title: Contribution of the Wolffian duct mesenchyme to the formation of the female reproductive tract

abstract:
The female reproductive tract develops from its embryonic precursor, the Mullerian duct.

In close proximity to the Mullerian duct lies the precursor for the male reproductive tract, the Wolffian duct, which is eliminated in the female embryo during sexual differentiation. We discovered that a component of the Wolffian duct, its mesenchyme, is not eliminated after sexual differentiation. Instead, the Wolffian duct mesenchyme underwent changes in transcriptome and chromatin accessibility from male tract to female tract identity, and became a unique mesenchymal population in the female reproductive tract with localization and transcriptome distinct from the mesenchyme derived from the Mullerian duct. Partial ablation of the Wolffian duct mesenchyme stunted the growth of the fetal female reproductive tract. These findings reveal a new fetal origin of mesenchymal tissues for female reproductive tract formation and reshape our understanding of sexual differentiation of reproductive tracts.

Significance statementA female embryo initially possesses both the primitive female and male reproductive tracts, also known as the Mullerian and Wolffian duct. During sexual differentiation, the female eliminates the Wolffian duct and maintains the Mullerian duct that eventually differentiates into the female reproductive tract organs. However, in this paper, we show the female embryo retains mesenchymal cells surrounding the Wolffian duct for its reproductive tract formation. When incorporated into the female reproductive tract organs, the Wolffian duct mesenchyme shows unique anatomical localization and transcriptome and plays critical roles in female reproductive tract growth. This discovery provides new insights into female reproductive tract development and advances our understanding of sexual differentiation of reproductive tracts.


title: Comparisons of cell proliferation and cell death across life histories in the hemichordate Schizocardium californicum

abstract:
BackgroundThere are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when the tissue that will give rise to the adult originates. Here we present patterns of cell proliferation and cell death during larval development, metamorphosis, and adult body plan formation, in the hemichordate Schizocardium californium to answer these questions.

ResultsWe identified distinct patterns of cell proliferation between larval and adult body plan formation of S. californicum. We found that some adult tissues proliferate prior to the start of any morphological metamorphosis. Additionally, we describe a genetic signature of proliferative cells with an irradiation approach that revealed markers shared between the life history states and others that are unique to larvae or juveniles. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis.

ConclusionsCell proliferation during the development of S. californicum has distinct patterns in the formation of larval and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferate and give rise to the larva and juvenile have a genetic signature that is more suggestive of a heterogeneous pool of proliferative progenitors versus a population of pluripotent set-aside cells. Taken together, we propose that S. californicum has a transformative metamorphosis that may be more representative of the development strategies that characterize metamorphosis in many metazoan animals.

category: developmental biology


title: Single-Cell Multi-Omic Roadmap of Human Fetal Pancreatic Development

abstract: The critical cellular transitions that govern human pancreas development are largely unknown. We performed large-scale single-cell RNA-sequencing (scRNA-Seq) to interrogate human fetal pancreas development from 8-20 weeks post conception. We identified 103 distinct cell types, including four novel endocrine progenitor subtypes displaying unique transcriptional features and differentiation potency. Integration with single-nucleus Assay for Transposase Accessible Chromatin Sequencing (snATAC-Seq) identified candidate regulators of human endocrine cell fate and revealed development-specific regulatory annotation at diabetes risk loci. Comparison of in vitro stem cell-derived and endogenous endocrine cells predicted aberrant genetic programs leading to the generation of off-target cells. Finally, knock-out studies revealed that the gene FEV regulates human endocrine differentiation. This work establishes a roadmap of human pancreatic development, highlights previously unappreciated cellular diversity and lineage dynamics, and provides a blueprint for understanding pancreatic disease and physiology, as well as generating human stem cell-derived islet cells in vitro for regenerative medicine purposes.


title: Mechanistic model of MAPK signaling reveals how allostery and rewiring contribute to drug resistance

abstract:
BRAFV600E is prototypical of oncogenic mutations that can be targeted therapeutically and treatment of BRAF-mutant melanomas with RAF and MEK inhibitors results in rapid tumor regression. However, drug-induced rewiring causes BRAFV600E melanoma cells to rapidly acquire a drug-adapted state. In patients this is thought to promote acquisition or selection for resistance mutations and disease recurrence. In this paper we use an energy-based implementation of ordinary differential equations in combination with proteomic, transcriptomic and imaging data from melanoma cells, to model the precise mechanisms responsible for adaptive rewiring. We demonstrate the presence of two parallel MAPK (RAF-MEK-ERK kinase) reaction channels in BRAFV600E melanoma cells that are differentially sensitive to RAF and MEK inhibitors. This arises from differences in protein oligomerization and allosteric regulation induced by oncogenic mutations and drug binding. As a result, the RAS-regulated MAPK channel can be active under conditions in which the BRAFV600E-driven channel is fully inhibited. Causal tracing demonstrates that this provides a sufficient quantitative explanation for initial and acquired responses to multiple different RAF and MEK inhibitors individually and in combination.

HighlightsO_LIA thermodynamic framework enables structure-based description of allosteric interactions in the EGFR and MAPK pathways
C_LIO_LICausal decomposition of efficacy of targeted drugs elucidates rewiring of MAPK channels
C_LIO_LIModel-based extrapolation from type I[1/2] RAF inhibitors to type II RAF inhibitors
C_LIO_LIA unified mechanistic explanation for adaptive and genetic resistance across BRAF-cancers
C_LI

category: systems biology


title: Mitochondrial membrane potential acts as a retrograde signal to regulate cell cycle progression

abstract: Mitochondria are central to numerous anabolic and catabolic pathways whereby mitochondrial dysfunction has a profound impact on metabolism and can manifest in disease. The consequences of mitochondrial dysfunction can be ameliorated by adaptive responses that rely on mito-cellular crosstalk to communicate mitochondrial distress to the rest of the cell. Such mito-cellular signaling slows cell cycle progression in mitochondrial-DNA deficient ({rho}0) Saccharomyces cerevisiae cells, but the initial trigger and the pathway mediating the response has remained unknown. Here, we show that decreased mitochondrial membrane potential ({Delta}{Psi}m) acts as the initial signal of mitochondrial stress that delays G1-to-S phase transition in both {rho}0 and control cells. Accordingly, experimentally increasing {Delta}{Psi}m was sufficient to restore timely cell cycle progression in {rho}0 cells. Neither the RTG retrograde pathway nor central DNA damage checkpoint kinases were involved in mediating this form of mito-cellular communication. The identification of {Delta}{Psi}m as a novel regulator of cell cycle progression may have implications for disease states involving mitochondrial dysfunction.

category: cell biology


title: Lens aquaporin-5 inserts into bovine fiber cell plasma membranes through mitochondria-associated lysosome secretion

abstract:
PURPOSETo spatially map aquaporin-5 (AQP5) expression in bovine lens, molecularly characterize cytoplasmic AQP5-containing vesicles in the outer cortex, and elucidate AQP5 membrane trafficking mechanisms.

METHODSImmunofluorescence was performed on bovine lens cryosections using AQP5, TOMM20, COX IV, calnexin, LC3B, LIMP-2, and connexin-50 antibodies and the fluorescent lipid membrane dye CM-DiI. AQP5 plasma membrane insertion was defined via line expression profile analysis. Transmission electron microscopy (TEM) was performed on bovine lens tissue sections to define cytoplasmic organelle identity, morphology, and subcellular localization in cortical fiber cells. Bovine lenses were treated with 10 nM bafilomycin A1 or 0.1% dimethyl sulfoxide vehicle control in ex vivo culture to determine changes in AQP5 plasma membrane expression.

RESULTSImmunofluorescence analysis revealed cytoplasmic AQP5 expression in bovine lens epithelial cells and differentiating fiber cells. In the bovine lens cortex, complete AQP5 plasma membrane insertion occurs at r/a 0.951 + 0.005. AQP5-containing cytoplasmic vesicles are spheroidal, tubular in morphology, express TOMM20, and contain LC3B and LIMP-2 as fiber cells mature. TEM analysis revealed spheroidal, tubular autophagosomes, autolysosomes, and lysosomes with degrading mitochondria. AQP5-containing cytoplasmic vesicles and autolysosomes dock and fuse with the plasma membrane. Bafiloymcin A1 treatment reduced AQP5 plasma membrane expression by 27%.

CONCLUSIONSAQP5 localizes to spheroidal, tubular cytoplasmic vesicles in the differentiating bovine lens fiber cells. During fiber cell differentiation, these vesicles incorporate LC3B and fuse with LIMP-2-positive lysosomes. AQP5 trafficking to the plasma membrane occurs through lysosome secretion as a novel mechanism of AQP5 trafficking.


title: In situ architecture of human kinetochore-microtubule interface visualized by cryo-electron tomography

abstract: Faithful segregation of chromosomes during mitosis relies on a carefully coordinated and intricate interplay between the centromere, kinetochore, and spindle microtubules. Despite its importance, the architecture of this interface remains elusive. Here we used in situ cryo-electron tomography to visualize the native architecture of the kinetochore-microtubule interface in human U2OS cells at different stages of mitosis. We find that the centromere forms a pocket-like structure around kinetochore microtubules. Two morphologically distinct fibrillar densities form end-on and side-on connections to the plus-ends of microtubules within this centromeric pocket. Our data suggest a dynamic kinetochore-microtubule interface with multiple interactions between outer kinetochore components and spindle microtubules.


title: Proteomic analysis of the actin cortex in interphase and mitosis

abstract: In animal cells, many shape changes are driven by gradients in the contractile tension of the actomyosin cortex, a thin cytoskeletal network that supports the plasma membrane. Elucidating how cortical tension is controlled is thus essential for understanding cell and tissue morphogenesis. Increasing evidence shows that alongside myosin II activity, actin network organisation and composition are key to cortex tension regulation. However, how cortex composition changes when cortical tension changes remains poorly understood. Here, we compared cortices from cells in interphase and in mitosis, as mitosis entry is associated with a strong increase in cortical tension. We purified cortex-enriched cellular fractions and analysed their composition using mass spectrometry, identifying 922 proteins consistently represented in both interphase and mitotic cortices. We curated this dataset by focusing on actin-related proteins, narrowing down to 238 candidate regulators of the cortex during the mitotic cortical tension increase. Among these candidates, our analysis pointed to a role for septins, and in particular septin 9, in the control of mitotic cell rounding. Overall, our study brings insight into the regulation of mitotic rounding, and paves the way for systematic investigations of the regulation of cell surface mechanics.


title: Follow that cell: leukocyte migration in L-plastin mutant zebrafish.

abstract: Actin assemblies are important in motile cells such as leukocytes which form dynamic plasma membrane extensions or podia. L-plastin (LCP1) is a leukocyte-specific calcium-dependent actin-bundling protein that, in mammals, is known to affect immune cell migration. Previously, we generated CRISPR/Cas9 engineered zebrafish lacking L-plastin (lcp1-/-) and reported that they had reduced survival to adulthood, suggesting that lack of L-plastin might negatively affect the immune system. To test this hypothesis, we examined the distribution and migration of neutrophils and macrophages in the transparent tail of early zebrafish larvae using cell-specific markers and an established wound-migration assay. Knockout larvae were similar to their heterozygous siblings in having equal body sizes and comparable numbers of neutrophils in caudal hematopoietic tissue at two days post-fertilization, indicating no gross defect in neutrophil production or developmental migration. When stimulated by a tail wound, all genotypes of neutrophils were equally migratory in a two-hour window. However for macrophages we observed both migration defects and morphological differences. L-plastin knockout macrophages still homed to wounds but were slower, less directional and had a star-like morphology with many leading and trailing projections. In contrast, wild type macrophages were faster, more directional, and had a more streamlined, slug-like morphology. Overall, these findings show that in larval zebrafish L-plastin knockout primarily affects the macrophage response with possible consequences for organismal immunity. Consistent with our observations, we propose a model in which cytoplasmic L-plastin negatively regulates macrophage integrin adhesion by holding these transmembrane heterodimers in a  clasped, inactive form and is a necessary part of establishing macrophage polarity during chemokine-induced motility.


title: A unique mineralizing pool of Gli1+ stem cells builds the tendon enthesis and demonstrates therapeutic potential

abstract:
The enthesis, a fibrocartilaginous transition between tendon and bone, is necessary for the transfer of force from muscle to bone to produce joint motion. The enthesis is prone to injury due to mechanical demands, and it cannot regenerate. A better understanding of how the enthesis develops will lead to more effective therapies to prevent pathology and promote regeneration. Here, we used single-cell RNA sequencing to define the development transcriptome of the entheses over postnatal stages. Six resident cell types, including enthesis progenitors and mineralizing chondrocytes, were identified along with their transcription factor regulons and temporal regulation. Following our prior discovery of the necessity of Gli1-lineage cells for enthesis development and healing, we then examined their transcriptomes at single cell resolution and demonstrated their clonogenicity and multipotency. Transplantation of these Gli1- lineage cells to enthesis injuries improved healing, demonstrating their therapeutic potential for enthesis regeneration.

HighlightsO_LIThe transcriptome and differentiation trajectory of enthesis stem cells during postnatal development are defined at single cell resolution.
C_LIO_LITranscription factor regulons drive enthesis stem cell differentiation.
C_LIO_LIGli1-lineage enthesis stem cells demonstrate in vivo and in vitro clonogenicity and multipotency.
C_LIO_LITransplantation of Gli1-lineage enthesis stem cells to enthesis injuries improves healing.
C_LI


title: The high-throughput perturbation of long non-coding RNA reveals functional features in stem cells and across cell-types

abstract: Long non-coding RNAs (lncRNAs) were shown to regulate pluripotency and differentiation, however, a comprehensive assessment of their regulatory roles in stem cells is currently lacking. By performing a large-scale knockdown of lncRNAs (n = 390) in human induced pluripotent stem (iPS) cells, we systematically characterized their roles in self-renewal and pluripotency. Cell growth and transcriptomic assays revealed respectively, 18.3% and 29.3% of these lncRNAs in altering cellular and molecular phenotypes upon knockdown. By integrating the molecular phenotypes with chromatin-interaction assays, we identified their cis- and trans- interacting partners as potential primary targets. In addition, cell-type enrichment revealed 16 lncRNAs associated with pluripotency and we highlighted LINC02595, CATG00000090305.1 and RP11-148B6.2. Next, we integrated fibroblasts phenotyping data (Ramilowski et al., 2020; Genome Research) and reported that only 2.9% lncRNAs exhibited consistent cell growth phenotype, while molecular phenotype showed 66.7% agreement. This highlights their consistent primary functional roles across cell-types and the distinct secondary effects under different cellular environments.

category: genomics


title: Molecular mechanisms of spontaneous curvature and softening in complex lipid bilayer mixtures

abstract: Membrane reshaping is an essential biological process. The chemical composition of lipid membranes determines their mechanical properties, and thus the energetics of their shape. Hundreds of distinct lipid species make up native bilayers, and this diversity complicates efforts to uncover what compositional factors drive membrane stability in cells. Simplifying assumptions, therefore, are used to generate quantitative predictions of bilayer dynamics based on lipid composition. One assumption commonly used is that "per lipid" mechanical properties are both additive and constant -- that they are an intrinsic property of lipids independent of the surrounding composition. Related to this, is the assumption that lipid bulkiness, or "shape" determines its curvature preference, independently of context. In this study, all-atom molecular dynamics simulations on three separate multi-lipid systems were used to explicitly test these assumptions, applying methodology recently developed to isolate properties of single lipids or nanometer-scale patches of lipids. The curvature preference of populations of lipid conformations were inferred from their redistribution on a dynamically fluctuating bilayer. Representative populations were extracted by both structural similarity and semi-automated hidden Markov model analysis. The curvature preferences of lipid dimers were then determined and compared to an additive model that combines the monomer curvature preference of both the individual lipids. In all three systems, we identified conformational subpopulations of lipid dimers that showed non-additive curvature preference, in each case mediated by a special chemical interaction (e.g., hydrogen bonding). Our study highlights the importance of specific chemical interactions between lipids in multicomponent bilayers and the impact of interactions on bilayer stiffness. We identify two mechanisms of bilayer softening: Diffusional softening, which is driven by the dynamic coupling between lipid distributions and membrane undulations, and conformational softening, which is driven by the inter-conversion between distinct dimeric conformations.

category: biophysics


title: Mechanism of Voltage Gating in the Voltage-Sensing Phosphatase Ci-VSP

abstract: The conformational changes in voltage-sensing domain (VSD) are driven by the transmembrane electric field acting on charges and countercharges. Yet, the overall energetics and detailed mechanism of this process are not fully understood. Here, we determined free energy and displacement charge landscapes, as well as major conformations corresponding to a complete functional gating cycle in the isolated voltage-sensing domain of the phosphatase Ci-VSP (Ci-VSD) comprising four transmembrane helices (segments S1-S4). Molecular dynamics simulations highlight the extent of S4 movements. In addition to the crystallographically determined activated  Up and resting  Down states, the simulations predict two novel Ci-VSD conformations: a deeper resting state ( Down-minus) and an extended activated ( Up-plus) state. These additional conformations were experimentally probed via systematic cysteine mutagenesis with metal-ion bridges and the engineering of proton conducting mutants at hyperpolarizing voltages. These results show that voltage activation involves sequentially populating these four states in a stepwise way, translating one arginine across the membrane electric field per step, transferring ~3 e0 charges.


title: Hyperactivity of indirect pathway-projecting spiny projection neurons drives compulsive behavior

abstract: Compulsive behaviors are a hallmark symptom of obsessive compulsive disorder (OCD). Striatal hyperactivity has been linked to compulsive behavior generation in correlative studies in humans and causal studies in rodents. However, the contribution of the two distinct striatal output populations to the generation and treatment of compulsive behavior is unknown. These populations of direct and indirect pathway-projecting spiny projection neurons (SPNs) have classically been thought to promote or suppress actions, respectively, leading to a long-held hypothesis that increased output of direct relative to indirect pathway promotes compulsive behavior. Contrary to this hypothesis, here we find that indirect pathway hyperactivity drives compulsive grooming in the Sapap3-knockout mouse model of OCD-relevant behavior. Furthermore, we show that suppression of indirect pathway activity using optogenetics or treatment with the first-line OCD pharmacotherapy fluoxetine is associated with reduced compulsive behavior. Together, these findings highlight the striatal indirect pathway as a potential new treatment target for compulsive behavior.

category: neuroscience


title: Atypical MAPK regulates translocation of GATA transcription factor in response to chemoattractant stimulation

abstract: The Dictyostelium atypical MAPK Erk2 (a.k.a. ErkB) is required for chemotactic responses to external cAMP as individual amoeba aggregate and undergo a multicellular developmental program. External cAMP also stimulates the translocation of the GATA transcription factor, GtaC, a key regulator of developmental gene expression and a developmental timer of repeated cAMP stimulation of developing cells. In this study, atypical MAPK Erk2 was found to be essential for the translocation of GtaC in response to external cAMP as indicated by the cellular distribution of GFP-GtaC reporters. Erk2 was also demonstrated to mediate the translocation of GtaC in response to external folate, a signal that directs the foraging of amoeba for bacterial food sources. Erk1, the only other Dictyostelium MAPK, was not required for the GtaC translocation to either chemoattractant, indicating that GFP-GtaC is a kinase translocation reporter specific for atypical MAPKs. The translocation of GFP-GtaC in response to folate was absent in mutants lacking the folate receptor, Far1 (a.k.a. GrlL) or the coupled G protein, G4 subunit. Loss of GtaC function resulted in enhanced chemotactic movement to folate. Inspection of the GtaC primary sequence revealed four sites recognized as Erk2 preferred phosphorylation sites. The creation and analysis of GFP-GtaC mutants with alterations at these sites determined that these sites are required for translocation in response to folate. Consistent with the role of translocation for GtaC function certain combinations of these phosphorylation site alterations interfered with the ability of GFP-GtaC reporter to rescue aggregation of gtaC- cells. These findings provide the first evidence that atypical MAPKs can regulate transcription factors through specific phosphorylation sites in response to chemoattractants. The ability of different stimuli to regulate GtaC translocation through Erk2 indicates atypical MAPKs can regulate different cell fates in part through the translocation of transcription factors.


title: The giant Mimivirus 1.2 Mb genome is elegantly organized into a 30 nm helical protein shield

abstract:
Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the [~]0.5 {micro}m icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography and proteomics revealed that it is encased into a [~]30 nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.

One-Sentence SummaryMimivirus genome organization in the icosahedral virion.

category: microbiology


title: LncRNA RUS shapes the gene expression program towards neurogenesis

abstract: The evolution of brain complexity correlates with an increased expression of long, non- coding (lnc) RNAs in neural tissues. Although prominent examples illustrate the potential of lncRNAs to scaffold and target epigenetic regulators to chromatin loci, only few cases have been described to function during brain development. We present a first functional characterization of the lncRNA LINC01322, which we term RUS for  RNA upstream of Slitrk3. The RUS gene is well conserved in mammals by sequence and synteny next to the neurodevelopmental gene Slitrk3. RUS is exclusively expressed in neural cells and its expression increases along with neuronal markers during neuronal differentiation of mouse embryonic cortical neural stem cells. Depletion of RUS locks neuronal precursors in an intermediate state towards neuronal differentiation resulting in arrested cell cycle and increased apoptosis. RUS associates with chromatin in the vicinity of genes involved in neurogenesis, most of which change their expression upon RUS depletion. The identification of a range of epigenetic regulators as specific RUS interactors suggests that the lncRNA may mediate gene activation and repression in a highly context-dependent manner.

category: molecular biology


title: E-cadherin is sorted by Rab7 and Snx16 for polarised secretion via Myosin V

abstract: E-cadherin has a fundamental role in epithelial tissues by providing cell-cell adhesion. Epithelial homeostasis relies on polarised E-cadherin exocytosis to the lateral plasma membrane, however the secretion mechanisms are unknown. Epithelial plasticity depends on constant E-cadherin endocytosis and recycling, but it is unclear how recycling is facilitated. Here we use the Drosophila follicular epithelium to analyse E- cadherin recycling and secretion. We identify endosomes in the apical region of the epithelium, in which newly translated and endocytosed E-cadherin converge for polarised E-cadherin secretion. Our data provide evidence that Rab7 recruits Snx16 to these endosomes, and that Snx16 moves E-cadherin via tubulation into the Rab11 compartment. Rab11 forms E-cadherin transport vesicles by recruiting its effector Myosin V. We show in living follicles how Myosin V transports E-cadherin along an apical actin network to the zonula adherence. An additional secretion pathway exists in the basal epithelium, where Myosin V moves E-cadherin vesicles along parallel actin bundles to the plasma membrane.


title: mRNA 5' terminal sequences drive 200-fold differences in expression through effects on synthesis, translation and decay

abstract: mRNA regulatory sequences control gene expression at multiple levels including translation initiation and mRNA decay. The 5' terminal sequences of mRNAs have unique regulatory potential because of their proximity to key post-transcriptional regulators. Here we have systematically probed the function of 5' terminal sequences in gene expression in human cells. Using a library of reporter mRNAs initiating with all possible 7-mer sequences at their 5' ends, we find an unexpected impact on transcription that underlies 200-fold differences in mRNA expression. Library sequences that promote high levels of transcription mirrored those found in native mRNAs and define two basic classes with similarities to classic Initiator (Inr) and TCT core promoter motifs. By comparing transcription, translation and decay rates, we identify sequences that are optimized for both efficient transcription and growth-regulated translation and stability, including variants of terminal oligopyrimidine (TOP) motifs. We further show that 5' sequences of endogenous mRNAs are enriched for multi-functional TCT/TOP hybrid sequences. Together, our results reveal how 5' sequences define two general classes of mRNAs with distinct growth-responsive profiles of expression across synthesis, translation and decay.

category: genetics


title: nPoRe: n-Polymer Realigner for improved pileup variant calling

abstract: Despite recent improvements in nanopore basecalling accuracy, germline variant calling of small insertions and deletions (INDELs) remains poor. Although precision and recall for single nucleotide polymorphisms (SNPs) now regularly exceeds 99.5%, INDEL recall at relatively high coverages (85x) remains below 80% for standard R9.4.1 flow cells [22, 23, 31]. Current nanopore variant callers work in two stages: an efficient pileup-based method identifies candidates of interest, and then a more expensive full-alignment model provides the final variant calls. Most false negative INDELs are lost during the first (pileup-based) step, particularly in low-complexity repeated regions. We show that read phasing and realignment can recover a significant portion of INDELs lost during this stage. In particular, we extend Needleman-Wunsch affine gap alignment by introducing new gap penalties for more accurately aligning repeated n-polymer sequences such as homopolymers (n = 1) and tandem repeats (2 [≤] n [≤] 6). On our dataset with 60.6x coverage, haplotype phasing improves INDEL recall in all evaluated high confidence regions from 63.76% to 70.66% and then nPoRe realignment improves it further to 73.04%, with no loss of precision.

category: bioinformatics


title: LabGym: A versatile computational tool for automatic quantification of user-defined animal behaviors

abstract: Quantifying animal behavior is important for many branches of biological research. Current computational tools for quantifying behaviors typically rely on a few pre-defined, simplified features to identify a behavior. However, such an approach underuses the behavioral information and restricts the tools applicability to limited behavior types or animal species. Here we report a new tool, LabGym, for quantifying animal behaviors without such limitations. Combining a novel approach for effective evaluation of animal motion with customizable convolutional recurrent networks for capturing spatiotemporal details, LabGym provides holistic behavioral assessment and accurately identify user-defined animal behaviors without restrictions on behavior types or animal species. It then calculates various parameters to provide quantitative measurements of the intensity and dynamics of each behavior. LabGym requires neither any intermediate step that causes information loss nor programming knowledge from users for post-hoc analysis. It tracks multiple animals simultaneously in various experimental settings for high-throughput and versatile analysis, and provides users a way to generate visualizable behavioral datasets that are valuable resources for research in neuroscience, behavioral science, and computer science. We demonstrate its efficacy in capturing subtle behavioral changes in animals ranging from soft-bodied invertebrates to mammals.

category: animal behavior and cognition


title: LabGym: quantification of user-defined animal behaviors using learning-based holistic assessment


title: Autophagy promotes programmed cell death and corpse clearance in specific cell types of the Arabidopsis root cap

abstract: Autophagy is a conserved quality control pathway that mediates the degradation of unnecessary or dysfunctional cellular components by targeting them to the lysosomes or central vacuoles. Autophagy has been implicated in the regulation or execution of regulated cell death processes in a wide range of eukaryotes. However, its function in developmentally controlled programmed cell death (dPCD) in plants remains little studied and controversial. Here, we investigated the role of autophagy in dPCD using the Arabidopsis root cap as an accessible and genetically tractable model system. We show that autophagic flux is induced prior to dPCD execution in both root cap tissues, the columella and the lateral root cap (LRC), and impaired in autophagy-deficient mutants. These mutants show a strongly delayed cell death and an absence of corpse clearance in the columella during and after their shedding into the rhizosphere. However, autophagy deficiency does not affect dPCD execution or corpse clearance in LRC cells at the distal end of the root cap. Our results demonstrate that autophagy promotes dPCD in a highly cell-type specific manner, and present the root cap as a powerful model system to study organ-specific autophagy in vivo.

category: plant biology


title: Autophagy promotes organelle clearance and organized cell separation of living root cap cells in Arabidopsis thaliana

abstract:
The root cap is a multi-layered tissue covering the tip of a plant root that directs root growth through its unique functions such as gravity-sensing and rhizosphere interaction. To prevent damages from the soil environment, cells in the root cap continuously turn over through balanced cell division and cell detachment at the inner and the outer cell layers, respectively. Upon displacement toward the outermost layer, columella cells at the central root cap domain functionally transition from gravity-sensing cells to secretory cells, but the mechanisms underlying this drastic cell fate transition are largely unknown. By using live-cell tracking microscopy, we here show that organelles in the outermost cell layer undergo dramatic rearrangements, and at least a part of this rearrangement depends on spatiotemporally regulated activation of autophagy. Notably, this root cap autophagy does not lead to immediate cell death, but rather is necessary for organized separation of living root cap cells, highlighting a previously undescribed role of developmentally regulated autophagy in plants.

Summary statementTime-lapse microscope imaging revealed spatiotemporal dynamics of intracellular reorganization associated with functional transition and cell separation in the Arabidopsis root cap and the roles of autophagy in this process.


title: Abscisic Acid modulates neighbor proximity-induced leaf hyponasty in Arabidopsis

abstract:
Leaves of shade avoiding plants like Arabidopsis thaliana change their growth pattern and position in response to a low red to far-red ratio (LRFR) that is encountered in dense plant communities. In LRFR, transcription factors of the phytochrome interacting family (PIFs) are de-repressed. PIFs induce auxin production, which is required to promote leaf hyponasty thereby favoring access to unfiltered sunlight. Abscisic acid (ABA) has also been implicated in the control of leaf hyponasty. In addition, gene expression patterns suggest that LRFR regulates the ABA response. Here we show that, in leaves, LRFR leads to a rapid increase in ABA levels. Changes in ABA levels depend on PIFs, which regulate the expression of genes encoding isoforms of the enzyme catalyzing a rate-limiting step in ABA biosynthesis. Interestingly, ABA biosynthesis and signaling mutants have more erect leaves in white light but respond less to LRFR. Consistent with this, ABA application decreases the leaf angle in white light but this response is inhibited in LRFR. Tissue-specific interference with ABA signaling indicates that an ABA response is required in different cell types for LRFR-induced hyponasty. Collectively, our data indicate LRFR triggers rapid PIF-mediated ABA production. ABA plays a different role in controlling hyponasty in white light compared to LRFR. Moreover, ABA exerts its activity in multiple cell types to control leaf position.

One sentence summaryAbscisic acid biosynthesis and signaling are required to promote leaf hyponasty in response to a drop in the red to far-red ratio.


title: Transcription factor retention through multiple polyploidisation steps in wheat

abstract:
O_LIWhole genome duplication (WGD) is widespread in plant evolutionary history, but the mechanisms of non-random gene loss after WGD are debated. The gene balance hypothesis proposes that dosage-sensitive genes such as regulatory genes are retained in polyploids. To test this hypothesis, we analysed the retention of transcription factors (TFs) in the recent allohexaploid bread wheat (Triticum aestivum).
C_LIO_LIWe annotated TFs in hexaploid, tetraploid and diploid wheats; compared the co-expression of homoeologous TF and non-TF triads; and analysed single nucleotide variation in TFs across cultivars.
C_LIO_LIWe found that, following each of two hybridisation and WGD events, the proportion of TFs in the genome increased. TFs were preferentially retained over other genes as homoeologous groups in tetraploid and hexaploid wheat. Across cultivars, TF triads contain fewer deleterious missense mutations than non-TFs.
C_LIO_LITFs are preferentially retained as three functional homoeologs in hexaploid wheat, in support of the gene balance hypothesis. High co-expression between TF homoeologs suggests that neo- and sub-functionalisation are not major drivers of TF retention in this young polyploid. Knocking out one TF homoeolog to alter gene dosage, using TILLING or CRISPR, could be a way to further test the gene balance hypothesis and generate new phenotypes for wheat breeding.
C_LI


title: Low Density Lipoprotein Receptor-Related Protein 1 (LRP1) is a host factor for RNA viruses including SARS-CoV-2

abstract: Viruses with an RNA genome are the main causes of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that rendered them resistant to the zoonotic Rift Valley fever virus (RVFV; family Phleboviridae, order Bunyavirales). This screen returned the Low Density Lipoprotein Receptor-Related protein 1 (LRP1, or CD91) as top hit, a 600 kDa plasma membrane protein known to be involved in a wide variety of cell activities. Inactivation of LRP1 expression in human cells reduced RVFV infection at the early stages of infection, including the particle attachment to the cell. In the highly LRP1-positive human HuH-7 cell line, LRP1 was required for the early infection stages also of Sandfly fever Sicilian virus (SFSV; family Phleboviridae, order Bunyavirales), vesicular stomatitis (VSV; family Rhabdoviridae, order Mononegavirales), Encephalomyocarditis virus (EMCV, family Picornaviridae), and the coronaviruses MERS-CoV, SARS-CoV-1, and SARS-CoV-2. While for RVFV, EMCV, and MERS-CoV the replication cycle could eventually catch up, LRP1 requirement for the late infection stage in HuH-7 cells was observed for SFSV, La Crosse virus (LACV; family Peribunyaviridae, order Bunyavirales), VSV, SARS-CoV-1, and SARS-CoV-2. For SARS-CoV-2, the absence of LRP1 stably reduced viral RNA levels in human lung Calu-3 cells, and both RNA levels and particle production in the hepatic HuH-7 cells. Thus, we identified LRP1 as a host factor that supports various infection cycle stages of a broad spectrum of RNA viruses.


title: Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity.

abstract: During the RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10 mediated modulation remains unclear in the absence of nsp14 structure. Here we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10 bound structures explain the modulatory role of the co-factor protein. Further, the structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.


title: On Correlation between Structural Properties and Viral Escape Measurements from Deep Mutational Scanning

abstract: Encouraged by recent efforts to map responses of SARS-CoV-2 mutations to various antibody treatments with deep mutational scanning, we explored the possibility of tying measurable structural contact information from the binding complexes of antibodies and their targets to experimentally determined viral escape responses. With just a single crystal structure for each binding complex, we find that the average correlation coefficient R is surprisingly high at 0.76. Our two methods for calculating contact information use binary contacts measured between all residues of two proteins. By varying the parameters to obtain binary contacts, we find that 3.6 [A] and 7 [A] are pivotal distances to toggle the binary step function when tallying the contacts for each method. The correlations are improved by short simulations ([~]25 ns), which increase average R to 0.78. With blind tests using the random forest model, we can further improve average R to 0.84. These easy-to-implement measurements can be utilized in computational screening of viral mutations that escape antibody treatments and potentially other protein-protein interaction problems.


title: Tensor decomposition reveals coordinated multicellular patterns of transcriptional variation that distinguish and stratify disease individuals

abstract: Tissue- and organism-level biological processes often involve coordinated action of multiple distinct cell types. Current computational methods for the analysis of single-cell RNA-sequencing (scRNA-seq) data, however, are not designed to capture co-variation of cell states across samples, in part due to the low number of biological samples in most scRNA-seq datasets. Recent advances in sample multiplexing have enabled population-scale scRNA-seq measurements of tens to hundreds of samples. To take advantage of such datasets, here we introduce a computational approach called single-cell Interpretable Tensor Decomposition (scITD). This method extracts "multicellular gene expression patterns" that vary across different biological samples. These patterns capture how changes in one cell type are connected to changes in other cell types. The multicellular patterns can be further associated with known covariates (e.g., disease, treatment, or technical batch effects) and used to stratify heterogeneous samples. We first validated the performance of scITD using in vitro experimental data and simulations. We then applied scITD to scRNA-seq data on peripheral blood mononuclear cells (PBMCs) from 115 patients with systemic lupus erythematosus and 56 healthy controls. We recapitulated a well-established pan-cell-type signature of interferon-signaling that was associated with the presence of anti-dsDNA autoantibodies and a disease activity index. We further identified a novel multicellular pattern that appears to potentiate renal involvement for patients with anti-dsDNA autoantibodies. This pattern was characterized by an expansion of activated memory B cells along with helper T cell activation and is predicted to be mediated by an increase in ICOSLG-ICOS interaction between monocytes and helper T cells. Finally, we applied scITD to two PBMC datasets from patients with COVID-19 and identified reproducible multicellular patterns that stratify patients by disease severity. Overall, scITD is a flexible method for exploring co-variation of cell states in multi-sample single-cell datasets, which can yield new insights into complex non-cell-autonomous dependencies that define and stratify disease.


title: Arbidol inhibits esophageal squamous cell carcinoma growth in vitro and in vivo through suppressing ataxia telangiectasia and Rad3-related protein kinase

abstract:
BackgroundEsophageal cancer has a global impact on human health due to its high incidence and mortality. Therefore, there is an urgent need to develop new drugs to treat or prevent the prominent pathological subtype of esophageal cancer, esophageal squamous cell carcinoma. Based upon a screening of drugs approved by the Food and Drug Administration, we discovered that Arbidol could effectively inhibit the proliferation of esophageal squamous cell carcinoma in vitro and in vivo.

MethodWe first conducted a series of cell-based assays and found that Arbidol treatment inhibited the proliferation and colony formation ability of ESCC cells and promoted G1 phase cell cycle arrest. Phospho-proteomics experiments and in vitro kinase assays were subsequently performed using cell lysates derived from Arbidol-treated cells in order to identify the underlying growth inhibitory mechanism. Finally, a PDX model was used to verify the inhibitory effect of Arbidol in vivo.

FindingArbidol inhibits proliferation of ESCC in vitro and in vivo through the DNA replication pathway and is associated with the cell cycle.

InterpretationArbidol is a potential ATR inhibitor via binding to ATR kinase to reduce the phosphorylation and activation of MCM2 at Ser108.

FundingThis research was supported by the National Natural Science Foundation of China (grant number 81872335), the National Natural Science Youth Foundation (grant number 81902486), and the Natural Science Foundation of Henan (grant number 161100510300).

category: cancer biology


title: Spatial patterns of biomass change across Finland in 2009 - 2015

abstract: Forest characteristics vary largely at the regional level and in smaller geographic areas in Finland. The amount of greenhouse gas emissions is related to changes in biomass and the soil type (e.g. upland soils vs. peatlands). Spatially accurate map data of forests and biomass changes could improve the ability to suggest optimal management alternatives for any patch of land, e.g. in terms of climate change mitigation. In our study, estimating and explaining spatial patterns of biomass change across Finland was the main interest. We analysed biomass changes on different soil and site types on forested land using the Finnish Multi-Source National Forest Inventory (MS-NFI) map layers of the 2009 and 2015 datasets. Silvicultural management and treatment units larger than individual pixels were created by automatic segmentation of the thematic maps. The segmented MS-NFI biomass maps of growing stocks, including above and below ground biomass resulted an average estimate of 77.7 tons ha-1 compared to the national forest inventory estimate of 76.5 tons ha-1 in 2015 for Finland. Forest soil type had a similar effect on average biomass in segmented MS-NFI and NFI data. Despite good regional and country-level results, at the segment level the biomass distributions were narrowed and averaging of biomass estimates was observed. Hence, biomass changes on segments can be considered only approximate values; also, those small differences in average biomass may accumulate when map layers from more than one time point are compared. MS-NFI classification results depend on the satellite images and field data used, causing variation in successive inventories. In addition, to avoid false biomass change observations due to the low growth rate of boreal forests, a six-year study period was set. A kappa of 0.44 was achieved for precision when comparing undisturbed and disturbed forest stands in the Global Forest Cover layer and MS-NFI segmented map, indicating the low ability of the global forest map to identify land cover changes for Finland. The segmented biomass maps provide a useful tool for forest owners to analyse carbon stock changes in their forests and how to affect the amount of carbon by forest management.

category: ecology


title: Ionic remodelling following myocardial infarction explains phenotypic variability in ECG and arrhythmic substrate but not in ejection fraction

abstract:
AimsSudden death after myocardial infarction (MI) is associated with electrophysiological heterogeneities and ionic remodelling, which are reflected as variable phenotypes. Low ejection fraction (EF) is used in risk stratification, but its mechanistic links with the post-MI electrophysiological heterogeneities are unknown. We aim to unravel how phenotypic ECG and EF variability in post-MI may be explained by the impact of ionic remodelling on spatio-temporal dispersion of repolarization using human biventricular electromechanical modelling and simulation.

Methods and ResultsMultiple post-MI ECG phenotypes observed clinically were investigated using multi-scale modelling and simulation, calibrated and evaluated using experimental and clinical data. Acute stage T-wave inversion was caused by delayed repolarization in the epicardial border zone (BZ), and Brugada phenocopy was generated by repolarization delay and activation failure in the BZ. Upright tall T-waves in chronic MI represented large repolarisation dispersion between BZ and surrounding tissue, which promoted ectopic propagation at fast pacing. T-wave morphology alternans were present at fast-pacing due to prolonged refractoriness in the mid-myocardium. Post-MI ionic remodelling reduced EF through inhibition of calcium transient amplitude, but the EF at resting heart rate was not sensitive to the extent of repolarisation heterogeneity and the risk of repolarisation abnormalities at fast pacing.

ConclusionsIn acute post-MI, ionic remodelling and its effect on refractoriness and propagation failure in the BZ have a strong impact on phenotypic ECG variability, whereas in chronic post-MI, the repolarisation dispersion across the BZ is crucial. T-wave and QT abnormalities are better indicators of repolarisation heterogeneities than EF in post-MI.

category: bioengineering


title: Depletion of Sun1/2 Induces Heterochromatin Accrual in Mesenchymal Stem Cells during Adipogenesis

abstract: Critical to the mechano-regulation of mesenchymal stem cells (MSC), Linker of the Nucleoskeleton and Cytoskeleton (LINC) complex transduces cytoskeletal forces to the nuclei. The LINC complex contains outer nuclear membrane Nesprin proteins that associate with the cytoskeleton and their inner nuclear membrane couplers, Sun proteins. In addition to coupling Nesprin-associated cytoskeletal elements to inner nuclear membrane, Sun proteins also function in regulating nuclear mechanics and chromatin tethering to inner nuclear membrane. This suggests that release of LINC-mediated cytoskeletal connections from cell nuclei may have different effects on chromatin organization and MSC differentiation than those due to ablation of intra-nuclear Sun proteins. To test this hypothesis we compared Sun1/2 depletion with expression of the dominant-negative KASH domain (dnKASH) that inhibits Nesprin-Sun association. In cells cultured under adipogenic conditions, disconnecting LINC from the cytoskeleton through dnKASH expression, increased adipogenic gene expression and fat droplet formation; heterochromatin H3K27 and H3K9 methylation was unaltered. In contrast, Sun1/2 depletion inhibited adipogenic gene expression and fat droplet formation; as well the anti-adipogenic effect of Sun1/2 depletion was accompanied by increased H3K9me3, which was enriched on Adipoq, silencing this fat locus. We conclude that releasing the nucleus from cytoskeletal constraints via dnKASH accelerates adipogenesis while depletion of Sun1/2 increases heterochromatin accrual on adipogenic genes in a fashion independent of LINC complex function. Therefore, while these two approaches both disable LINC functions, their divergent effects on the epigenetic landscape indicate they cannot be used interchangeably to study mechanical regulation of cell differentiation.


title: Depletion of Sun1/2 induces heterochromatin accrual in mesenchymal stem cells during adipogenesis


title: Global hypo-methylation in a subgroup of glioblastoma enriched for an astrocytic signature is associated with increased invasion and altered immune landscape

abstract: We describe a new molecular subgroup of glioblastoma, the most prevalent malignant adult brain tumour, harbouring a bias towards hypomethylation at defined differentially methylated regions. This epigenetic signature correlates with an enrichment for an astrocytic gene signature, which together with the identification of enriched predicted binding sites of transcription factors known to cause demethylation and to be involved in astrocytic/glial lineage specification, point to a shared ontogeny between this glioblastoma subgroup and astroglial progenitors. At functional level, increased invasiveness, at least in part mediated by SRPX2, and macrophage infiltration characterise this glioblastoma subgroup.


title: Comparative evaluation of glomerular morphometric techniques reveals differential technical artefacts between FSGS and normal glomeruli

abstract:
Morphometric estimates of mean glomerular volume (MGV) have clinical implications, over and above histologic data. However, MGV estimation is time-consuming, could waste tissue sections and requires expertise limiting its utility in retrospective clinical studies.

MethodsWe evaluated MGV using both plastic and paraffin-embedded tissue from control and FSGS mice (n=10 each) using the gold-standard Disector/Cavalieri technique (Vglom-Cav) and other reported techniques [2- or 3-profile technique, Weibel-Gomez method (W-G)]. Within Vglom-Cav we examined the precision of MGV estimation while using MGVs obtained from 5- or 10-individual glomeruli measurements vs the true mean (20 glomeruli).

ResultsIn both FSGS and controls, we identified an acceptable precision of 10-glomerular sampling vs true MGV within Vglom-Cav technique [88 (79-94) % of MGV obtained were within 10% of the true MGV]. The 5-glomerular sampling was less precise [70 (56, 81) % of MGV obtained were within 10% of true MGV]. In plastic based techniques, 2- or 3-profile MGVs showed greater concordance with Vglom-Cav, than W-G MGV. The new 3-profile technique offered incremental benefit to the existing 2-profile method (improved Lins concordance in control and FSGS animals). We observed a consistent reduction of Vglom values within control animals (52+/-0.06%) in paraffin-embedded tissue (vs corresponding methods in plastic) demonstrating a clear shrinkage artefact due to tissue processing. FSGS glomeruli showed significantly less and more variable shrinkage artefact likely due to glomerular fibrosis.

ConclusionWe report the precision of 5- or 10-glomerular sampling for MGV estimation using controls and FSGS animals. We demonstrate and quantify the shrinkage bias in MGV during tissue processing for paraffin-embedding that also differentiated control animals and FSGS. Our findings have implications for experimental studies using glomerular morphometry.

category: pathology


title: Fibrosis resolution involves more than collagen; new potential players of fibrosis recovery in mouse liver

abstract: Although most work has focused on resolution of collagen ECM, fibrosis resolution involves changes to several ECM proteins. The purpose of the current study was two-fold: 1) to examine the role of MMP12 and elastin; and 2) to investigate the changes in degraded proteins in plasma (i.e., the "degradome ") in a preclinical model of fibrosis resolution. Fibrosis was induced by 4 weeks carbon tetrachloride (CCl4) exposure, and recovery was monitored for an additional 4 weeks. Some mice were treated with daily MMP12 inhibitor (MMP408) during the resolution phase. Liver injury and fibrosis was monitored by clinical chemistry, histology and gene expression. The release of degraded ECM peptides in the plasma was analyzed using by 1D-LC-MS/MS, coupled with PEAKS Studio (v10) peptide identification. Hepatic fibrosis and liver injury rapidly resolved in this mouse model. However, some collagen fibrils were still present 28d after cessation of CCl4. Despite this persistent collagen presence, expression of canonical markers of fibrosis were also normalized. The inhibition of MMP12 dramatically delayed fibrosis resolution under these conditions. LC-MS/MS analysis identified that several proteins were being degraded even at late stages of fibrosis resolution. Calpains 1/2 were identified as potential new proteases involved in fibrosis resolution. CONCLUSION. The results of this study indicate that remodeling of the liver during recovery from fibrosis is a complex and highly coordinated process that extends well beyond the degradation of the collagenous scar. These results also indicate that analysis of the plasma degradome may yield new insight into the mechanisms of fibrosis recovery, and by extension, new "theragnostic " targets. Lastly, a novel potential role for calpain activation in the degradation and turnover of proteins was identified.


title: Cold-inducible promoter-driven knockdown of Brachypodium antifreeze proteins confers freeze sensitivity

abstract: The model forage crop, Brachypodium distachyon, has a family of ice recrystallization inhibition (BdIRI) genes, which encode antifreeze proteins that function by adsorbing to ice crystals and inhibiting their growth. The genes were previously targeted for knockdown using a constitutive CaMV 35S promoter and the resulting transgenic Brachypodium showed reduced antifreeze activity and a greater susceptibility to freezing. However, the transgenic plants also showed developmental defects with shortened stem lengths and were almost completely sterile, raising the possibility that their reduced freeze tolerance could be attributed to developmental deficits. A cold-induced promoter from rice (prOsMYB1R35) has now been substituted for the constitutive promoter to generate temporal miRNA-mediated Brachypodium antifreeze protein knockdowns. Although transgenic lines showed no apparent pleiotropic developmental defects, they demonstrated reduced antifreeze activity as assessed by assays for ice-recrystallization inhibition, thermal hysteresis, electrolyte leakage, leaf infrared thermography, and leaf damage after infection with an ice nucleating phytopathogen. Strikingly, the number of cold-acclimated transgenic plants that survived freezing at -8 {degrees}C was reduced by half or killed entirely, depending on the line, compared to cold-acclimated wild type plants. Although these proteins have been studied for almost 60 years, this is the first unequivocal demonstration in any organism of the utility of antifreeze protein function and their contribution to freeze protection, independent of obvious developmental defects. These proteins are thus of potential interest in a wide range of biotechnological applications from accessible cryopreservation, to frozen product additives, to the engineering of transgenic crops with enhanced freezing tolerance.


title: A two-gene strategy increases the iron and zinc concentration of wheat flour and improves mineral bioaccesibility for human nutrition.

abstract: Dietary deficiencies of iron and zinc cause human malnutrition globally, which can be mitigated by biofortified staple crops. Conventional breeding approaches to increase grain mineral concentrations in wheat (Triticum aestivum L.) have had only limited success so far due to relatively low genetic variation. Here we demonstrate that a transgenic approach combining endosperm-specific expression of the wheat vacuolar iron transporter gene TaVIT2-D with constitutive expression of the rice nicotianamine synthase gene OsNAS2 has the potential to dramatically improve mineral micronutrient intake from wheat products. In two distinct bread wheat cultivars, we show that the VIT-NAS construct led to a two-fold increase in zinc to [~]50 {micro}g g-1 in wholemeal flour and a two-fold increase in both zinc and iron in hand-milled white flour. In highly pure, roller-milled white flour, the concentration of iron was enhanced three-fold to [~]25 {micro}g g-1. A greater than three-fold increase in the level of the natural plant metal chelator nicotianamine in the grain of VIT-NAS lines was associated with improved iron and zinc bioaccessibility in white flour. The growth of VIT-NAS plants in the greenhouse was indistinguishable from untransformed controls. We conclude that the effects of each gene cassette are additive in altering the total concentration and distribution of iron and zinc in wheat grains. This demonstrates the potential of a transgenic approach to enhance the nutritional quality of wheat well beyond what is possible by breeding approaches in order to alleviate dietary mineral deficiencies.


title: Leishmania amazonensis infection induces PD-L1 expression on dendritic cells in an mTor-dependent manner and impairs Th1 responses in vitro and in vivo

abstract: Leishmania amazonensis is one of the etiological agents of diffuse cutaneous leishmaniasis in South America. In murine models of this infection, dysregulated expansion of effector T cells or exhaustion of Th1 responses are known to be related to pathogenesis, while the induction of regulatory T cells (Tregs) promotes lesion resolution. Recent research has identified several important co-stimulator/receptor pairs, including PD-1/PD-L1, for modulating Th1 responses and Treg induction. In this study, we examined the roles of these molecules in L. amazonensis-infected C57BL/6 mice. We found a significant and selective increase in the expression of PD-1 and PD-L1 (20-fold and 5-fold, respectively), in infected footpad tissues, which correlated with an increased percentage of PD-L1+CD11c+ dendritic cells (DCs) and PD-1+CD4+ T cells in the draining lymph nodes. To evaluate the mechanism of parasite-induced PD-L1 expression, we infected bone marrow-derived DCs (BMDCs) with promastigotes and amastigotes in vitro in the presence of small molecular inhibitors of critical signaling pathways. While L. amazonensis infection decreased PD-L2 expression on the surface of BMDCs, infection-mediated PD-L1 up-regulation was consistently detected, which was dependent on mTOR and partially dependent on STAT3, PI3K, and MAPK. Infected BMDCs also significantly inhibited the expansion of Th1 cells, but were more competent in inducing CD25+FoxP3+ Tregs in vitro than the non-infected BMDCs; such effects were PD-L1-dependent, as BMDCs from PD-L1-/- mice failed to do the same. In vivo experiments revealed that infected PD-L1-/- mouse tissues showed increased Th1 responses and IFN-{gamma} production, as well as reduced lesion sizes and parasite loads, without affecting Tregs cell expansion. Together, these results support a protective role for PD-1/PD-L1 signaling in regulating local immune responses during L. amazonensis infection. This study provides new insights on immune regulation in New World cutaneous leishmaniasis.

category: immunology


title: IKK2/NFkB signaling controls lung resident CD8 T cell memory during influenza infection

abstract: CD8 tissue resident memory (TRM) cells are especially suited to control pathogen spread at mucosal sites. However, their maintenance in lung is limited. Here, we found that enhancing NFkB signaling in T cells once memory to influenza is established increased pro-survival Bcl-2 and CD122 levels boosting lung CD8 TRM maintenance. By contrast, enhancing NFkB signals during the contraction phase of the response led to a defect in TRM differentiation without impairing recirculating memory subsets. Specifically, inducible activation of NFkB via constitutive active IKK2 or tumor necrosis factor (TNF) interfered with tumor growth factor beta (TGF{beta}) signaling resulting in defects of lung CD8 TRM imprinting molecules CD69, CD103, Runx3 and Eomes. Conversely, inhibiting NFkB signals not only recovered but improved the transcriptional signature and generation of lung CD8 TRM. Thus, NFkB signaling is a critical regulator of tissue resident memory, whose levels can be tuned at specific times during infection to boost lung CD8 TRM.


title: Neocentromere formation through Robertsonian fusion and centromere repositioning during the evolution of zebras

abstract: Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated to highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and E. asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras E. burchelli (15 of 22) and E. grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine neocenstromeres are shared between the two species in agreement with their recent separation. We de novo assembled all neocentromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a neocentromere was seeded on an inversion breakpoint. At eleven chromosomes, whose primary constrictions seemed to be associated to satellite repeats by cytogenetic analysis, neocentromeres were instead located near the ancestral inactivated satellite-based centromeres, therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.


title: Design of a Versatile Microfluidic Device for Imaging Precision-Cut-Tissue Slices

abstract: Precision-cut-tissues (PCT), which preserve many aspects of a tissues microenvironment, are typically imaged using conventional sample dishes and chambers. These can require large amounts of reagent and, when used for flow-through experiments, the shear forces applied on the tissues are often ill-defined. Their physical design also makes it difficult to image large volumes and repetitively image smaller regions of interest in the living slice. We report here on the design of a versatile microfluidic device capable of holding mouse or human pancreas PCTs for 3D fluorescence imaging using confocal and selective plane illumination microscopy (SPIM). Our design positions PCTs within a 5 x 5 mm x 140 {micro}m deep chamber fitted with 150 {micro}m tall channels to facilitate media exchange. Shear stress in the device is localized to small regions on the surface of the tissue and can be easily controlled. This design allows for media exchange at flow rates ~10-fold lower than those required for conventional chambers. Finally, this design allows for imaging the same immunofluorescently labelled PCT with high resolution on a confocal and with large field of view on a SPIM, without adversely affecting image quality.


title: Reclassifying Cancer: Defining tumour cell cycle activity in terms of its tissue of origin in over 13,000 samples

abstract: Genomic alterations resulting in loss of control over the cell cycle is a fundamental hallmark of human malignancies. Whilst pan-cancer studies have broadly assessed tumour genomics and their impact on oncogenic pathways, analyses taking the baseline signalling levels in normal tissue into account are lacking. To this end, we aimed to reclassify the cell cycle activity of tumours in terms of their tissue of origin and determine the DNA mutations, chromosome arm-level changes and signalling pathways driving cell cycle activity. Combining normal tissue and pan-cancer data from over 13,000 samples we demonstrate that tumours of gynaecological origin show the highest levels of baseline corrected cell cycle activity, partially owing to hormonal signalling and gene expression changes. We also show that normal and tumour tissues can be separated into groups (quadrants) of low/high cell cycle activity and propose the novel hypothesis of an upper limit on these activity levels in tumours.


title: Genetic and epigenetic driven variation in regulatory regions activitycontribute to adaptation and evolution under endocrine treatment

abstract: Comprehensive profiling of hormone-dependent breast cancer (HDBC) has identified hundreds of protein-coding alterations contributing to cancer initiation1, 2, but only a handful have been linked to endocrine therapy resistance, potentially contributing to 40% of relapses1, 3-9. If other mechanisms underlie the evolution of HDBC under adjuvant therapy is currently unknown. In this work, we employ integrative functional genomics to dissect the contribution of cis-regulatory elements (CREs) to cancer evolution by focusing on 12 megabases of non-coding DNA, including clonal enhancers10, gene promoters, and boundaries of topologically associating domains11. Massive parallel perturbation in vitro reveals context-dependent roles for many of these CREs, with a specific impact on dormancy entrance12, 13 and endocrine therapy resistance9. Profiling of CRE somatic alterations in a unique, longitudinal cohort of patients treated with endocrine therapies identifies non-coding changes involved in therapy resistance. Overall, our data uncover actionable transient transcriptional programs critical for dormant persister cells and unveil new regulatory nodes driving evolutionary trajectories towards disease progression.


title: Nuclear Receptor 5A2 Regulation of Agrp underlies Olanzapine-induced Hyperphagia

abstract: Summary/AbstractAntipsychotic (AP) drugs are highly efficacious treatments for psychiatric disorders, but a serious side effect of their use is excessive weight gain and subsequent development of metabolic disease. Increased food intake is the underlying driver of AP-induced weight gain, although the underlying mechanisms remain unknown. In previous studies, we identified hypothalamic genes whose expression level was altered following APs-induced hyperphagia. Among these genes, the orexigenic peptide Agrp and the transcription factor nuclear receptor subfamily 5 group A member 2 (Nr5a2) were two of the most significantly upregulated genes by APs. NR5a2 is broadly expressed throughout the body, but little is known about its role in the brain. In this study, we investigated the role of hypothalamic NR5a2 in AP-induced hyperphagia and weight gain. In hypothalamic cell lines, OLZ treatment resulted in a dose-dependent increase in gene expression of NR5a2 and Agrp. In mice, administration of a specific Nr5a2 inhibitor decreased olanzapine-induced hyperphagia and weight gain, while knockdown of Nr5a2 in the arcuate nucleus (ARC) partially reversed olanzapine-induced hyperphagia. Chromatin-immunoprecipitation-PCR studies showed for the first time that NR5a2 directly binds to the Agrp promoter region. In addition, in situ hybridization studies confirm that NR5a2 and Agrp are co-localized in a subset of cells in the arcuate nucleus. In summary, we identify Nr5a2 as a key mechanistic driver of AP-induced food intake and these findings can be used to inform future clinical development of APs that do not activate hyperphagia and weight gain.

category: physiology


title: Blood Concentrations of Per- and Polyfluoroalkyl Substances are Associated with Lupus-like Autoimmunity in the American Alligator

abstract:
Surface and ground water of the Cape Fear River basin in central and coastal North Carolina is contaminated with high levels of per- and polyfluoroalkyl substances (PFAS). Elevated levels of PFAS have also been found in blood of fish and wildlife living in and around the Cape Fear River, and in the blood of human populations reliant on contaminated well or surface water from the Cape Fear River basin as a sources of drinking water. While the public and environmental health impacts of long-term PFAS exposures are poorly understood, elevated blood concentrations of some PFAS are linked with immunotoxicity and increased incidence of some chronic autoimmune diseases in human populations. The goal of this study was to evaluate PFAS exposure and biomarkers related to immune health in populations of American alligators (Alligator mississippiensis), a protected and predictive sentinel species of adverse effects caused by persistent toxic pollutants. We found that serum PFAS concentrations in a representative population of alligators from the Cape Fear River were increased compared to a reference population of alligators from the adjoining Lumber River basin. The elevated serum PFAS concentrations in the Cape Fear River alligators were associated with increased innate immune activities and hallmark phenotypes of the human autoimmune disease systemic lupus erythematosus. In addition to significant increases in double stranded-DNA binding autoantibodies in adult Cape Fear River alligators, our qRT-PCR analysis found remarkably high induction of Interferon- signature genes implicated in the pathology of human autoimmune disease. We interpret the association of increased PFAS exposure with disrupted immune functions to indicate that PFAS broadly alters immune activities resulting in autoimmune disease in American Alligators. This work substantiates and extends evidence from experimental models and human epidemiology studies showing that some PFAS are immune toxicants.

HighlightsO_LIPopulations of American Alligator (Alligator mississippiensis) from the Cape Fear River in NC have elevated PFAS exposures compared to a nearby reference population
C_LIO_LIIncreased blood PFAS concentrations are associated with increased innate immune activities and hallmark phenotypes of autoimmune disease including increases in autoantibodies
C_LIO_LICompared the reference population, expression of interferon alpha responsive signature genes that are predictive of lupus erythematosus in humans was increased [~] 60- to 577- fold in the Cape Fear River Alligators with elevated blood PFAS
C_LI

category: pharmacology and toxicology


title: Relative positioning of B and T cell epitopes drives immunodominance.

abstract:
Humoral immunity is crucial for protection against invading pathogens. Broadly neutralizing antibodies (bnAbs) provide sterilizing immunity by targeting conserved regions of viral variants and represent the goal of most vaccination approaches. While antibodies can be selected to bind virtually any region of a given antigen, consistent induction of bnAbs in the context of influenza and HIV has been representing a major roadblock. Many possible explanations have been considered, however, none of the arguments proposed so far seems to fully recapitulate the observed counter-selection for broadly protective antibodies.

Antibodies can influence antigen presentation by enhancing the processing of CD4 epitopes adjacent to the binding region while suppressing the overlapping ones. We analyzed the relative positioning of dominant B and T cell epitopes in published antigens that elicit strong and poor humoral responses. In strong immunogenic antigens, regions bound by immunodominant antibodies are frequently adjacent to CD4 epitopes, potentially boosting their presentation. Conversely, poorly immunogenic regions targeted by bnAbs in HIV and influenza overlap with clusters of dominant CD4 epitopes, potentially conferring an intrinsic disadvantage for bnAb-bearing B cells in germinal centers.

Here we propose the theory of immunodominance relativity, according to which relative positioning of immunodominant B and CD4 epitopes within a given antigen drives immunodominance. Thus, we suggest that relative positioning of B-T epitopes may be one additional mechanism that cooperates with other previously described processes to influence immunodominance. If demonstrated, this theory can improve the current understanding of immunodominance, provide a novel explanation on HIV and influenza escape from humoral responses, and pave the way for new rational design of universal vaccines.


title: Preparing for the next COVID: Deep Reinforcement Learning trained Artificial Intelligence discovery of multi-modal immunomodulatory control of systemic inflammation in the absence of effective anti-microbials

abstract:
BackgroundDespite a great deal of interest in the application of artificial intelligence (AI) to sepsis/critical illness, most current approaches are limited in their potential impact: prediction models do not (and cannot) address the lack of effective therapeutics and current approaches to enhancing the treatment of sepsis focus on optimizing the application of existing interventions, and thus cannot address the development of new treatment options/modalities. The inability to test new therapeutic applications was highlighted by the generally unsatisfactory results from drug repurposing efforts in COVID-19.

HypothesisAddressing this challenge requires the application of simulation-based, model-free deep reinforcement learning (DRL) in a fashion akin to training the game-playing AIs. We have previously demonstrated the potential of this method in the context of bacterial sepsis in which the microbial infection is responsive to antibiotic therapy. The current work addresses the control problem of multi-modal, adaptive immunomodulation in the circumstance where there is no effective anti-pathogen therapy (e.g., in a novel viral pandemic or in the face of resistant microbes).

MethodsThis is a proof-of-concept study that determines the controllability of sepsis without the ability to pharmacologically suppress the pathogen. We use as a surrogate system a previously validated agent-based model, the Innate Immune Response Agent-based Model (IIRABM), for control discovery using DRL. The DRL algorithm  trains an AI on simulations of infection where both the control and observation spaces are limited to operating upon the defined immune mediators included in the IIRABM (a total of 11). Policies were learned using the Deep Deterministic Policy Gradient approach, with the objective function being a return to baseline system health.

ResultsDRL trained an AI policy that improved system mortality from 85% to 10.4%. Control actions affected every one of the 11 targetable cytokines and could be divided into those with static/unchanging controls and those with variable/adaptive controls. Adaptive controls primarily targeted 3 different aspects of the immune response: 2nd order pro-inflammation governing TH1/TH2 balance, primary anti-inflammation, and inflammatory cell proliferation.

DiscussionThe current treatment of sepsis is hampered by limitations in therapeutic options able to affect the biology of sepsis. This is heightened in circumstances where no effective antimicrobials exist, as was the case for COVID-19. Current AI methods are intrinsically unable to address this problem; doing so requires training AIs in contexts that fully represent the counterfactual space of potential treatments. The synthetic data needed for this task is only possible through the use of high-resolution, mechanism-based simulations. Finally, being able to treat sepsis will require a reorientation as to the sensing and actuating requirements needed to develop these simulations and bring them to the bedside.


title: SARS-CoV-2 has not emerged in roe, red or fallow deer in Germany or Austria during the COVID 19 pandemic

abstract:
Spillover of SARS-CoV-2 to North American white tailed deer (Odocoileus virginianus) has been documented. We evaluated pre and pandemic exposure of German and Austrian deer species using a SARS-CoV-2 pseudoneutralization assay. In stark contrast to North American white tailed deer, we found no evidence of SARS-CoV-2 exposure.

Article Summary LineUsing a sensitive serological assay, 433 pre and pandemic deer samples from Germany and Austria tested negative for SARS-CoV-2-specific antibodies highlighting a major difference between central European and North American deer exposure and in their epidemiologic roles.


title: Metagenomic analysis reveals the abundance and diversity of opportunistic fungal pathogens in the nasopharyngeal tract of COVID-19 patients

abstract:
The nasopharyngeal tract (NT) of human is a habitat of a diverse microbial community that work together with other gut microbes to maintain the host immunity. In our previous study, we reported that SARS-CoV-2 infection reduces human nasopharyngeal commensal microbiome (bacteria, archaea and commensal respiratory viruses) but increases the abundance of pathobionts. This study aimed to assess the possible changes in the resident fungal diversity by the inclusion of opportunistic fungi due to the infection of SARS-CoV-2 in the NT of humans. Twenty-two (n = 22) nasopharyngeal swab samples (including COVID-19 = 8, Recovered = 7, and Healthy = 7) were collected for RNAseq-based metagenomics analyses. Our results indicate that SARS-CoV-2 infection significantly increased (p < 0.05, Wilcoxon test) the population and diversity of NT fungi with a high inclusion of opportunistic pathogens. We detected 863 fungal species including 533, 445, and 188 species in COVID-19, Recovered, and Healthy individuals, respectively that indicate a distinct microbiome dysbiosis due to the SARS-CoV-2 infection. Remarkably, 37% of the fungal species were exclusively associated with SARS-CoV-2 infection, where S. cerevisiae (88.62%) and Phaffia rhodozyma (10.30%) were two top abundant species in the NT of COVID-19 patients. Importantly, 16% commensal fungal species found in the Healthy control were not detected in either COVID-19 patients or when they were recovered from the COVID-19. Pairwise Spearmans correlation test showed that several altered metabolic pathways had significant positive correlations (r > 0.5, p < 0.01) with dominant fungal species detected in three metagenomes. Taken together, our results indicate that SARS-CoV-2 infection causes significant dysbiosis of fungal microbiome and alters some metabolic pathways and expression of genes in the NT of human. Findings of our study might be helpful for developing microbiome-based diagnostics, and also devising appropriate therapeutic regimens including antifungal drugs for prevention and control of concurrent fungal coinfections in COVID-19 patients.

Author summaryThe SARS-CoV-2 is a highly transmissible and pathogenic betacoronavirus that primarily enters into the human body through NT to cause fearsome COVID-19 disease. Recent high throughput sequencing and downstream bioinformatic analyses revealed that microbiome dysbiosis associated with SARS-CoV-2 infection are not limited to bacteria, and fungi are also implicated in COVID-19 development in susceptible individuals. This study demonstrates that SARS-CoV-2 infection results in remarkable depletion of NT commensal fungal microbiomes with inclusion of various opportunistic fungal pathogens. We discussed the role of these altered fungal microbiomes in the pathophysiology of the SARS-CoV-2 infection. Our results suggest that dysbiosis in fungal microbiomes and associated altered metabolic functional pathways (or genes) possibly play a determining role in the progression of SARS-CoV-2 pathogenesis. Thus, the identifiable changes in the diversity and composition of the NT fungal population and their related genomic features demonstrated in this study might lay a foundation for better understanding of the underlying mechanism of co-pathogenesis, and the ongoing development of therapeutic agents including antifungal drugs for the resolution of COVID-19 pandemic.


title: Genomic Characterization of Sars-Cov-2 from Islamabad Pakistan by Rapid Nanopore Sequencing

abstract: Since the start of COVID-19 pandemic, Pakistan has experienced four waves of pandemic. The fourth wave ended in October, 2021 while the fifth wave of pandemic starts in January, 2022. The data regarding the circulating strains after the fourth wave of pandemic from Pakistan is not available. The current study explore the genomic diversity of SARS-CoV-2 after fourth wave and before fifth wave of pandemic through whole genome sequencing. The results showed the circulation of different strains of SARS-CoV-2 during November-December, 2021. We have Omicron BA.1 (n=4), Lineage A (n=2) and delta AY.27 (n=1) variants of SARS-CoV-2 in the population of Islamabad. All the isolates harbors characteristics mutations of omicron and delta variant in the genome. The lineage A isolate harbors a nine amino acid (68-76) and a ten amino acid (679-688) deletion in the genome. The circulation of omicron in the population before the fifth wave of pandemic and subsequent upsurges of COVID-19 positive cases in Pakistan highlights the importance of genomic surveillance.


title: Systematic identification of conditionally folded intrinsically disordered regions by AlphaFold2

abstract: Deep learning-based approaches to protein structure prediction, such as AlphaFold2 and RoseTTAFold, can now define many protein structures with atomic-level accuracy. The AlphaFold Protein Structure Database (AFDB) contains a predicted structure for nearly every protein in the human proteome, including proteins that have intrinsically disordered regions (IDRs), which do not adopt a stable structure and rapidly interconvert between conformations. Although it is generally assumed that IDRs have very low AlphaFold2 confidence scores that reflect low-confidence structural predictions, we show here that AlphaFold2 assigns confident structures to nearly 15% of human IDRs. The amino-acid sequences of IDRs with high-confidence structures do not show significant similarity to the Protein Data Bank; instead, these IDR sequences exhibit a higher degree of positional amino-acid sequence conservation and are more enriched in charged and hydrophobic residues than IDRs with low-confidence structures. We compared the AlphaFold2 predictions to experimental NMR data for a subset of IDRs known to fold under specific conditions, finding that AlphaFold2 tends to capture the folded state structure. We note, however, that these AlphaFold2 predictions cannot detect functionally relevant structural plasticity within IDRs and cannot offer an ensemble representation of IDRs. Nevertheless, AlphaFold2 assigns high-confidence scores to about 60% of a set of 350 IDRs that have been reported to conditionally fold, suggesting that AlphaFold2 has learned to identify conditionally folded IDRs, which is unexpected, since IDRs were minimally represented in the training data. Leveraging this ability to discover IDRs that conditionally fold, we find that up to 80% of IDRs in archaea and bacteria are predicted to conditionally fold, but less than 20% of eukaryotic IDRs. Our results suggest that a large majority of IDRs in the proteomes of human and other eukaryotes would be expected to function in the absence of conditional folding.

category: biochemistry


title: Sequential action of a tRNA base editor in conversion of cytidine to pseudouridine

abstract: Post-transcriptional RNA editing modulates gene expression in a condition-dependent fashion. We recently discovered C-to-{Psi} editing in Vibrio cholerae tRNA. Here, we characterize the biogenesis, regulation, and functions of this previously undescribed RNA editing process. We show that an enzyme, TrcP, mediates the editing of C-to-U followed by the conversion of U to {Psi}, consecutively. AlphaFold-2 predicts that TrcP consists of two globular domains, including a novel cytidine deaminase and a pseudouridylase, along with a long helical domain. The latter domain tethers tRNA substrates during both the C-to-U editing and pseudouridylation, likely enabling a substrate channeling mechanism for efficient catalysis all the way to the terminal product. C-to-{Psi} editing both requires and suppresses other modifications, creating an interdependent network of modifications in the tRNA anticodon loop that facilitates coupling of tRNA modification states to iron availability. Our findings provide mechanistic insights into an RNA editing process that likely promotes environmental adaptation.


title: Mosaic receptor-binding domain nanoparticles induce protective immunity against SARS-CoV-2 challenges

abstract:
Recurrent spillovers of - and {beta}-coronaviruses (CoV) such as acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, SARS-CoV-2, and possibly human CoV (NL63, 229E, OC43, and HKU1) have caused serious morbidity and mortality worldwide. Six receptor binding domains (RBDs) derived from - and {beta}-CoV that are considered to have originated from animals and cross-infected humans were linked to proliferating cell nuclear antigen (PCNA) heterotrimeric subunits, PCNA1, PCNA2, and PCNA3. These were used to form a scaffold-based mosaic multivalent antigen, 6RBD-np. Electron microscopic and atomic force microscopic images show a ring-shaped disk with six protruding RBDs, like jewels in a crown, with a size of 40 nm. Prime-boost immunizations with 6RBD-np in BALB/c mice elicited strong, dose-dependent antibody responses. In human angiotensin converting enzyme 2-transgenic mice, the same immunization induced full-protection against SARS-CoV-2 wild type and Delta challenges, resulting in a 100% survival rate. The mosaic 6RBD-np provides a potential platform for developing a pan-CoV vaccine against newly emerging SARS-CoV-2 variants and future CoV spillovers.

SignificanceDespite the arsenal of COVID-19 vaccines, hospitalization and mortality associated with SARS-CoV-2 (acute respiratory syndrome coronavirus 2) variants remain high. There is an urgent need to develop next-generation COVID vaccines that provide broad protection against diseases by current and newly emerging SARS-CoV-2 variants. In this study, six receptor binding domains (RBDs) derived from - and {beta}-CoV were linked to proliferating cell nuclear antigen (PCNA) heterotrimeric scaffolds. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens. The prime-boost immunization in BALB/c and human angiotensin converting enzyme 2-transgenic mice with the 6RBD-np elicited strong, dose-dependent antibody responses and induced full-protection against both the SARS-CoV-2 wild type (WT) and Delta challenges. This study provides proof-of-concept that the mosaic 6RBD-np induces 100% protection against SARS-CoV-2 WT and Delta. It provides the potential of co-displaying heterologous antigens for novel vaccine designs, which can be deployed countering future pandemics.


title: Furin-cleavage site is present in an antiparallel β-strand in SARS-CoV2 Spike protein

abstract: Furin cleavage-site (CS) present between the S1/S2 junction in SARS-CoV2 spike (S) protein is critical to drive the fusion of SARS-CoV2 with the host cell. SARS-CoV2 falls in the sarbecovirus lineage that doesnt comprise of furin CS and therefore makes its origin enigmatic. The available wild-type (Wt) SARS-CoV2 S protein with PDB ID: 6yvb lacks a stretch of amino acid including furin CS as well. All investigators till date have shown this stretch existing in the form of a loop. We are for the first time reporting that this stretch comprises of 14 amino acid residues (677QTNSPRRARSVASQ689), forming an antiparallel {beta}-sheet comprising of PRRAR furin CS. We observed the presence of this antiparallel {beta}-sheet in MERS spike protein as well. While switching over from Wt. SARS-CoV2 with PRRAR furin CS to B.1.1.7 variant with HRRAR furin CS, we found 3% increase in the percentage content of {beta} stands. Interestingly, we found that the change of B.1.1.7 to B.1.617 variant comprising of RRRAR furin CS shifted the percentage secondary structure back to that found in Wt. SARS-CoV2. We anticipate that this {beta}-sheet is used as a docking site by host cell proteases to act on furin-CS. Additionally, we studied the interaction of modeled SARS-CoV2 S protein with transmembrane protease, serine 2 (TMPRSS2), and furin proteases, which clearly highlighted that these proteases exclusively uses furin CS located in {beta}-sheet to cleave the SARS-CoV2 S protein at its S1/S2 junction.


title: Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains

abstract: The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human VH domain, F6, which we generated by sequentially panning large phage displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC50) of 4.8 nM in vitro. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.


title: Transcriptome analysis of diverse Plasmodium falciparum clinical isolates identifies genes correlating with highly variable expression of merozoite surface protein MSPDBL2

abstract: The merozoite surface protein MSPDBL2 of Plasmodium falciparum is under strong balancing selection and is a target of naturally acquired antibodies. Remarkably, MSPDBL2 is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene transcription increases in response to overexpression of the gametocyte development inducer GDV1, so it is important to understand its natural expression. Here, MSPDBL2 in mature schizonts was analysed in the first ex vivo culture cycle of 96 clinical isolates from four populations with varying levels of infection endemicity in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2 (median of 0.6% overall), but the frequency distribution was highly skewed as nine isolates had more than 3% schizonts positive and one had 73% positive. To investigate whether expression of other gene loci correlated with MSPDBL2 expression, whole transcriptome sequencing was performed on schizont-enriched material from 17 of the clinical isolates with a wide range of proportions of schizonts positive. Transcripts of particular parasite genes were highly significantly positively correlated with MSPDBL2 positivity in schizonts as well as with mspdbl2 gene transcript levels, with overrepresentation of genes previously implicated as likely to be involved in gametocytogenesis, but not including the gametocytogenesis master regulator ap2g. Although MSPDBL2 is apparently not directly involved in sexual commitment, it marks a co-occurring developmental subpopulation that may be functionally distinct within blood stage infections.