title: A subset of Memory B-derived antibody repertoire from 3-dose vaccinees is ultrapotent against diverse and highly transmissible SARS-CoV-2 variants, including Omicron

abstract:
Omicron, the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. We examined whether sera from individuals who received two or three doses of inactivated vaccine, could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2/60) and 95% (57/60) for 2- and 3-dose vaccinees, respectively. For three-dose recipients, the geometric mean neutralization antibody titer (GMT) of Omicron was 15, 16.5-fold lower than that of the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in 3-dose vaccinees, half of which recognize the receptor binding domain (RBD) and show that a subset of them (24/163) neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron, potently. Therapeutic treatments with representative broadly neutralizing mAbs individually or antibody cocktails were highly protective against SARS-CoV-2 Beta infection in mice. Atomic structures of the Omicron S in complex with three types of all five VOC-reactive antibodies defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to one major class of antibodies bound at the right shoulder of RBD through altering local conformation at the binding interface. Our results rationalize the use of 3-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are a rational target for a universal sarbecovirus vaccine.

One sentence summaryA sub-set of antibodies derived from memory B cells of volunteers vaccinated with 3 doses of an inactivated SARS-CoV-2 vaccine work individually as well as synergistically to keep variants, including Omicron, at bay.


title: Reactive oxygen species signalling is involved in alkamide-induced altered root development.

abstract:
Alkamides are alpha unsaturated N-acylamides structurally related to N-acyl ethanolamides (NAEs) and N-acyl-L-homoserine lactones (AHLs). Studies have shown that alkamides induce prominent changes in root architecture, a significant metabolic readjustment, and transcriptional reprogramming. Some alkamide responses have been associated with redox signalling; however, this involvement and ROS sources have not been fully described. We utilized a genetic approach to address ROS signalling in alkamide-induced processes and found that in Arabidopsis, treatment with the alkamide affinin (50{micro}M) increased the in-situ accumulation of H2O2 in lateral root emergence sites and reduced H2O2 accumulation in primary root meristems implying that altered root growth was dependent on endogenous H2O2. Results show that ROS sourced from PRX34, RBOHC and RBOHD were involved in promotion of lateral root emergence by alkamides. RBOHC was required for affinin-induced enhanced root hair expansion. Furthermore, affinin-induced changes in lateral root emergence, but not root hair length, were dependent on a change in extracellular pH. Finally, reverse genetic experiments suggest heterotrimeric G-proteins were involved in plant response to alkamides; nevertheless, further studies with additional higher order G-protein mutants will be required to resolve this question. These results support that alkamides recruit specific ROS signaling programs to mediate alterations in root architecture.

HighlightReactive oxygen species (ROS) are involved in alkamide-induced altered root development. Heterotrimeric G-protein complex, extracellular acidification, and ROS sourced from peroxidases and NADPH-oxidases are involved in these processes.

category: plant biology


title: Neocortical pyramidal neurons with axons emerging from dendrites are frequent in non-primates, but rare in monkey and human

abstract: The canonical view of neuronal function is that inputs are received by dendrites and somata, become integrated in the somatodendritic compartment and upon reaching a sufficient threshold, generate axonal output with axons emerging from the cell body. The latter is not necessarily the case. Instead, axons may originate from dendrites. The terms "axon carrying dendrite" (AcD) and "AcD neurons" have been coined to describe this feature. Here, we report on the diversity of axon origins in neocortical pyramidal cells. We found that in non-primates (rodent, cat, ferret, pig), 10-21% of pyramidal cells of layers II-VI had an AcD. In marked contrast, in macaque and human, this proportion was lower, and it was particularly low for supragranular neurons. Unexpectedly, pyramidal cells in the white matter of postnatal cat and aged human cortex exhibit AcDs to much higher percentages. In rodent hippocampus, AcD cells are functionally  privileged, since inputs here can circumvent somatic integration and lead to immediate action potential initiation in the axon. Our findings expand the current knowledge regarding the distribution and proportion of AcD cells in neocortial regions of non-primate taxa, which strikingly differs from primates where these cells are mainly found in deeper layers and white matter.

category: neuroscience


title: Rapid selection of P323L in the SARS-CoV-2 polymerase (NSP12) in humans and non-human primate models and confers a large plaque phenotype

abstract: The mutational landscape of SARS-CoV-2 varies at both the dominant viral genome sequence and minor genomic variant population. An early change associated with transmissibility was the D614G substitution in the spike protein. This appeared to be accompanied by a P323L substitution in the viral polymerase (NSP12), but this latter change was not under strong selective pressure. Investigation of P323L/D614G changes in the human population showed rapid emergence during the containment phase and early surge phase of wave 1 in the UK. This rapid substitution was from minor genomic variants to become part of the dominant viral genome sequence. A rapid emergence of 323L but not 614G was observed in a non-human primate model of COVID-19 using a starting virus with P323 and D614 in the dominant genome sequence and 323L and 614G in the minor variant population. In cell culture, a recombinant virus with 323L in NSP12 had a larger plaque size than the same recombinant virus with P323. These data suggest that it may be possible to predict the emergence of a new variant based on tracking the distribution and frequency of minor variant genomes at a population level, rather than just focusing on providing information on the dominant viral genome sequence e.g., consensus level reporting. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

category: microbiology


title: Elevated ozone concentration and nitrogen addition increase poplar rust severity by shifting the phyllosphere microbial community

abstract:
The tropospheric ozone and nitrogen deposition are two major environmental pollutants. Numerous studies have focused on the negative impacts of elevated O3 and the complementary effect of soil N addition to tree physiological characteristics. However, it was notoriously ignored of how elevated O3 with N addition affect tree immunity in face of pathogen infection, as well as of the important roles of phyllosphere microbiome community in host-pathogen-environment interplay. Here, we examined the effects of elevated O3 and soil N addition on poplar leaf rust (Melampsora larici-populina) severity of two susceptible hybrid poplars (clone  107: Populus euramericana cv.  74/76; clone  546: P. deltoides {subseteq} P. cathayana) in Free-Air-Controlled-Environment plots, besides, the link between Mlp-susceptibility and changes in microbial community was determined using Miseq amplicon sequencing. Rust severity of clone  107 significantly increased under elevated O3 or N addition only, however, the negative impact of elevated O3 could be significantly alleviated when simultaneously conducting N addition, likewise, this trade-off was also found in its phyllosphere microbial -diversity responding to elevated O3 and N addition. However, the rust severity of clone  546 did not significantly differ in the cases of elevated O3 and N addition. Mlp infection altered microbial community composition and increased its sensitivity to elevated O3 assessed by significantly different abundance of taxa. Elevated O3 and N addition reduced the complexity of microbial community, which may explain the increased severity of poplar rust. These findings demonstrated that poplars need shifting phyllosphere microbial associations to optimize plant immunity in response to environmental changes.

ImportanceExploitation of the interaction mechanisms between host plants and pathogens is the essential basis in disease control. However, while much was known about the molecular determinants in pathogenesis process in the past decades, less is known about the role of nonpathogenic microbial community in plant-pathogen interaction, especially when some host plants are currently encountering severe environmental stresses, such as elevated ozone concentration and superfluous nitrogen addition. Thus, we targeted at the widespread and detriment rust disease (poplar-poplar rust) to dissect the influences of elevated ozone and nitrogen addition on rust disease severity and how phyllosphere microbial groups interacting with both poplars and rust pathogen under these biotic stresses. Our findings could be used to prescribe ecological information about poplar plantation in areas with high levels of ozone pollution and better understand the important roles of microbiome in plant heath.


title: Zooplankton as a transitional host for Escherichia coli in freshwater

abstract:
This study shows that Escherichia coli can be temporarily enriched in zooplankton in natural conditions and that these bacteria can belong to different phylogroups and sequence types including environmental as well as clinical and animal isolates. We isolated 10 E. coli strains and sequenced the genomes of two of them. Phylogenetically the two isolates were closer to strains isolated from poultry meat than with freshwater E. coli, albeit their genomes were smaller than those from poultry. After isolation and fluorescent protein tagging of strains ED1 and ED157 we show that Daphnia sp. can take up these strains and release them alive again, thus forming a temporary host for E. coli. In a chemostat experiment we show that the association does not prolong the bacterial long-term survival, but that at low abundances it does also not significantly reduce the bacterial numbers. We demonstrate that E. coli does not belong to the core microbiota of Daphnia, suffers from competition by the natural microbiota of Daphnia, but can profit from its carapax to survive in water. All in all, this study suggests that the association of E. coli to Daphnia is only temporary but that the cells are viable therein and this might allow encounters with other bacteria for genetic exchange and potential genomic adaptations to the freshwater environment.

ImportanceThe contamination of freshwaters with faecal derived bacteria is of major concern regarding drinking water acquisition and recreational activities. Ecological interactions promoting their persistence are still very scarcely studied. This study, which analyses the survival of E. coli in the presence of zooplankton, is thus of ecological as well as water safety relevance.

category: ecology


title: Developmental resilience of synaptome architecture

abstract: Neurodevelopmental disorders of genetic origin delay the acquisition of normal abilities and cause disabling phenotypes. Spontaneous attenuation and even complete amelioration of symptoms in early childhood and adolescence occur in many disorders1-10, suggesting that brain circuits possess an intrinsic capacity to repair themselves. We examined the molecular composition of almost a trillion excitatory synapses on a brain-wide scale between birth and adulthood in mice carrying a mutation in the homeobox transcription factor Pax6, a neurodevelopmental disorder model11. Pax6 haploinsufficiency had no impact on total synapse number at any age. By contrast, the postnatal expansion of synapse diversity and acquisition of normal synaptome architecture were delayed in all brain regions, interfering with network and cognitive functions. Specific excitatory synapse types and subtypes were affected in two key developmental age-windows. These phenotypes were reversed within 2-3 weeks of onset, restoring synaptome architecture to its normal developmental trajectory. Synapse subtypes with high rates of protein turnover mediated these events. These results show synaptome remodelling confers resilience to neurodevelopmental disorders.


title: Harnessing the Intrinsic Photochemistry of Isoxazoles for the Development of Chemoproteomic Crosslinking Methods

abstract: Photo-crosslinking is a powerful technique for identifying both coarse- and fine-grained information on protein binding by small molecules. However, the scope of useful functional groups remains limited, with most studies focusing on diazirine, aryl azide, or benzophenone-containing molecules. Here, we report a unique method for photo-crosslinking, employing the intrinsic photochemistry of the isoxazole, a common heterocycle in medicinal chemistry, to offer an alternative to existing strategies using more perturbing, extrinsic crosslinkers. In this initial report, this technique is applied both in vitro and ex vivo, used in a variety of common chemoproteomic workflows, and validated across multiple proteins, demonstrating the utility of isoxazole photo-crosslinking in a wide range of biologically relevant experiments.

category: biochemistry


title: OTUD1 deubiquitylase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways

abstract: Deubiquitylating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-{kappa}B activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-{kappa}B pathway by hydrolysing K63-linked ubiquitin chains from NF-{kappa}B signalling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-{kappa}B activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. The N-terminal intrinsically disordered region of OTUD1, which contains an EGTE motif, is indispensable for KEAP1-binding and NF-{kappa}B suppression. OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1-/--mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.

category: cell biology


title: Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase

abstract:
Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining protein structure. Despite recent advances in instruments and algorithms, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air/water interface and the presence of preferred orientations and nonuniform ice layers. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently attracted attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Graphene minimizes background noise and provides a stable platform for specimens under a high-voltage electron beam and cryogenic conditions. Here, we introduce a reliable, easily implemented, and reproducible method of producing 36 graphene-coated grids at once within 1.5 days. The quality of the graphene grids was assessed using various tools such as scanning EM, Raman spectroscopy, and atomic force microscopy. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.4 [A] using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages; for example, it requires less protein, enables easy control of the ice thickness, and prevents protein denaturation at the air/water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we revealed subtle yet significant geometrical differences at the non-heme di-iron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.

Table of Contents (Graphical abstract) O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: ABA represses TOR and root meristem activity through nuclear exit of the SnRK1 kinase

abstract: The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses like drought, partly by modulating plant growth. However, the underlying mechanisms are poorly understood. Here, we show that cell proliferation in the Arabidopsis thaliana root meristem is controlled by the interplay between three kinases, SNF1-RELATED KINASE 2 (SnRK2), the main driver of ABA signaling, the SnRK1 energy sensor, and the growth-promoting TARGET OF RAPAMYCIN (TOR) kinase. Under favorable conditions, the SnRK11 catalytic subunit is enriched in the nuclei of root cells and this is accompanied by normal cell proliferation and meristem size. Depletion of SnRK2s in a snrk2.2 snrk2.3 double mutant causes constitutive cytoplasmic localization of SnRK11 and a reduction in meristem size, suggesting that, under non-stress conditions, SnRK2s enable growth by retaining SnRK11 in the nucleus. In response to elevated ABA levels, SnRK11 translocates to the cytoplasm and this is accompanied by inhibition of TOR, decreased cell proliferation and meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK11 relocalization to the cytoplasm and the inhibition of TOR. Fusion of SnRK11 to an SV40 nuclear localization signal leads to defective TOR repression in response to ABA, demonstrating that SnRK11 nuclear exit is a premise for this repression. Altogether, we demonstrate that SnRK2-dependent changes in SnRK11 subcellular localization are crucial for the regulation of TOR activity and root growth in response to ABA. Such swift relocalization of key regulators may represent a more general strategy of sessile organisms like plants to rapidly respond to environmental changes.


title: Tonic IKK signalling regulates naive T cell survival in vivo by both repressing RIPK1 dependent extrinsic cell death pathways and independent activation of NF-κB

abstract:
The Inhibitor of Kappa B Kinase (IKK) complex is a critical regulator of NF-{kappa}B activation. In addition, IKK has recently been shown to repress RIPK1 dependent extrinsic cell death pathways by directly phosphorylating RIPK1. Our previous work shows that normal thymopoiesis relies on IKK exclusively for repression of TNF triggered cell death pathways, and that NF-{kappa}B activation by IKK is redundant for development. The role of these pathways in mature naive T cells has not previously been reported. Here, we show that, like thymocytes, naive peripheral T cells require continued IKK1/2 expression for survival. In contrast, however, cell loss is only partially prevented by blocking extrinsic cell death pathways by either deleting Casp8 or inhibiting RIPK1 kinase activity. Inducible deletion of Rela in mature CD4+ T cells also results in a significant loss of naive CD4+ T cells and loss of IL7R expression, revealing an additional reliance upon NF-{kappa}B for long term survival of mature T cells. Together, these data show that IKK dependent survival of naive T cells depends upon both repression of extrinsic cell death pathways and activation of NF-{kappa}B survival programme.

Summary statementThe IKK complex performs two essential functions required for long term survival of naive T cells: i) to activate NF-{kappa}B dependent survival pathways, ii) direct repression of RIPK1 induced apoptosis

Caption"Seddon and colleagues show that the IKK complex is essential for long term survival of peripheral naive T cells, required for both NF-{kappa}B activation and repression of extrinsic cell death"

category: immunology


title: Clinical grade ACE2 effectively inhibits SARS-CoV-2 Omicron infections

abstract: The recent emergence of the SARS-CoV-2 variant Omicron has caused considerable concern due to reduced vaccine efficacy and escape from neutralizing antibody therapeutics. Omicron is spreading rapidly around the globe and is suspected to account for most new COVID-19 cases in several countries, though the severity of Omicron-mediated disease is still under debate. It is therefore paramount to identify therapeutic strategies that inhibit the Omicron SARS-CoV-2 variant. Here we report using 3D structural modelling that Spike of Omicron can still associate with human ACE2. Sera collected after the second mRNA-vaccination did not exhibit a protective effect against Omicron while strongly neutralizing infection of VeroE6 cells with the reference Wuhan strain, confirming recent data by other groups on limited vaccine and convalescent sera neutralization efficacy against Omicron. Importantly, clinical grade recombinant human soluble ACE2, a drug candidate currently in clinical development, potently neutralized Omicron infection of VeroE6 cells with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. These data show that SARS-CoV-2 variant Omicron can be readily inhibited by soluble ACE2, providing proof of principle of a viable and effective therapeutic approach against Omicron infections.

category: molecular biology


title: Chloroplast-localized translation for protein targeting in Chlamydomonas reinhardtii

abstract:
Translation is localized within cells to target proteins to their proper locations. We asked whether translation occurs on the chloroplast surface in Chlamydomonas and, if so, whether it is involved in co-translational protein targeting, aligned spatially with localized translation by the bacterial-type ribosomes within this organelle, or both. Our results reveal a domain of the chloroplast envelope which is bound by translating ribosomes. Purified chloroplasts retained ribosomes and mRNAs encoding two chloroplast proteins specifically on this "translation domain", but not a mRNA encoding a cytoplasmic protein. Ribosomes clusters were seen on this domain by electron tomography. Activity of the chloroplast-bound ribosomes is supported by results of the ribopuromycylation and puromycin-release assays. Co-translational chloroplast protein import is supported by nascent polypeptide dependency of the ribosome-chloroplast associations. This cytoplasmic translation domain aligns localized translation by organellar bacterial-type ribosomes in the chloroplast. This juxtaposition the dual translation systems facilitates the targeting and assembly of the polypeptide products.

One-Sentence SummaryTranslation is localized to a domain of the chloroplast envelope for co-translational protein targeting in Chlamydomonas.


title: Biological characteristics of two mesenchymal stem cell lines isolated from the umbilical cord and adipose tissue of a neonatal common hippo (Hippopotamus amphibius)

abstract: Mesenchymal stem cells (MSCs) are multipotent adult stem cells and can be isolated from many tissues of the body. Due to their potentials to treat various diseases and be applied in animal breeding, MSCs have been isolated and identified regarding their biological properties. Common hippos (Hippopotamus amphibius) are a vulnerable species and yet the cryopreservation of their genetic materials is scare. In this study, we successfully established two MSC lines (UC-MSCs and AT-MSCs) from the umbilical cord and adipose tissue of a neonatal common hippo and comparatively described their features. Both UC-MSCs and AT-MSCs showed fibroblastoid morphology and could be continuously passaged for over 17 passages without dramatic signs of senescence. The cell cultures had normal chromosome composition, say, 17 pairs of autosomes and 1 pair of X chromosomes. UC-MSCs and AT-MSCs displayed similar gene expression profiles. They were positive for CD45, CD73, CD90 and CD105 and negative for HLA-DR. They demonstrated stemness maintenance by expression of classical stem cell markers. UC-MSCs and AT-MSCs manifested different differentiation potentials into other cell lineages. In summary, these two cell lines demonstrated the essential properties of mesenchymal stem cells and might play a role in the future research.


title: Considering variance in pollinator responses to stressors can reveal potential for resilience

abstract:
O_LIEnvironmental stressors have sublethal consequences on animals, often affecting the mean of phenotypic traits in a population. However, potential effects on variance are poorly understood. Since phenotypic variance is the basis for adaptation, any influence of stressors may have important implications for population resilience.
C_LIO_LIHere we explored this possibility in insect pollinators by analysing raw datasets from 24 studies (6,913 bees) in which individuals were first exposed to stressors and then tested for cognitive tasks.
C_LIO_LIWhile all types of stressors decreased the mean cognitive performance of bees, their effect on variance was complex. Focusing on 15 pesticide studies, we found that the dose and the mode of exposure to stressors were critical. At low pesticide doses, cognitive variance decreased following chronic exposures but not for acute exposures. Acute exposure to low doses thus seems less damaging at the population level. In all cases however, the variance decreased with increasing doses.
C_LIO_LIPolicy implications. Current guidelines for the authorization of plant protection products on the European market prioritize acute over chronic toxicity assessments on non-target organisms. By overlooking the consequences of a chronic exposure, regulatory authorities may register new products that are harmful to bee populations. Our findings thus call for more research on stress-induced phenotypic variance and its incorporation to policy guidelines to help identify levels and modes of exposure animals can cope with.
C_LI

category: animal behavior and cognition


title: Moving bar of light evokes vectorial spatial selectivity in the immobile rat hippocampus

abstract: Visual cortical neurons encode the position and motion direction of specific stimuli retrospectively, without any locomotion or task demand1. Hippocampus, a part of visual system, is hypothesized to require self-motion or cognitive task to generate allocentric spatial selectivity that is scalar, abstract2,3, and prospective4-7. To bridge these seeming disparities, we measured rodent hippocampal selectivity to a moving bar of light in a body-fixed rat. About 70% of dorsal CA1 neurons showed stable activity modulation as a function of the bars angular position, independent of behavior and rewards. A third of tuned cells also encoded the direction of revolution. In other experiments, neurons encoded the distance of bar, with preference for approaching motion. Collectively, these demonstrate visually evoked vectorial selectivity (VEVS). Unlike place cells, VEVS was retrospective. Changes in the visual stimulus or its trajectory did not cause remapping but only caused gradual changes. Most VEVS tuned neurons behaved like place cells during spatial exploration and the two selectivity were correlated. Thus, VEVS could form the basic building block of hippocampal activity. When combined with self-motion, reward, or multisensory stimuli8, it can generate the complexity of prospective representations including allocentric space9, time10,11, and episodes12.


title: Stability of feature selection utilizing Graph Convolutional Neural Network and Layer-wise Relevance Propagation

abstract:
MotivationHigh-throughput technologies play a more and more significant role in discovering prognostic molecular signatures and identifying novel drug targets. It is common to apply Machine Learning (ML) methods to classify high-dimensional gene expression data and to determine a subset of features (genes) that is important for decisions of a ML model. One feature subset of important genes corresponds to one dataset and it is essential to sustain the stability of feature sets across different datasets with the same clinical endpoint since the selected genes are candidates for prognostic biomarkers. The stability of feature selection can be improved by including information of molecular networks into ML methods. Gene expression data can be assigned to the vertices of a molecular networks graph and then classified by a Graph Convolutional Neural Network (GCNN). GCNN is a contemporary deep learning approach that can be applied to graph-structured data. Layer-wise Relevance Propagation (LRP) is a technique to explain decisions of deep learning methods. In our recent work we developed Graph Layer-wise Relevance Propagation (GLRP) -- a method that adapts LRP to a graph convolution and explains patient-specific decisions of GCNN. GLRP delivers individual molecular signatures as patient-specific subnetworks that are parts of a molecular network representing background knowledge about biological mechanisms. GLRP gives a possibility to deliver the subset of features corresponding to a dataset as well, so that the stability of feature selection performed by GLRP can be measured and compared to that of other methods.

ResultsUtilizing two large breast cancer datasets, we analysed properties of feature sets selected by GLRP (GCNN+LRP) such as stability and permutation importance. We have implemented a graph convolutional layer of GCNN as a Keras layer so that the SHAP (SHapley Additive exPlanation) explanation method could be also applied to a Keras version of a GCNN model. We compare the stability of feature selection performed by GCNN+LRP to the stability of GCNN+SHAP and to other ML based feature selection methods. We conclude, that GCNN+LRP shows the highest stability among other feature selection methods including GCNN+SHAP. It was established that the permutation importance of features among GLRP subnetworks is lower than among GCNN+SHAP subnetworks, but in the context of the utilized molecular network, a GLRP subnetwork of an individual patient is on average substantially more connected (and interpretable) than a GCNN+SHAP subnetwork, which consists mainly of single vertices.

Availabilityhttps://gitlab.gwdg.de/UKEBpublic/graph-lrp

Contacttim.beissbarth@bioinf.med.uni-goettingen.de

category: bioinformatics


title: Simple and cost-effective SNP genotyping method for discriminating subpopulations of the fish pathogen, Nocardia seriolae

abstract: Nocardia seriolae has caused significant fish losses in Asia and the Americas in recent decades, including in Vietnam, which has witnessed devastating economic and social impacts due to this bacterial pathogen. Surveillance strategies are urgently needed to mitigate N. seriolae dissemination in Vietnamese aquaculture and mariculture industries. Whole-genome sequencing (WGS) offers the highest level of resolution to discriminate closely related strains and to determine their putative origin and transmission routes. However, WGS is impractical for epidemiological investigations and pathogen surveillance due to its time-consuming and costly nature, putting this technology out-of-reach for many industry end-users. To overcome this issue, we targeted two previously characterised, phylogenetically informative single-nucleotide polymorphisms (SNPs) in N. seriolae that accurately distinguish: i) Vietnamese from non-Vietnamese strains, and ii) the two Vietnamese subclades. Using the mismatch amplification mutation assay (MAMA) format, we developed assays that genotype strains based on differences in amplicon melting temperature (melt-MAMA) and size (agarose-MAMA). Our MAMA assays accurately genotyped strains both from culture and fish tissues at low cost, using either real-time (~AUD$1/per sample) or conventional (~AUD$0.50/per sample) PCR instrumentation. Our novel assays provide a rapid, reproducible, and cost-effective tool for routine genotyping of this pathogen, allowing faster identification and treatment of nocardiosis-effected permit fish within Vietnamese aquaculture/mariculture facilities, an essential step in mitigating N. seriolae-associated losses.


title: Early Computational Detection of Potential High Risk SARS-CoV-2 Variants

abstract:
The ongoing COVID-19 pandemic is leading to the discovery of hundreds of novel SARS-CoV-2 variants on a daily basis. While most variants do not impact the course of the pandemic, some variants pose significantly increased risk when the acquired mutations allow better evasion of antibody neutralisation in previously infected or vaccinated subjects, or increased transmissibility. Early detection of such high risk variants (HRVs) is paramount for the proper management of the pandemic. However, experimental assays to determine immune evasion and transmissibility characteristics of new variants are resource-intensive and time-consuming, potentially leading to delayed appropriate responses by decision makers. Here we present a novel in silico approach combining Spike protein structure modelling and large protein transformer language models on Spike protein sequences, to accurately rank SARS-CoV-2 variants for immune escape and fitness potential. We validate our immune escape and fitness metrics with in vitro pVNT and binding assays. These metrics can be combined into an automated Early Warning System (EWS) capable of evaluating new variants in minutes and risk monitoring variant lineages in near real-time. The EWS flagged 12 out of 13 variants, designated by the World Health Organisation (WHO, Alpha-Omicron) as potentially dangerous, on average two months ahead of them being designated as such, demonstrating its ability to help increase preparedness against future variants. Omicron was flagged by the EWS on the day its sequence was made available, with immune evasion and binding metrics subsequently confirmed through our in vitro experiments.

One-Sentence SummaryA COVID-19 Early Warning System combining structural modelling with AI to detect and monitor high risk SARS-CoV-2 variants, identifying >90% of WHO designated variants on average two months in advance.


title: mRNA-1273 vaccination protects against SARS-CoV-2 elicited lung inflammation in non-human primates

abstract:
Vaccine-elicited SARS-CoV-2 antibody responses are an established correlate of protection against viral infection in humans and non-human primates. However, it is less clear that vaccine-induced immunity is able to limit infection-elicited inflammation in the lower respiratory tract. To assess this, we collected bronchoalveolar lavage fluid samples post-SARS-CoV-2 strain USA-WA1/2020 challenge from rhesus macaques vaccinated with mRNA-1273 in a dose-reduction study. Single-cell transcriptomic profiling revealed a broad cellular landscape 48 hours post-challenge with distinct inflammatory signatures that correlated with viral RNA burden in the lower respiratory tract. These inflammatory signatures included phagocyte-restricted expression of chemokines such as CXCL10 (IP10) and CCL3 (MIP-1A) and the broad expression of interferon-induced genes such as MX1, ISG15, and IFIT1. Induction of these inflammatory profiles was suppressed by prior mRNA-1273 vaccination in a dose-dependent manner, and negatively correlated with pre-challenge serum and lung antibody titers against SARS-CoV-2 spike. These observations were replicated and validated in a second independent macaque challenge study using the B.1.351/beta-variant of SARS-CoV-2. These data support a model wherein vaccine-elicited antibody responses restrict viral replication following SARS-CoV-2 exposure, including limiting viral dissemination to the lower respiratory tract and infection-mediated inflammation and pathogenesis.

One Sentence SummarySingle cell RNA sequencing analysis demonstrates that mRNA-1273 vaccination limits the development of lower respiratory tract inflammation in SARS-CoV-2 challenged rhesus macaques


title: Multivalent Interactions between Molecular Components Involved in Clathrin Independent Endocytosis Drive Protein Phase Separation

abstract: Endocytosis of transmembrane receptors initiates via molecular interactions between the activated receptor and the endocytic machinery. A specific group of receptors, including the {beta}1-adrenergic receptor ({beta}1-AR), is internalized through a non-clathrin pathway known as Fast Endophilin Mediated Endocytosis (FEME). A key question is: how does the endocytic machinery assemble and how is it modulated by activated receptors during FEME. Here we show that endophilin, a major regulator of FEME, undergoes a phase transition into liquid-like condensates, which facilitates the formation of multi-protein assemblies by enabling the phase partitioning of endophilin binding proteins. The phase transition can be triggered by specific multivalent binding partners of endophilin in the FEME pathway such as the third intracellular loop (TIL) of the {beta}1-AR, and the proline-rich-motifs of lamellipodin (LPD-PRMs). Other endocytic accessory proteins can either partition into, or target interfacial regions of, these condensate droplets. On the membrane, TIL promotes protein clustering in the presence of endophilin and LPD-PRMs. Our results demonstrate how the multivalent interactions between endophilin, LPD-PRMs and TIL regulate protein assembly formation on the membrane, providing mechanistic insights into the priming and initiation steps of FEME.

category: biophysics


title: Structural development of amyloid precursors in insulin B chain and the inhibition effect by fibrinogen

abstract: Amyloid fibrils are abnormal protein aggregates that relate to a large number of amyloidoses and neurodegenerative diseases. The oligomeric precursors, or prefibrillar intermediates, which emerge prior to the amyloid fibril formation, have been known to play a crucial role for the formation. Therefore, it is essential to elucidate the mechanisms of the structural development of the prefibrillar intermediates and ways to prevent its fibril formation. An insulin-derived peptide, insulin B chain, has been known for its stable accumulation of the prefibrillar intermediates. In this study, structural development of B chain prefibrillar intermediates was monitored by transmission electron microscopy and small-angle X-ray scattering combined with size exclusion chromatography and solid-state NMR spectroscopy to elucidate the stability and secondary structure. We further tracked its inhibition process by fibrinogen (Fg), which has been known to effectively prevent the amyloid fibril formation of B chain. We demonstrated that prefibrillar intermediates are wavy structures with low {beta}-sheet content, growing in a multistep manner toward the nucleation for the amyloid fibril formation. In the presence of Fg, the formation of the prefibrillar intermediates slowed down by forming specific complexes. These observations suggest that the prefibrillar intermediates serve as reaction fields for the nucleation and its propagation for the amyloid fibril formation, whereas the inhibition of prefibrillar intermediate elongation by Fg is the significant factor to suppress the fibril formation. We propose that the obtained molecular picture could be a general inhibition mechanism of the amyloid fibril formation by the inhibitors.


title: Hybrid Gaussian Process Models for continuous time series in bolus fed-batch cultures

abstract:
Hybrid modeling, meaning the integration of data-driven and knowledge-based methods, is quickly gaining popularity among many research fields, including bioprocess engineering and development. Recently, the data-driven part of hybrid methods have been largely extended with machine learning algorithms (e.g., artificial neural network, support vector regression), while the mechanistic part is typically using differential equations to describe the dynamics of the process based on its current state. In this work we present an alternative hybrid model formulation that merges the advantages of Gaussian Process State Space Models and the numerical approximation of differential equation systems through full discretization.

The use of Gaussian Process Models to describe complex bioprocesses in batch, fed-batch, has been reported in several applications. Nevertheless, handling the dynamics of the states of the system, known to have a continuous time-dependent evolution governed by implicit dynamics, has proven to be a major challenge. Discretization of the process on the sampling steps is a source of several complications, as are: 1) not being able to handle multi-rate date sets, 2) the step-size of the derivative approximation is defined by the sampling frequency, and 3) a high sensitivity to sampling and addition errors. We present a coupling of polynomial regression with Gaussian Process Models as representation of the right-hand side of the ordinary differential equation system and demonstrate the advantages in a typical fed-batch cultivation for monoclonal antibody production.

category: bioengineering


title: NetREx - Network-based Rice Expression Analysis Server for Abiotic Stress Conditions

abstract: Recent focus on transcriptomic studies in food crops like rice, wheat and maize provide new opportunities to address issues related to agriculture and climate change. Re-analysis of such data available in public domain supplemented with annotations across molecular hierarchy can be of immense help to the plant research community, particularly co-expression networks representing transcriptionally coordinated genes that are often part of the same biological process. With this objective we have developed NetREx, a Network based Rice Expression Analysis Server, that hosts ranked co-expression networks of Oryza sativa using publicly available mRNA-seq data across uniform experimental conditions. It provides a range of interactable data viewers and modules for analysing user queried genes across different stress conditions (drought, flood, cold and osmosis) and hormonal treatments (abscisic and jasmonic acid) and tissues (root and shoot). Subnetworks of user-defined genes can be queried in preconstructed tissue-specific networks, allowing users to view the fold-change, module memberships, gene annotations and analysis of their neighborhood genes and associated pathways. The webserver also allows querying of orthologous genes from Arabidopsis, wheat, maize, barley, and sorghum. Here we demonstrate that NetREx can be used to identify novel candidate genes and tissue-specific interactions under stress conditions and can aid in the analysis and understanding of complex phenotypes linked to stress response in rice. Available at: https://bioinf.iiit.ac.in/netrex/index.html


title: Histone neutralization in a rat model of acute lung injury induced by double-hit lipopolysaccharide

abstract:
BackgroundAcute respiratory distress syndrome (ARDS) remains a challenge because of its high morbidity and mortality. Circulation histones levels in ARDS patients were correlated to disease severity and mortality. This study examined the impact of histone neutralization in a rat model of acute lung injury (ALI) induced by a lipopolysaccharide (LPS) double-hit.

MethodsSixty-eight male Sprague-Dawley rats were randomized to sham (N=8, received saline only) or LPS (N=60). The LPS double-hit consisted of a 0.8 mg/kg intraperitoneal injection followed after 16 hours by 5 mg/kg intra-tracheal nebulized LPS. The LPS group was then randomized into five groups: LPS only (N=12); LPS + 5, 25, or 100 mg/kg intravenous STC3141 every 8 hours (LPS+L, LPS+M, LPS+H, respectively, each N=12); or LPS + intraperitoneal dexamethasone 2.5 mg/kg every 24 hours for 56 hours (LPS+D, N=12) The animals were observed for 72 hours.

ResultsLPS animals developed ALI as suggested by lower oxygenation, lung edema formation, and histological changes compared to the sham animals. Compared to the LPS group, LPS+H and +D animals had significantly lower circulating histone levels; only the LPS+D group had significantly lower bronchoalveolar lavage fluid (BALF) histone concentrations. The LPS+L, +M, +H and +D groups had improved oxygenation compared to the LPS group and the LPS+H and +D groups had a lower lung wet-to-dry ratio. All animals survived.

ConclusionNeutralization of histone using STC3141, especially at high dose, had similar therapeutic effects to dexamethasone in this LPS double-hit rat ALI model, with significantly decreased circulating histone concentration, improved oxygenation, and decreased lung edema formation.

category: pharmacology and toxicology


title: Encoding of odors by mammalian olfactory receptors

abstract:
O_LIIdentified ligands for > 500 mouse ORs
C_LIO_LIORs are specifically tuned towards individual odorants and their molecular properties
C_LIO_LIOdor molecular properties are informative of odor responses
C_LIO_LIPredictive modeling and convergent evolution analyses suggest specific residues within a canonical location for odorant binding
C_LI

Olfactory receptors (ORs) constitute the largest multi-gene family in the mammalian genome, with hundreds to thousands of loci in humans and mice respectively1. The rapid expansion of this massive family of genes has been generated by numerous duplication and diversification events throughout evolutionary history. This size, similarity, and diversity has made it challenging to define the principles by which ORs encode olfactory stimuli. Here, we performed a broad surveying of OR responses, using an in vivo strategy, against a diverse panel of odorants. We then used the resulting interaction profiles to uncover relationships between OR responses, odorants, odor molecular properties, and OR sequences. Our data and analyses revealed that ORs generally exhibited sparse tuning towards odorants and their molecular properties. Odor molecular property similarity between pairs of odorants was informative of odor response similarity. Finally, ORs sharing response to an odorant possessed amino acids at poorly conserved sites that exhibited both, predictive power towards odorant selectivity and convergent evolution. The localization of these residues occurred primarily at the interface of the upper halves of the transmembrane domains, implying that canonical positions govern odor selectivity across ORs. Altogether, our results provide a basis for translating odorants into receptor neuron responses for the unraveling of mammalian odor coding.


title: Nuclear localization and transactivation of SYS-1/β-catenin is the result of serial gene duplications and subfunctionalizations

abstract: {beta}-catenin is a multifunctional protein capable of mediating cell adhesion via E-cadherin and transactivation of target genes of the canonical Wnt signaling pathway. The nematode, C. elegans contains four paralogs of {beta}-catenin which are highly specific in their functions. Though similar in overall structure, the four beta-catenins are functionally distinct, each regulating different aspects of development. Of the four, SYS-1 is a key player in Wnt dependent asymmetric cell division (ACD). In ACD, a polarized mother will give rise to a daughter with high nuclear SYS-1 and another with low nuclear SYS-1. Despite sequence dissimilarity, SYS-1 shares a close structural resemblance with human {beta}-catenin where it retains an unstructured amino-terminus (NTD) and 12 armadillo repeats. Using existing genome sequence data from several nematode species, we find that the four {beta}-catenin paralogs result from 3 sequential gene duplications and neofunctionalizations during nematode evolution. SYS-1, however, lacks an unstructured carboxyl-terminus (CTD) that is essential for human {beta}-catenin transactivation processes. This work supports the hypothesis that SYS-1 compensated for the lack of CTD by acquiring novel transactivation domains with cryptic nuclear localization signals in the NTD and the first four armadillo repeats, as shown by transactivation assays in worms and yeast. Furthermore, SYS-1 regulatory domains are not localized to the NTD as in canonical {beta}-catenin and instead spans the entire length of the protein. Truncating SYS-1 abolishes the classical SYS-1 nuclear asymmetry, resulting in daughter cells with symmetrical SYS-1 truncation localization. A screen for SYS-1 physical interactors followed by in vivo cell fate and SYS-1 localization analyses suggest that proper SYS-1 nuclear export is facilitated by XPO-1, while an interaction with IMB-3, an importin {beta}-like protein, suggests import mechanisms. Interestingly, XPO-1 is especially required for lowering SYS-1 in the Wnt-unsignaled nucleus, suggesting a distinct mechanism for regulating asymmetric nuclear SYS-1. In summary, we provide insights on the mechanism of {beta}-catenin evolution within nematodes and inform SYS-1 transactivation and nuclear transport.

category: developmental biology


title: Denitrification in foraminifera has ancient origins and is complemented by associated bacteria

abstract: Benthic foraminifera are unicellular eukaryotes that inhabit sediments of aquatic environments. Several foraminifera of the order Rotaliida are known to store and use nitrate for denitrification, a unique energy metabolism among eukaryotes. The rotaliid Globobulimina spp. has been shown to encode an incomplete denitrification pathway of bacterial origins. However, the prevalence of denitrification genes in foraminifera remains unknown and the missing denitrification pathway components are elusive. Analysing transcriptomes and metagenomes of ten foraminifera species from the Peruvian oxygen minimum zone, we show that denitrification genes are highly conserved in foraminifera. We infer of the last common ancestor of denitrifying foraminifera, which enables us to predict further denitrifying species. Additionally, an examination of the foraminifera microbiota reveals evidence for a stable interaction with Desulfobacteracea, which harbour genes that complement the foraminifera denitrification pathway. Our results provide evidence that foraminiferal denitrification is complemented by the foraminifera microbiome. The interaction of Foraminifera with their resident bacteria is at the basis of foraminifera adaptation to anaerobic environments that manifested in ecological success within oxygen depleted habitats.

category: evolutionary biology


title: Improving the computation efficiency of polygenic risk score modeling: Faster in Julia

abstract: To enable large-scale application of polygenic risk scores in a computationally efficient manner we translate a widely used polygenic risk score construction method, Polygenic Risk Score - Continuous Shrinkage (PRS-CS), to the Julia programing language, PRS.jl. On nine different traits with varying genetic architectures, we demonstrate that PRS.jl maintains accuracy of prediction while decreasing the average run time by 5.5x. Additional programmatic modifications improve usability and robustness. This freely available software substantially improves work flow and democratizes utilization of polygenic risk scores by lowering the computational burden of the PRS-CS method.

category: genetics


title: Genomic evidence of speciation by fusion linked to trophic niche expansion in a recent radiation of grasshoppers

abstract: Post-divergence gene flow can trigger a number of creative evolutionary outcomes, ranging from the transfer of beneficial alleles across species boundaries (i.e., adaptive introgression) to the formation of new species (i.e., hybrid speciation). While neutral and adaptive introgression has been broadly documented in nature, hybrid speciation is assumed to be rare and the evolutionary and ecological context facilitating this phenomenon still remains controversial. Through combining genomic and phenotypic data, we evaluate the hypothesis that the dual feeding regime (scrub legumes and gramineous herbs) of the taxonomically controversial grasshopper Chorthippus saulcyi algoaldensis resulted from hybridization between two sister taxa that exhibit contrasting host-plant specializations: C. binotatus (scrub legumes) and C. saulcyi (gramineous herbs). Genetic clustering analyses and inferences from coalescent-based demographic simulations confirmed that C. s. algoaldensis represents a uniquely evolving lineage and supported the ancient hybrid origin of this taxon (ca. 1.4 Ma), which provides a mechanistic explanation for its broader trophic niche and sheds light on its uncertain phylogenetic position. We propose a Pleistocene hybrid speciation model where range shifts resulting from climatic oscillations can promote the formation of hybrid swarms and facilitate its long-term persistence through geographic isolation from parental forms in topographically complex landscapes.


title: Influence of sequencing depth on the fidelity and sensitivity of 1%-5% low-frequency mutation detection and recommendation for standardization of sequencing depth

abstract:
Sequencing depth has always played an important role in the accurate detection of low-frequency mutations. The increase of sequencing depth and the reasonable setting of threshold can maximize the probability of true positive mutation, or sensitivity. Here, we found that when the threshold was set as a fixed number of positive mutated reads, the probability of both true and false-positive mutations increased with depth. However, When the number of positive mutated reads increased in an equal proportion with depth (the threshold was transformed from a fixed number to a fixed percentage of mutated reads), the true positive probability still increased while false positive probability decreased. Through binomial distribution simulation and experimental test, it is found that the "fidelity" of detected-VAFs is the cause of this phenomenon. Firstly, we used the binomial distribution to construct a model that can easily calculate the relationship between sequencing depth and probability of true positive (or false positive), which can standardize the minimum sequencing depth for different low-frequency mutation detection. Then, the effect of sequencing depth on the fidelity of NA12878 with 3% mutation frequency and circulating tumor DNA (ctDNA of 1%, 3% and 5%) showed that the increase of sequencing depth reduced the fluctuation range of detected-VAFs around the expected VAFs, that is, the fidelity was improved. Finally, based on our experiment result, the consistency of single-nucleotide variants (SNVs) between paired FF and FFPE samples of mice increased with increasing depth, suggesting that increasing depth can improve the precision and sensitivity of low-frequency mutations.

HighlightsThe normalized relationship between sequencing depth and the probability of true positive mutation (sensitivity) is established based on binomial distribution.

The probability of true positive increases and the probability of false positive decreases when the number of positive mutated reads increases (threshold) in an equal proportion with depth.

Detected-VAFs fluctuates regularly around expected-VAFs. The amplitude of detected-VAFs fluctuation decreases with sequencing depth and the "fidelity" increases.

The increase of "fidelity" leads to a higher degree of differentiation between true and false positive mutations, which ultimately increases the true positive probability and decreases the false positive probability.

category: genomics


title: Simultaneous Optimization of Excitation Wavelength and Emission Window for Semiconducting Polymer Nanoparticles to Improve Imaging Quality

abstract: Optimized excitation wavelength and emission window are essential for fluorescence imaging with high quality. Semiconducting polymer nanoparticles (SPNs) as fluorescent contrast agents have been extensively studied, but their imaging abilities in the second near-infrared IIb window (NIR-IIb, 1500-1700 nm) with long excitation wavelength have not been reported yet. Herein, as a proof-of-concept, we demonstrate for the first time that an SPN named L1057 nanoparticles (NPs) exhibit intense NIR-IIb signal due to its ultra-high brightness and broad emission spectrum. After screening 915 nm as an optimal excitation wavelength, we applied L1057 NPs to visualize the whole-body vessels, cerebral vessels, gastrointestinal tract, and tumor progression in different stages, achieving superior spatial resolution and signal to background ratio in the NIR-IIb window with respect to NIR-II window (1000-1700 nm). This study reveals that simultaneous optimization of excitation wavelength and emission window is an efficient strategy to enhance imaging quality and that L1057 NPs can serve as a promising NIR-IIb contrast agent for high-resolution and deep-tissue imaging.


title: Comparative analysis of two NGS platforms and different databases for analysis of AMR genes

abstract: The use of antibiotics in human medicine and livestock production has contributed to the widespread occurrence of antimicrobial resistance (AMR). Recognizing the relevance of AMR to human and livestock health, it is important to assess the occurrence of genetic determinants of resistance in medical, veterinary, and public health settings in order to understand risks of transmission and treatment failure. Advances in Next Generation Sequencing (NGS) technologies have had a significant impact on research in microbial genetics and microbiome analyses. Now, strategies for high throughput sequencing from panels of PCR amplicons representing known AMR genes offer opportunities for targeted characterization of complex microbial populations. Aim of the present study was to compare the Illumina MiSeq and Ion Torrent S5 Plus sequencing platforms for use with the Ion AmpliSeq AMR Research Panel in a veterinary/public health setting. All samples were processed in parallel for the two sequencing technologies, subsequently following a common bioinformatics workflow to define the occurrence and abundance of AMR gene sequences. Regardless of sequencing platform, the results were closely comparable with minor differences. The Comprehensive Antibiotic Resistance Database (CARD), QIAGEN Microbial Insight - Antimicrobial Resistance (QMI-AR), Antimicrobial resistance database (AR), and CARD-CLC databases were compared for analysis, with the most genes identified using CARD. Drawing on these results we describe an end-to-end workflow for AMR gene analysis using NGS.


title: DeepMHCII: A Novel Binding Core-Aware Deep InteractionModel for Accurate MHC II-peptide Binding Affinity Prediction

abstract: Computationally predicting MHC-peptide binding affinity is an important problem in immunological bioinformatics. Recent cutting-edge deep learning-based methods for this problem are unable to achieve satisfactory performance for MHC class II molecules. This is because such methods generate the input by simply concatenating the two given sequences: (the estimated binding core of) a peptide and (the pseudo sequence of) an MHC class II molecule, ignoring the biological knowledge behind the interactions of the two molecules. We thus propose a binding core-aware deep learning-based model, DeepMHCII, with binding interaction convolution layer (BICL), which allows integrating all potential binding cores (in a given peptide) and the MHC pseudo (binding) sequence, through modeling the interaction with multiple convolutional kernels. Extensive empirical experiments with four large-scale datasets demonstrate that DeepMHCII significantly outperformed four state-of-the-art methods under numerous settings, such as five-fold cross-validation, leave one molecule out, validation with independent testing sets, and binding core prediction. All these results with visualization of the predicted binding cores indicate the effectiveness and importance of properly modeling biological facts in deep learning for high performance and knowledge discovery. DeepMHCII is publicly available at https://weilab.sjtu.edu.cn/DeepMHCII/.


title: The Relationship Between Soldier Height, Weight and Army Combat Fitness Scores

abstract: The United States Army recently implemented the Army Combat Fitness Test (ACFT) which was designed to more accurately measure functional-combat fitness constructs. The ACFT replaced the former Army Physical Fitness Test (APFT). The three advent APFT consisted of: two-minute push-ups (PU), two-minute sit-ups (SU), and a timed two-mile run (RUN). The ACFT consists of six events; 3 Rep Max Deadlift (MDL), Standing Power Throw (SPT), Hand Release Push-up (HRP), Sprint-Drag-Carry (SDC), Hanging Leg Tuck (LTK), and a timed two-mile run (2MR). This study investigated the relationship between Soldier height (1.79{+/-}0.07 m) and weight (body mass 86.8{+/-}14.2 kg, BMI 27.1{+/-}3.9) on ACFT scores (442.3{+/-}54.4) of 655 male U.S. Army National Guard Soldiers in a Field Artillery Brigade. For the purpose of the investigation body mass index (BMI) was calculated as the metric representing the Soldiers height and weight. The mean and standard deviation (sd) were calculated for the ACFT event and total scores. Pearson correlation coefficients (PCCs or r) were calculated between BMI and ACFT event and total scores. Likewise, PCCs were calculated between the ACFT event and total scores. The ACFT mean{+/-}sd scores were as follows: MDL=92.2{+/-}31.8 (3 maximum repetitions), SPT=9.5{+/-}2.2 (meters), HRP=24.6{+/-}13.1 (repetitions), SDC=119.8{+/-}21.7 (seconds), LTK=6.2{+/-}5.4 (repetitions), 2MR=1095.0{+/-}233.7 (seconds), ACFT total score=442.3{+/-}54.4 (points). Significant positive correlations were found between the ACFT total score: MDL (r=0.70), SPT (r=0.50), HRP (r=0.74), and LTK (r=0.76) events. Conversely, significant negative correlations were identified between ACFT total score: SDC (r=-0.68) and 2MR (r=-0.53) events. Within the parameters of this study, Soldier BMI demonstrated "no to weak" association with individual ACFT event or ACFT total scores. Further, the range of PCCs between the ACFT event scores were "no to moderately high". Military leaders may consider the results provided as combat and fitness tests continue to evolve.

category: physiology


title: Phytoremediation of EMS Induced Brassica juncea Heavy Metal Hyperaccumulator Genotypes

abstract: Buriganga, an economically important river of Dhaka, Bangladesh, is highly polluted by different toxic heavy metals. In this study, phytoremediation of EMS induced Indian mustard (Brassica juncea L) genotypes against three pollutants viz. lead (Pb), chromium (Cr) and cadmium (Cd) of Buriganga riverbank soil was assessed in field condition. Among 1-, 2- and 3% EMS induced genotypes, better seed germination rate, germination speed and plant survival rate were observed in 1% EMS induced genotype, BE21. The highest concentration of Pb, Cr and Cd were also obtained in the leaf of BE21 genotype and therefore was considered as a super-hyperaccumulator genotype. Concentration of Pb in the next generation of this genotype was approximately two-fold higher in the root (91.53{+/-}6.59 mg/kg dry weight, DW); three-fold higher in the shoot (33.31{+/-}1.01 mg/kg DW) and leaf (28.35{+/-}3.61 mg/kg DW), and more in the fruit (5.59{+/-}0.93 mg/kg DW) than the control. Concentration of Cr was approximately two-fold in the root (57.02{+/-}3.24 mg/kg DW), shoot (18.51{+/-}1.36 mg/kg DW) and leaf (14.98{+/-}2.01 mg/kg DW), and more in the fruit (6.15{+/-}1.92 mg/kg DW) of BE21 genotype compared to the control. Cd concentration was more in the root (1.96{+/-}0.92 mg/kg DW), leaf (0.52{+/-}0.32 mg/kg DW) and fruit (0.19{+/-}0.02 mg/kg DW) and less in the shoot (0.19{+/-}0.01 mg/kg DW) of BE21 genotype than the control. Root, shoot, leaf and fruit of BE21 altogether accumulated 98-, 73- and 87% Pb, Cr and Cd, respectively and can thus be utilized to remove heavy metals of Buriganga River. As like root, shoot and leaf, fruit also accumulated heavy metals; hence those plants which are used in phytoremediation should not be used as food or fodder. To the best of our knowledge, this is the first report of developing EMS induced hyperaccumulator genotype of B. juncea for phytoremediation of Buriganga riverbank soil of Bangladesh.

category: plant biology


title: A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 delta variant

abstract:
Although efficacious vaccines have significantly reduced the morbidity and mortality due to COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of two highly potently neutralizing mAbs (THSC20.HVTR04 and THSC20.HVTR26) from an Indian convalescent donor, that neutralize SARS-CoV-2 VOCs at picomolar concentrations including the delta variant (B.1.617.2). These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein thereby preventing the virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness.

HighlightsO_LIIdentification of an Indian convalescent donor prior to emergence of SARS-CoV-2 Delta variant whose plasma demonstrated neutralization breadth across SARS-CoV-2 variants of concern (VOCs).
C_LIO_LITwo (THSC20.HVTR04 and THSC20.HVTR26) monoclonal antibodies isolated from peripheral memory B cells potently neutralize SARS-CoV-2 VOCs: Alpha, Beta, Gamma, Delta and VOIs: Kappa and Delta Plus.
C_LIO_LITHSC20.HVTR04 and THSC20.HVTR26 target non-competing epitopes on the receptor binding domain (RBD) and represent distinct germline lineages.
C_LIO_LIPassive transfer of THSC20.HVTR04 and THSC20.HVTR26 mAbs demonstrated protection against Delta virus challenge in K18-hACE2 mice at low antibody doses.
C_LI

Graphical Abstract

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title: Stronger density-dependent growth of Japanese sardine with lower food availability: Comparison of growth and zooplankton biomass between a historical and current stock-increase period in the western North Pacific

abstract: Density dependence is a fundamental concept for fish population dynamics. Although density-dependent growth and maturity among older juveniles and adults is important for regulating fish population size and for fisheries management, the mechanism of density dependence for marine fishes remains unclear. Here, we examined changes in Japanese sardine growth with increasing abundance beginning in the 2010s and how the current pattern of density-dependent growth differs from that of a previous stock-increase period from the 1970s to early 1980s. During the current period of increasing abundance, mean standard length has already dropped to the lowest level yet observed and growth has declined more sharply with increased abundance than in the 1970s and 1980s. Mesozooplankton biomass in July in the summer feeding grounds was also lower during the current period. Therefore, our results suggest that summer food availability in the western North Pacific controls the strength of density-dependent growth. A lower carrying capacity for Japanese sardine could account for the stronger density dependence of growth observed in the 2010s; this indicates that future Japanese sardine abundance might not increase as much as in the 1980s unless food availability improves.


title: Estimation of the biosorption potential of certain representatives of the genus Bacillus in interaction with lead cation in vitro

abstract: The article presents data on the physicochemical and metabolically dependent mechanisms of detoxification by microorganisms of heavy metals that enter the environment during anthropogenic pollution. The taxonomic and physiological-biochemical diversity of microorganisms capable of neutralizing toxicants has been demonstrated. In the experimental part of the study, the combination of the methods used made it possible to fully assess the degree of toxicity and the effect of lead cations on the growth of bacteria of the genus Bacillus in a model experiment. Thus, the use of atomic absorption spectrophotometry and atomic force microscopy gave an idea of the level of biosorption of a given xenobiotic element from a substrate with localization of inactive forms of lead. The data obtained indicate the presence of an inhibitory effect of Pb(NO3)2 and Pb(CH3COO)2 in concentrations from 1 mM to 0.063 mM about the test organisms under study. The presence of cations with a nutrient substrate gives a false-positive idea of the degree of influence of lead on the growth of the studied microorganisms, since an increase in optical density in test samples is due to high sorption characteristics and, as a consequence, is characterized by an increase in relative optical density. An assessment of the detoxification mechanisms, which is expressed by active bioaccumulation of lead on the surface elements of the microbial cell up to 65% at the point of entering the stationary growth phase, indicates the promising use of representatives of this group of microorganisms as microbial bioremediation and correctors of excess content of this element in the body or ecosystem as a whole.


title: Discovery of a class of giant virus relatives displaying unusual functional traits and prevalent within plankton: the Mirusviricetes

abstract:
Large and giant DNA viruses of the phylum Nucleocytoviricota have a profound influence on the ecology and evolution of planktonic eukaryotes. Recently, various Nucleocytoviricota genomes have been characterized from environmental metagenomes based on the occurrence of hallmark genes identified from cultures. However, lineages diverging from the culture genomics functional principles have been overlooked thus far. Here, we developed a phylogeny-guided genome-resolved metagenomic framework using a single hallmark gene as compass, a subunit of DNA-dependent RNA polymerase encoded by most Nucleocytoviricota. We applied this method to large metagenomic data sets from the surface of five oceans and two seas and characterized 697 non-redundant Nucleocytoviricota genomes up to 1.45 Mbp in length. This database expands the known diversity of the class Megaviricetes and revealed two additional putative classes we named Proculviricetes and Mirusviricetes. Critically, the diverse and prevalent Mirusviricetes population genomes seemingly lack several hallmark genes, in particular those related to viral particle morphogenesis. Instead, they share various genes of known (e.g., TATA-binding proteins, histones, proteases and viral rhodopsins) and unknown functions rarely detected if not entirely missing in other characterized Nucleocytoviricota classes. Phylogenomics, comparative genomics, functional trends and the signal among planktonic cellular size fractions point to Mirusviricetes being a major, functionally divergent class of large DNA viruses that actively infect eukaryotes in the sunlit ocean using an enigmatic functional life style. Finally, we built a comprehensive marine genomic database for Nucleocytoviricota by combining multiple environmental surveys that might contribute to future endeavors exploring the ecology and evolution of plankton.

CoverThis survey took advantage of the environmental DNA sequencing and bioinformatics legacies of Tara Oceans and anvio to discover new classes of giant virus relatives in the sunlit ocean by means of "Phylogeny-guided Genome-Resolved Metagenomics".

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title: Defective linear and circular RNAs biogenesis in Huntington's disease: CAG repeat expansion hijacks neuronal splicing

abstract:
Alternative splicing (AS) appears to be altered in Huntingtons disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected.

Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a strong correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, expanded Htt CAG repeats reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors.

Though the mechanisms of this dysregulation remain uncertain, our study unveils network of transcriptionally altered micro-RNAs and RNA-binding proteins (CELF, hnRNPS, PTBP, SRSF) which, in turn, might influence the AS machinery, primarily in neural cells.

We suggest that this unbalanced expression of linear and circular RNAs might result in altered neural fitness, contributing to HD striatal vulnerability.


title: TOP-2 is differentially required for the proper maintenance of cohesin on meiotic chromosomes in C. elegans spermatogenesis and oogenesis

abstract: During meiotic prophase I, accurate segregation of homologous chromosomes requires the establishment of a chromosomes with a meiosis-specific architecture. Sister chromatid cohesins and the enzyme Topoisomerase II are important components of meiotic chromosome axes, but the relationship of these proteins in the context of meiotic chromosome segregation is poorly defined. Here, we analyzed the role of Topoisomerase II (TOP-2) in the timely release of sister chromatid cohesins during spermatogenesis and oogenesis of Caenorhabditis elegans. We show that there is a different requirement for TOP-2 in meiosis of spermatogenesis and oogenesis. The loss-of-function mutation top-2(it7) results in premature REC-8 removal in spermatogenesis, but not oogenesis. This is due to a failure to maintain the HORMA- domain proteins HTP-1 and HTP-2 (HTP-1/2) on chromosome axes at diakinesis and mislocalization of the downstream components that control sister chromatid cohesion release including Aurora B kinase. In oogenesis, top-2(it7) causes a delay in the localization of Aurora B to oocyte chromosomes but can be rescued through premature activation of the maturation promoting factor via knock-down of the inhibitor kinase WEE-1.3. The delay in Aurora B localization is associated with an increase in the length of diakinesis chromosomes and wee-1.3 RNAi mediated rescue of Auorora B localization in top-2(it7) is associated with a decrease in chromosome length. Our results imply that the sex-specific effects of Topoisomerase II on sister chromatid cohesion release are due to differences in the temporal regulation of meiosis and chromosome structure in late prophase I in spermatogenesis and oogenesis.


title: The SARS-CoV-2 nucleocapsid protein preferentially binds long and structured RNAs

abstract:
The SARS-CoV-2 nucleocapsid protein (NCAP) functions in viral RNA genome packaging, virion assembly, RNA synthesis and translation, and regulation of host immune response. RNA-binding is central to these processes. Little is known how NCAP selects its binding partners in the myriad of host and viral RNAs. To address this fundamental question, we employed electrophoresis mobility shift and competition assays to compare NCAP binding to RNAs that are of SARS-CoV-2 vs. non-SARS-CoV-2, long vs. short, and structured vs. unstructured. We found that although NCAP can bind all RNAs tested, it primarily binds structured RNAs, and their association suppresses strong interaction with single-stranded RNAs. NCAP prefers long RNAs, especially those containing multiple structures separated by single-stranded linkers that presumably offer conformational flexibility. Additionally, all three major regions of NCAP bind RNA, including the low complexity domain and dimerization domain that promote formation of NCAP oligomers, amyloid fibrils and liquid-liquid phase separation. Combining these observations, we propose that NCAP-NCAP interactions that mediate higher-order structures during packaging also drive recognition of the genomic RNA and call this mechanism recognition-by-packaging. This study provides a biochemical basis for understanding the complex NCAP-RNA interactions in the viral life cycle and a broad range of similar biological processes.

HIGHLIGHTSO_LINCAP primarily binds structured RNAs.
C_LIO_LINCAP prefers multiple RNA structures separated by single-stranded linkers.
C_LIO_LINCAP favors binding to long RNAs.
C_LI


title: Structural basis for broad anti-phage immunity by DISARM

abstract: In the evolutionary arms race against phage, bacteria have assembled a diverse arsenal of antiviral immune strategies. While the recently discovered DISARM (Defense Island System Associated with Restriction-Modification) systems can provide protection against a wide range of phage, the molecular mechanisms that underpin broad antiviral targeting but avoiding autoimmunity remain enigmatic. Here, we report cryo-EM structures of the core DISARM complex, DrmAB, both alone and in complex with an unmethylated phage DNA mimetic. These structures reveal that DrmAB core complex is autoinhibited by a trigger loop (TL) within DrmA and binding to DNA substrates containing a 5 overhang dislodges the TL, initiating a long-range structural rearrangement for DrmAB activation. Together with structure-guided in vivo studies, our work provides insights into the mechanism of phage DNA recognition and specific activation of this widespread antiviral defense system.


title: Vendor-neutral sequences (VENUS) and fully transparent workflows improve inter-vendor reproducibility of quantitative MRI

abstract:
PurposeWe developed an end-to-end workflow that starts with a vendor-neutral acquisition and tested the hypothesis that vendor-neutral sequences decrease inter-vendor variability of T1, MTR and MTsat measurements.

MethodsWe developed and deployed a vendor-neutral 3D spoiled gradient-echo (SPGR) sequence on three clinical scanners by two MRI vendors. We then acquired T1 maps on the ISMRM-NIST system phantom, as well as T1, MTR and MTsat maps in three healthy participants. We performed hierarchical shift function analysis in vivo to characterize the differences between scanners when the vendor-neutral sequence is used instead of commercial vendor implementations. Inter-vendor deviations were compared for statistical significance to test the hypothesis.

ResultsIn the phantom, the vendor-neutral sequence reduced inter-vendor differences from 8 - 19.4% to 0.2 - 5% with an overall accuracy improvement, reducing ground truth T1 deviations from 7 - 11% to 0.2 - 4%. In vivo we found that the variability between vendors is significantly reduced (p = 0.015) for all maps (T1, MTR and MTsat) using the vendor-neutral sequence.

ConclusionWe conclude that vendor-neutral workflows are feasible and compatible with clinical MRI scanners. The significant reduction of inter-vendor variability using vendor-neutral sequences has important implications for qMRI research and for the reliability of multicenter clinical trials.


title: SynLethDB 2.0: A web-based knowledge graph database on synthetic lethality for novel anticancer drug discovery

abstract: Two genes are synthetic lethal if mutations in both genes result in impaired cell viability, while mutation of either gene does not affect the cell survival. The potential usage of synthetic lethality (SL) in anticancer therapeutics has attracted many researchers to identify synthetic lethal gene pairs. To include newly identified SLs and more related knowledge, we present a new version of the SynLethDB database to facilitate the discovery of clinically relevant SLs. We extended the first version of SynLethDB database significantly by including new SLs identified through CRISPR screening, a knowledge graph about human SLs, and new web interface, etc. Over 16,000 new SLs and 26 types of other relationships have been added, encompassing relationships among 14,100 genes, 53 cancers, and 1,898 drugs, etc. Moreover, a brand-new web interface has been developed to include modules such as SL query by disease or compound, SL partner gene set enrichment analysis and knowledge graph browsing through a dynamic graph viewer. The data can be downloaded directly from the website or through the RESTful APIs. The database is accessible online at http://synlethdb.sist.shanghaitech.edu.cn/v2.


title: Comparative analysis of single cell lung atlas of bat, cat, tiger and pangolin

abstract: Horseshoe bats (Rhinolophus sinicus) might help maintain coronaviruses severely affecting human health, such as SARS-CoV and SARS-CoV-2. It has long been suggested that bats may be more tolerant of viral infection than other mammals due to their unique immune system, but the exact mechanism remains to be fully explored. During the COVID-19 pandemic, multiple animal species were diseased by SARS-CoV-2 infection, especially in the respiratory system. Herein, single-cell transcriptomic data of the lungs of a horseshoe bat, a cat, a tiger, and a pangolin were generated. The receptor distribution of twenty-eight respiratory viruses belonging to fourteen viral families were characterized for the four species. Comparison on the immune-related transcripts further revealed limited cytokine activations in bats, which might explain the reason why bats experienced only mild diseases or even no symptoms upon virus infection. Our findings might increase our understanding of the immune background of horseshoe bats and their insensitivity to virus infections.


title: PyLiger: Scalable single-cell multi-omic data integration in Python

abstract:
MotivationLIGER is a widely-used R package for single-cell multi-omic data integration. However, many users prefer to analyze their single-cell datasets in Python, which offers an attractive syntax and highly-optimized scientific computing libraries for increased efficiency.

ResultsWe developed PyLiger, a Python package for integrating single-cell multi-omic datasets. PyLiger offers faster performance than the previous R implementation (2-5x speedup), interoperability with AnnData format, flexible on-disk or in-memory analysis capability, and new functionality for gene ontology enrichment analysis. The on-disk capability enables analysis of arbitrarily large single-cell datasets using fixed memory.

AvailabilityPyLiger is available on Github at https://github.com/welch-lab/pyliger and on the Python Package Index.

Contactwelchjd@umich.edu

Supplementary informationSupplementary data are available at Bioinformatics online.


title: Towards More Realistic Simulated Datasets for Benchmarking Deep Learning Models in Regulatory Genomics

abstract: Deep neural networks and support vector machines have been shown to accurately predict genome-wide signals of regulatory activity from raw DNA sequences. These models are appealing in part because they can learn predictive DNA sequence features without prior assumptions. Several methods such as in-silico mutagenesis, GradCAM, DeepLIFT, Integrated Gradients and Gkm-Explain have been developed to reveal these learned features. However, the behavior of these methods on regulatory genomic data remains an area of active research. Although prior work has benchmarked these methods on simulated datasets with known ground-truth motifs, these simulations employed highly simplified regulatory logic that is not representative of the genome. In this work, we propose a novel pipeline for designing simulated data that comes closer to modeling the complexity of regulatory genomic DNA. We apply the pipeline to build simulated datasets based on publicly-available chromatin accessibility experiments and use these datasets to bench-mark different interpretation methods based on their ability to identify ground-truth motifs. We find that a GradCAM-based method, which was reported to perform well on a more simplified dataset, does not do well on this dataset (particularly when using an architecture with shorter convolutional kernels in the first layer), and we theoretically show that this is expected based on the nature of regulatory genomic data. We also show that Integrated Gradients sometimes performs worse than gradient-times-input, likely owing to its linear interpolation path. We additionally explore the impact of user-defined settings on the interpretation methods, such as the choice of "reference"/"baseline", and identify recommended settings for genomics. Our analysis suggests several promising directions for future research on these model interpretation methods. Code and links to data are available at https://github.com/kundajelab/interpret-benchmark.


title: ABCnet : Self-Attention based Atom, Bond Message Passing Network for Predicting Drug-Target Interaction

abstract: Drug-target interaction (DTI) is a methodology for predicting the binding affinity between a compound and a target protein, and a key technology in the derivation of candidate substances in drug discovery. As DTI experiments have progressed for a long time, a substantial volume of chemical, biomedical, and pharmaceutical data have accumulated. This accumulation of data has occurred contemporaneously with the advent of the field of big data, and data-based machine learning methods could significantly reduce the time and cost of drug development. In particular, the deep learning method shows potential when applied to the fields of vision and speech recognition, and studies to apply deep learning to various other fields have emerged. Research applying deep learning is underway in drug development, and among various deep learning models, a graph-based model that can effectively learn molecular structures has received more attention as the SOTA in experimental results were achieved. Our study focused on molecular structure information among graph-based models in message passing neural networks. In this paper, we propose a self-attention-based bond and atom message passing neural network which predicts DTI by extracting molecular features through a graph model using an attention mechanism. Model validation experiments were performed after defining binding affinity as a regression and classification problem: binary classification to predict the presence or absence of binding to the drug-target, and regression to predict binding affinity to the drug-target. Classification was performed with BindingDB, and regression was performed with the DAVIS dataset. In the classification problem, ABCnet showed higher performance than MPNN, as it does in the existing study, and in regression, the potential of ABCnet was checked compared to that of SOTA. According to experiments, for Binary classification ABCnet have an average performance improvement of 1% for higher performance on DTI task than other MPNN and for regresssion ABCnet have CI with an average 0.01 to 0.02 performance degradation compared to SOTA. https://www.overleaf.com/project/618a05533676801d8f68ccf6


title: Structural Evolution of the Ancient Enzyme, Dissimilatory Sulfite Reductase

abstract: Dissimilatory sulfite reductase is an ancient enzyme that has linked the global sulfur and carbon biogeochemical cycles since at least 3.47 Gya. While much has been learned about the phylogenetic distribution and diversity of DsrAB across environmental gradients, far less is known about the structural changes that occurred to maintain DsrAB function as the enzyme accompanied diversification of sulfate/sulfite reducing organisms (SRO) into new environments. Analyses of available crystal structures of DsrAB from Archaeoglobus fulgidus and Desulfovibrio vulgaris, representing early and late evolving lineages, respectively, show that certain features of DsrAB are structurally conserved, including active siro-heme binding motifs. Whether such structural features are conserved among DsrAB recovered from varied environments, including hot spring environments that host representatives of the earliest evolving SRO lineage (e.g., MV2-Eury), is not known. To begin to overcome these gaps in our understanding of the evolution of DsrAB, structural models from MV2.Eury were generated and evolutionary sequence co-variance analyses were conducted on a curated DsrAB database. Phylogenetically diverse DsrAB harbor many conserved functional residues including those that ligate active siro-heme(s). However, evolutionary co-variance analysis of monomeric DsrAB subunits revealed several False Positive Evolutionary Couplings (FPEC) that correspond to residues that have co-evolved despite being too spatially distant in the monomeric structure to allow for direct contact. One set of FPECs corresponds to residues that form a structural path between the two active siro-heme moieties across the interface between heterodimers, suggesting the potential for allostery or electron transfer within the enzyme complex. Other FPECs correspond to structural loops and gaps that may have been selected to stabilize enzyme function in different environments. These structural bioinformatics results suggest that DsrAB has maintained allosteric communication pathways between subunits as SRO diversified into new environments. The observations outlined here provide a framework for future biochemical and structural analyses of DsrAB to examine potential allosteric control of this enzyme.


title: Strain dependent structural effects and in vivo efficacy of enterovirus-D68 inhibitors

abstract:
Acute flaccid myelitis (AFM) leads to loss of limb control in young children and is likely due to Enterovirus-D68 (EV-D68), for which there is no current treatment. We have developed a lead isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of the pleconaril-resistant CVB3-Woodruff (IC50 6-20 nM), EV-D68 (IC50 58 nM), and other enteroviruses. A mouse respiratory model of EV-D68 infection, in which pleconaril is inactive, showed decreased viremia of 3 log units as well as statistically significant 1 log reduction in lung titer reduction at day 5 after treatment with 11526092. A cryo-electron microscopy (cryo-EM) structure of EV-D68 in complex with 11526092 suggests that the increased potency may be due to additional hydrophobic interactions. Cryo-EM structures of 11526092 and pleconaril demonstrate destabilization of EV-D68 (MO strain) compared to the previously described stabilization of EV-D68 (Fermon strain) with pleconaril, illustrating clear strain dependent mechanisms of this molecule. 11526092 represents a more potent inhibitor in vitro with in vivo efficacy providing a potential future treatment for EV-D68 and AFM, suggesting an improvement over pleconaril for further optimization.

One-Sentence Summary11526092 demonstrates protein destabilization, improved in vitro potency and in vivo efficacy when compared with pleconaril against EV-D68.


title: Revealing the secrets beneath grapevine and Plasmopara viticola early communication: a picture of host and pathogen proteomes

abstract: Plant apoplast is the first hub of plant-pathogen communication where pathogen effectors are recognized by plant defensive proteins and cell receptors and several signal transduction pathways are activated. As a result of this first contact, the host triggers a defence response that involves the modulation of several extra and intracellular proteins. In grapevine-pathogen interactions, little is known about the communication between cells and apoplast. Also, the role of apoplastic proteins in response to pathogens still remains a blackbox. In this study we focused on the first 6 hours after Plasmopara viticola inoculation to evaluate grapevine proteome modulation in the apoplastic fluid (APF) and whole leaf tissue. Plasmopara viticola proteome was also assessed enabling a deeper understanding of plant and pathogen communication. Our results showed that oomycete recognition, plant cell wall modifications, ROS signalling and disruption of oomycete structures are triggered in Regent after P. viticola inoculation. Our results highlight a strict relation between the apoplastic pathways modulated and the proteins identified in the whole leaf proteome. On the other hand, P. viticola proteins related to growth/morphogenesis and virulence mechanisms were the most predominant. This pioneer study highlights the early dynamics of extra and intracellular communication in grapevine defence activation that leads to the successful establishment of an incompatible interaction.


title: Dichotomous Feedback: A Signal Sequestration-based Feedback Mechanism for Biocontroller Design

abstract: We introduce a new design framework for implementing negative feedback regulation in Synthetic Biology, which we term  dichotomous feedback. Our approach is different from current methods, in that it sequesters existing fluxes in the process to be controlled, and in this way takes advantage of the processs architecture to design the control law. This signal sequestration mechanism appears in many natural biological systems and can potentially be easier to realise than  molecular sequestration and other comparison motifs that are nowadays common in biomolecular feedback control design. The loop is closed by linking the strength of signal sequestration to the process output. Our feedback regulation mechanism is motivated by two-component signalling systems, where we introduce a second response regulator competing with the natural response regulator thus sequestering kinase activity. Here, dichotomous feedback is established by increasing the concentration of the second response regulator as the level of the output of the natural process increases. Extensive analysis demonstrates how this type of feedback shapes the signal response, attenuates intrinsic noise while increasing robustness and reducing crosstalk.

category: synthetic biology


title: Techniques for visualizing fibroblast-vessel interactions in the developing and adult CNS

abstract: Fibroblasts are found associated with blood vessels in various locations across the CNS: in the meninges, the choroid plexus, and in the parenchyma within perivascular spaces. CNS fibroblasts have been characterized using transcriptional profiling and a Col1a1-GFP mouse line used to identify CNS fibroblasts in vivo. However, current methods for visualizing CNS fibroblasts are lacking and, in particular, prevent adequate assessment of fibroblast-vessel interactions. Here, we describe methods for whole mount visualization of meningeal and choroid plexus fibroblasts, and optical tissue clearing methods for visualization of parenchymal vessel-associated fibroblasts. Importantly, these techniques can be combined with immunohistochemistry methods for labeling different cell types in the meninges and blood vasculature as well as EdU-based cell proliferation assays. These methods are ideal for visualization of vessel-fibroblast interactions in these CNS structures and provide significant improvement over traditional sectioning and staining methods. We expect these methods will advance studies of CNS fibroblast development and functions in homeostasis, injury, and disease.

category: neuroscience


title: The mitochondrial calcium uniporter regulates mitochondrial mass and dendritic localization in distinct hippocampal circuits

abstract: CA2 is an understudied subregion of the hippocampus that is critical for social memory. Previous studies identified multiple components of the mitochondrial calcium uniporter (MCU) complex as selectively enriched in CA2, however the functional significance of this enrichment remains unclear. The MCU complex regulates calcium entry into mitochondria, which in turn regulates mitochondrial transport and localization to active synapses. We found that MCU is strikingly enriched in CA2 distal apical dendrites, precisely where CA2 neurons receive entorhinal cortical input carrying social information. Further, MCU-enriched mitochondria in CA2 distal dendrites are larger compared to mitochondria in CA2 proximal apical dendrites and neighboring CA1 apical dendrites. Genetic knockdown of MCU in CA2 resulted in smaller mitochondria in CA2 distal dendrites, indicating that MCU expression plays a role in regulating mitochondrial mass in CA2. MCU overexpression in neighboring CA1 led to larger mitochondria preferentially in proximal dendrites compared to distal dendrites and GFP controls. Our findings demonstrate that mitochondria are molecularly and structurally diverse across hippocampal cell types and circuits, and that MCU expression cell-autonomously regulates mitochondrial mass, but layer-specific dendritic localization depends on cell type. Our data support the idea that CA2 mitochondria are functionally distinct from CA1 mitochondria, which may confer unique synaptic and circuit properties underlying CA2 function in social memory.