimentally.

DiscussionTITAN is a general tool for network reconstruction in high dimensional datasets. It could be used to study co-transcriptional networks and reveal connectivity patterns in any large omics datasets. Considering the universal nature of MI as a measure of correlation, TITAN can potentially be used to vertically integrate datasets for a cross-omics analysis that covers a system on every level, from genes to metabolites. This universality can be extended to cross-species analysis and evolutionary genetics. Other applications include molecular fingerprinting of cells, tissues, and organs, and tracing developmental trajectories for gene and protein networks.


title: US-align: Universal Structure Alignments of Proteins, Nucleic Acids, and Macromolecular Complexes

abstract: Structure comparison and alignment are of fundamental importance in structural biology studies. We developed the first universal platform, US-align, to uniformly align monomer and complex structures of different macromolecules (proteins, RNAs, and DNAs). The pipeline is built on a uniform TM-score objective function coupled with a heuristic alignment searching algorithm. Large-scale benchmarks demonstrated significant advantages of US-align over state-of-the-art methods in pairwise and multiple structure alignments of different molecules. Detailed analyses showed that the major advantage of US-align lies in the extensive optimization of the unified objective function powered by efficient heuristic search iterations, which significantly improve the accuracy and speed of the structural alignment process. Meanwhile, the universal protocol fusing different molecular and structural types helps facilitate the heterogeneous oligomer structure comparison and template-based protein-protein and protein-RNA/DNA docking.


title: A deep reinforcement learning approach to reconstructing quaternary structures of protein dimers through self-learning

abstract: Predicted interchain residue-residue contacts can be used to build the quaternary structure of protein complexes from scratch. However, only a small number of methods have been developed to reconstruct protein quaternary structures using predicted interchain contacts. Here, we present an agent-based self-learning method based on deep reinforcement learning (DRLComplex) to build protein complex structures using interchain contacts as distance constraints. We rigorously tested the DRLComplex on two standard datasets of homodimeric and heterodimeric dimers (the CASP-CAPRI homodimer dataset and Std_32 heterodimer dataset) using both true and predicted contacts. Utilizing true contacts as input, the DRLComplex achieved a high average TM-score of 0.9895 and 0.9881 and a low average interface RMSD (I_RMSD) of 0.2197 and 0.92 on the two datasets, respectively. When predicted contacts are used, the method achieves the TM-score of 0.73 and 0.76 for homodimers and heterodimers respectively. The accuracy of reconstructed quaternary structures depends on the accuracy of contact predictions. Compared with other optimization methods of reconstructing quaternary structures from interchain contacts, DRLComplex performs similarly to an advanced gradient descent method and better than a Markov Chain Monte Carlo simulation method and a simulated annealing-based method. The source code of DRLComplex is available at: https://github.com/jianlin-cheng/DRLComplex