title: Measuring host immune response status by simultaneous quantitative measurement of activity of signal transduction pathways that coordinate functional activity of immune cells from innate and adaptive immune system

abstract:
For many diseases, including cancer, viral infections such as COVID-19, bacterial infections, and auto-immune diseases, the immune response is a major determinant of progression, response to therapy, and clinical outcome. Innate and adaptive immune response are controlled by coordinated activity of multiple immune cell types. The functional activity state of immune cells is determined by cellular signal transduction pathways (STPs). A novel mRNA-based signaling pathway assay platform has been developed to quantitatively measure relevant STP activities in all types of immune cells and mixed immune cell samples for experimental and diagnostic purposes. We generated a STP activity profile, termed Immune-Pathway Activity Profile (I-PAP), for a variety of immune cell types in resting and activated state, and provide a first example for use in patient samples.

MethodsThe technology to measure STP activity has been described for androgen and estrogen receptor, PI3K, MAPK, TGF{beta}, Notch, NF{kappa}B, JAK-STAT1/2, and JAK-STAT3 pathways. STP activity was measured on Affymetrix expression microarray data from preclinical studies containing public data from different types of immune cells, resting/naive or immune-activated in vitro, to establish I-PAPs. Subsequently data from a clinical study on rheumatoid arthritis were analyzed.

ResultsI-PAPs of naive/resting and immune-activated CD4+ and CD8+ T cells, T helper cells, B cells, NK cells, monocytes, macrophages, and dendritic cells were established and in agreement with known experimental immunobiology. In whole blood samples of rheumatoid arthritis patients TGF{beta} pathway activity was increased; JAK-STAT3 pathway activity was selectively increased in female patients. In naive CD4+ Tregs TGF{beta} pathway activity was increased, while in memory T effector cells JAK-STAT3 pathway activity tended to increase, suggesting that these immune cell types contributed to whole blood analysis results.

ConclusionSTP assay technology (currently being converted to qPCR-based assays) makes it possible to directly measure functional activity of cells of the innate and adaptive immune response enabling quantitative assessment of the immune response of an individual patient. Envisioned utility lies in (1) prediction and monitoring of response to immunomodulatory treatments for a variety of immune-mediated diseases, including RA; (2) uncovering novel treatment targets; (3) improvement and standardization of in vitro immunology research and drug development.

category: immunology


title: Male White-shouldered Fairywrens (Malurus alboscapulatus) elevate testosterone when courting females but not during territorial challenges

abstract: Testosterone mediates suites of physical and behavioral traits across vertebrates, and circulation varies considerably across and within taxa. However, an understanding of the causal factors of variation in circulating testosterone has proven difficult despite decades of research. According to the challenge hypothesis, agonistic interactions between males immediately prior to the breeding season produce the highest levels of testosterone measured during this period. While many studies have provided support for this hypothesis, most species do not respond to male-male competition by elevating testosterone. As a result, a recent revision of the hypothesis ( challenge hypothesis 2.0) places male-female interactions as the primary cause of rapid elevations in testosterone circulation in male vertebrates. Here, we offer a test of both iterations of the challenge hypothesis in a tropical bird species. We first illustrate that male White-shouldered Fairywrens (Malurus alboscapulatus) differ by subspecies in plasma testosterone concentrations. Then we use a social network approach to find that males of the subspecies with higher testosterone are characterized by greater social interaction scores, including more time aggregating to perform sexual displays. Next, we use a controlled experiment to test whether males respond to simulated territorial intrusion or courtship interaction contexts by elevating testosterone. Males sampled during courtship had greater plasma testosterone both relative to flushed controls and males sampled during simulated intrusion. Ultimately, our results are consistent with challenge hypothesis 2.0, as males rapidly elevated testosterone following interactions with females, but not during territorial challenges.

category: animal behavior and cognition


title: High-throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2 Mac1 Macrodomain

abstract: Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodomain inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here, we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen which identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib does not inhibit MacroD2, the closest Mac1 homolog in humans. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for screening large compound libraries to identify improved macrodomain inhibitors and explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.

category: biochemistry


title: Minimal required exposures reveal the primacy of awareness in human face perception

abstract: The human visual system is capable of detecting extremely small light signals, as little as one single photon. But what is the visual systems minimal exposure threshold to detect meaningful stimuli? Due to hardware limitations, studies examining fast visual processing typically present stimuli for suprathreshold durations and disrupt processing with a mask. Here, we employed a newly developed tachistoscope that enables sub-millisecond presentations. Combining measures of perceptual sensitivity and recordings of neural activity, we find that both the holistic configuration and the emotional expression of a human face have the same minimal required exposure as the minimal required exposure for faces to reach awareness. These findings indicate that conscious awareness is required for processing meaningful visual information.

category: neuroscience


title: Multiple regulatory mechanisms control the production of CmrRST, an atypical signal transduction system in Clostridioides difficile

abstract:
Clostridioides difficile, an intestinal pathogen and leading cause of nosocomial infection, exhibits extensive phenotypic heterogeneity through phase variation by site-specific recombination. The signal transduction system CmrRST, which encodes two response regulators (CmrR and CmrT) and a sensor kinase (CmrS), impacts C. difficile cell and colony morphology, surface and swimming motility, biofilm formation, and virulence in an animal model. CmrRST is subject to phase variation through site-specific recombination and reversible inversion of the  cmr switch, and expression of cmrRST is also regulated by c-di-GMP through a riboswitch. The goal of this study was to determine how the cmr switch and c-di-GMP work together to regulate cmrRST expression. We generated "phase locked" strains by mutating key residues in the right inverted repeat flanking the cmr switch. Phenotypic characterization of these phase locked cmr-ON and -OFF strains demonstrates that they cannot switch between rough and smooth colony morphologies, respectively, or other CmrRST-associated phenotypes. Manipulation of c-di-GMP levels in these mutants showed that c-di-GMP promotes cmrRST expression and associated phenotypes independent of cmr switch orientation. We identified multiple promoters controlling cmrRST transcription, including one within the ON orientation of cmr switch and another that is positively autoregulated by CmrR. Overall, this work reveals a complex regulatory network that governs cmrRST expression and a unique intersection of phase variation and c-di-GMP signaling. These findings suggest that multiple environmental signals impact the production of this signaling transduction system.

IMPORTANCEClostridioides difficile is a leading cause of hospital-acquired intestinal infections in the U.S. The CmrRST signal transduction system controls numerous physiological traits and processes in C. difficile, including cell and colony morphology, motility, biofilm formation, and virulence. Here we define the complex, multi-level regulation of cmrRST expression, including stochastic control through phase variation, modulation by the second messenger c-di-GMP, and positive autoregulation by CmrR. The results of this study suggest that multiple, distinct environmental stimuli and selective pressures must be integrated to appropriately control cmrRST expression.

category: microbiology


title: Genomic analysis of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli colonising adults in Blantyre, Malawi reveals previously undescribed diversity.

abstract:
Escherichia coli is a ubiquitous bacterial species, associated with drug resistant infections; hundreds of thousands of genomes are now available, but are biased towards high-income countries and clinical isolates. Data from sub-Saharan Africa (sSA) are underrepresented in global sequencing efforts and may represent a major source of genetic diversity with respect to transmissible antimicrobial resistance (AMR). We carried out a genomic investigation of extended-spectrum beta-lactamase (ESBL)-producing E. coli colonising adults in Blantyre, Malawi to assess the diversity and AMR determinants and to place these isolates in the context of globally available genomes. We carried out short-read whole-genome sequencing of 473 colonising ESBL E. coli isolated from stool and placed them in the context of a previous curated species wide collection of 10,146 isolates using the popPUNK clustering algorithm and by constructing a core gene phylogeny. The most frequently identified STs in Malawian isolates were the globally successful ST131 and ST410, and blaCTX-M were the dominant ESBL genes, mirroring global trends. However, 37% of Malawian isolates did not cluster with any isolates in the global collection, and the core gene phylogeny was consistent with local subclades including in ST410 and several phylogroup A lineages. Apparent undescribed diversity in Malawian E. coli could be due to local selection pressures or sampling biases in global E. coli collections. Taking a one health approach to further sampling of E. coli from Malawi and sSA, and principled incorporation into unbiased global collections is necessary to understand local, regional and global transmission of both E. coli and priority AMR genes.

Data SummaryAll data and code to replicate this analysis is available as the blantyreESBL v1.0.0 R package (https://doi.org/10.5281/zenodo.5554082) available at https://github.com/joelewis101/blantyreESBL. Reads from all isolates sequenced as part of this study have been deposited in the European Nucleotide Archive, and accession numbers (as well as accession numbers of publicly available genomes used in this analysis) are provided in the R package.


title: High throughput identification of genes conferring resistance or sensitivity to toxic effectors delivered by the type VI secretion system

abstract:
The type six secretion system (T6SS) is a prevalent bacterial weapon delivering toxic effector proteins into nearby competitors. In addition to immunity genes that protect against a particular effector, alternate yet crucial nonspecific defences have also recently been identified. To systematically identify genes influencing T6SS susceptibility in numerous species, we designed a Tn-Seq-based competition assay. Combined with follow-up analyses using E. coli and V. cholerae gene knockout collections, we demonstrate that our Tn-Seq competition technique can be used to identify both immunity and non-immunity defences against the T6SS. We also identify E. coli proteins that facilitate T6SS-mediated cell death, including metabolic genes such as cyaA and gltA, where mutant strains were resistant to attack. Our findings act as a proof-of-concept for the technique while also illuminating novel genes of interest. Since Tn-Seq can be applied in numerous species, our method has broad potential for identifying diverse T6SS defence genes across genomes in a high-throughput manner.

ImportanceThe type six secretion system (T6SS) is a molecular poison-tipped spear that bacteria use to kill nearby competitors. To prevent self killing, they use antitoxins called immunity genes that specifically neutralize the poisons. Beyond immunity genes, multiple additional defences have recently been discovered but there are likely many more across the genomes of diverse species. To help discover these novel mechanisms, we designed a high-throughput method that can be used in numerous different species to rapidly identify genes involved in sensitivity to T6SS attacks. Using T6SS  killers delivering individual poisons and two commonly studied  prey bacteria, we show proof-of-principle that the technique can discover proteins that make the prey cells more resistant or sensitive to particular poisons. This will greatly improve the speed at which genes influencing the T6SS can be identified and selected for further study in follow-up analyses.


title: Heterologous expression of Dehalobacter spp. respiratory reductive dehalogenases in Escherichia coli

abstract:
Reductive dehalogenases (RDases) are a family of redox enzymes that are required for anaerobic organohalide respiration, a microbial process that is useful in bioremediation. Structural and mechanistic studies of these enzymes have been greatly impeded due to challenges in RDase heterologous expression, primarily because of their cobamide-dependence. There have been a few successful attempts at RDase production in unconventional heterologous hosts, but a robust method has yet to be developed. In this work we outline a novel respiratory RDase expression system using Escherichia coli as the host. The overexpression of E. colis cobamide transport system, btu, and RDase expression under anaerobic conditions were established to be essential for the expression of active RDases from Dehalobacter - an obligate organohalide respiring bacterium. The expression system was validated on six RDase enzymes with amino acid sequence identities ranging from >30-95%. Dehalogenation activity was verified for each RDase by assaying cell-free extracts of small-scale expression cultures on various chlorinated substrates including chloroalkanes, chloroethenes, and hexachlorocyclohexanes. Two RDases, TmrA from Dehalobacter sp. UNSWDHB and HchA from Dehalobacter sp. HCH1, were purified by nickel affinity chromatography. Incorporation of both the cobamide and iron-sulfur cluster cofactors was verified, and the specific activity of TmrA was found to be consistent with that of the native enzyme. The heterologous expression of respiratory RDases, particularly from obligate organohalide respiring bacteria, has been extremely challenging and unreliable. Here we present a relatively straightforward E. coli expression system that has performed well for a variety of Dehalobacter spp. RDases.

IMPORTANCEUnderstanding microbial reductive dehalogenation is important to refine the global halogen cycle and to improve bioremediation of halogenated contaminants; however, studies of the family of enzymes responsible are limited. Characterization of reductive dehalogenase enzymes has largely eluded researchers due to the lack of a reliable and high-yielding production method. We are presenting an approach to express reductive dehalogenase enzymes from Dehalobacter, a key group of organisms used in bioremediation, in E. coli. This expression system will propel the study of reductive dehalogenases by facilitating their production and isolation, allowing researchers to pursue more in-depth questions about the activity and structure of these enzymes. This platform will also provide a starting point to improve the expression of reductive dehalogenases from many other organisms.


title: Genomic and metabolic adaptations of biofilms to ecological windows of opportunities in glacier-fed streams

abstract: Microorganisms dominate life in cryospheric ecosystems. In glacier-fed streams (GFSs), ecological windows of opportunities allow complex microbial biofilms to develop and transiently form the basis of the food web, thereby controlling key ecosystem processes. Here, using high-resolution metagenomics, we unravel strategies that allow biofilms to seize this opportunity in an ecosystem otherwise characterized by harsh environmental conditions. We found a diverse microbiome spanning the entire tree of life and including a rich virome. Various and co-existing energy acquisition pathways point to diverse niches and the simultaneous exploitation of available resources, likely fostering the establishment of complex biofilms in GFSs during windows of opportunity. The wide occurrence of rhodopsins across metagenome-assembled genomes (MAGs), besides chlorophyll, highlights the role of solar energy capture in these biofilms. Concomitantly, internal carbon and nutrient cycling between photoautotrophs and heterotrophs may help overcome constraints imposed by the high oligotrophy in GFSs. MAGs also revealed mechanisms potentially protecting bacteria against low temperatures and high UV-radiation. The selective pressure of the GFS environment is further highlighted by the phylogenomic analysis, differentiating the representatives of the genus Polaromonas, an important component of the GFS microbiome, from those found in other ecosystems. Our findings reveal key genomic underpinnings of adaptive traits that contribute to the success of complex biofilms to exploit environmental opportunities in GFSs, now rapidly changing owing to global warming.


title: Large-scale discovery of candidate type VI secretion effectors with antibacterial activity

abstract: Type VI secretion systems (T6SS) are common bacterial contractile injection systems that inject toxic "effector" proteins into neighboring cells. Effector discovery is generally done manually, and computational approaches used for effector discovery depend on genetic linkage to T6SS genes and/or sequence similarity to known effectors. We bioinformatically investigated T6SS in more than 11,832 genomes of Gram negative bacteria. We found that T6SS encoding bacteria are host-associated and pathogenic, enriched in specific human and plant tissues, while depleted in marine, soil, and engineered environments. Analysis of T6SS cores with C-terminal domains ("evolved" cores) showed "evolved" HCP are rare, overwhelmingly encoded in orphan operons, and are largely restricted to Escherichia. Using the wealth of data generated from our bioinformatic analysis, we developed two algorithms for large-scale discovery of T6SS effector proteins (T6Es). We experimentally validated ten putative antibacterial T6SS effector proteins and one cognate immunity gene from a diverse species. This study provides a systematic genomic perspective of the role of the T6SS in nature, a thorough analysis of T6E evolution and genomic properties, and discovery of a large number of candidate T6Es using new approaches.


title: Low potential electron generating enzymes serve different functions during aerobic nitrogen fixation in Azotobacter vinelandii

abstract:
Biological nitrogen fixation requires large amounts of energy in the form of ATP and low potential electrons to overcome the high activation barrier for cleavage of the dinitrogen triple bond. The model aerobic nitrogen-fixing bacteria, Azotobacter vinelandii, generates low potential electrons in the form of reduced ferredoxin (Fd) and flavodoxin (Fld) using two distinct mechanisms via the enzyme complexes Rnf and Fix. Both Rnf and Fix are expressed during nitrogen fixation, and deleting either rnf1 or fix genes has little effect on diazotrophic growth. However, deleting both rnf1 and fix eliminates the ability to grow diazotrophically. Rnf and Fix both use NADH as a source of electrons, but overcoming the energetics of NADHs endergonic reduction of Fd/Fld is accomplished through different mechanisms. Rnf harnesses free energy from the proton motive force, whereas Fix uses electron bifurcation to effectively couple the endergonic reduction of Fd/Fld to the exergonic reduction of quinone. Different stoichiometries and gene expression analyses indicate specific roles for the two reactions under different conditions. In this work, complementary physiological studies and thermodynamic modeling reveal how Rnf and Fix simultaneously balance redox homeostasis in various conditions. Specifically, the Fix complex is required for efficient growth under low oxygen concentrations, while Rnf sustains homeostasis and delivers sufficient reduced Fd to nitrogenase under standard conditions. This work provides a framework for understanding how the production of low potential electrons sustains robust nitrogen fixation in various conditions.

ImportanceThe availability of fixed nitrogen is critical for life in many ecosystems, from hot springs to agriculture. Due to the energy demands of biological nitrogen fixation, organisms must tailor their metabolism during diazotrophic growth to deliver energy requirements to nitrogenase in the form of ATP and low potential electrons. Therefore, a complete understanding of diazotrophic energy metabolism and redox homeostasis is required to understand the impact on ecological communities or to promote crop growth in agriculture through engineered diazotrophs.


title: Bacterial invasion across the human skin barrier - mechanisms and ensuing tissue degradation.

abstract:
Atopic Dermatitis (AD) is associated with a deficiency of skin lipids, increased populations of Staphylococcus aureus in the microbiome, and structural defects in the stratum corneum (SC), the outermost layer of human skin. However, the pathogenesis of AD is ambiguous as it is unclear whether observed changes are the result of AD or contribute to the pathogenesis of the disease. Previous studies have shown that S. aureus is capable of permeating across isolated human SC tissue when lipids are depleted to levels consistent with AD conditions. In this study, we expand upon this discovery to determine the mechanisms of bacterial penetration into the SC barrier. Specifically, we establish whether bacteria are permeating intercellularly, between corneocytes, or employing a combination pathway of both inter- and intra-cellular travel. The mechanical implications of bacterial invasion, lipid depletion, and media immersion are also evaluated using a newly developed, physiologically relevant, temperature-controlled drip chamber. Results reveal that S. aureus can be internalized by corneocytes, indicating transcellular movement through the tissue during permeation, consistent with previous theoretical models. S. aureus also degrades the mechanical integrity of human SC, particularly when the tissue is partially depleted of lipids. These observed mechanical changes are likely the cause of broken or ruptured tissue seen as exudative lesions in AD flares. This work further highlights the necessity of lipids in skin microbial barrier function.

ImportanceMillions of people suffer from the chronic inflammatory skin disease Atopic Dermatitis (AD), whose symptoms are associated with a deficiency of skin lipids that exhibit antimicrobial functions, and increased populations of the opportunistic pathogen Staphylococcus aureus. However, the pathogenesis of AD is ambiguous, and it remains unclear if these observed changes are merely the result of AD, or contribute to the pathogenesis of the disease. In this article, we demonstrate the necessity of skin lipids in preventing S. aureus from penetrating the outermost barrier of human skin thereby causing a degradation in tissue integrity. In terms of AD, this bacterial permeation into the viable epidermis could act as an inflammatory trigger of the disease and could also explain tissue fragility and lesion formation seen with AD patients. Moreover, bacterial induced degradation could lead to increased pathways and further allergen intervention creating chronic irritation.


title: Transcriptional differences of lipid-metabolizing enzymes in sebocytes derived from sebaceous glands of the skin and pre-putial glands

abstract: Sebaceous glands are adnexal structures, which critically contribute to skin homeostasis and the establishment of a functional epidermal barrier. Sebocytes, the main cell population found within the sebaceous glands, are highly specialized lipid-producing cells. Sebaceous gland-resembling tissue structures are also found in male rodents in form of preputial glands. Similar to sebaceous glands, they are composed of lipid-specialized sebocytes. Due to a lack of adequate organ culture models for skin sebaceous glands and the fact that preputial glands are much larger and easier to handle, previous studies have used preputial glands as a model for skin sebaceous glands. Here, we compared both types of sebocytes, using a single cell RNA sequencing approach, to unravel potential similarities and differences between the two sebocyte populations. In spite of common gene expression patterns due to general lipid-producing properties, we found significant differences in the expression levels of genes encoding enzymes involved in the biogenesis of specialized lipid classes. Specifically, genes critically involved in the mevalonate pathway, including squalene synthase, as well as the sphingolipid salvage pathway, such as ceramide synthase, (acid) sphingomyelinase or acid and alkaline ceramidases, were significantly less expressed by preputial gland sebocytes. Together, our data revealed tissue-specific sebocyte populations, indicating major developmental, functional as well as biosynthetic differences between both glands. The use of preputial glands as surrogate model to study skin sebaceous glands is therefore limited, and major differences between both glands need to be carefully considered before planning an experiment.

category: molecular biology


title: Polymorphism at four-fold degenerate site, but not at the intergenic regions, explains nucleotide compositional strand asymmetry in bacteria

abstract: We investigated single nucleotide polymorphism in intergenic regions (IRs) and four-fold degenerate sites (FFS) in genomes of three {gamma}-Proteobacteria and two Firmicutes to understand the mechanism of nucleotide compositional asymmetry between the leading and the lagging strands. Pattern of the polymorphism spectra were alike regarding transitions but variable regarding transversions in the IRs of these bacteria. Contrasting trends of complementary polymorphisms such as C[->]T vs G[->]A as well as A[->]G vs T[->]C in the IRs vindicated similar replication-associated strand asymmetry regarding cytosine and adenine deamination, respectively, across these bacteria. Surprisingly, the polymorphism pattern at FFS was different from that of the IRs and its frequency was always more than the IRs in these bacteria. Further, the polymorphism patterns within a bacterium were inconsistent across the five amino acids, which neither the replication nor the transcription-associated mutations could explain. However, the polymorphism at FFS coincided with amino acid specific codon usage bias in the five bacteria. Further, strand asymmetry in nucleotide composition could be explained by the polymorphism at FFS, not at the IRs. Therefore, polymorphisms at FFS might not be treated as nearly neutral unlike that in IRs in these bacteria.


title: Processive translocation of cohesive and non-cohesive cohesin in vivo

abstract:
Cohesin is a central architectural element of chromosomes that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops. Purified cohesin translocates along DNA in a diffusive fashion that can be propelled by transcribing RNA polymerase. The complex also extrudes DNA loops in a process that consumes ATP. In this study we examine processive genomic translocation of cohesin in vivo. To this end, obstacles of increasing size were tethered to DNA to act as roadblocks to complexes mobilized by transcription in yeast. The obstacles were built from a GFP-lacI core fused to one or more mCherries. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by as few as three mCherries. Furthermore, cohesive complexes that were stalled at obstacles, in turn, blocked the passage of non-cohesive complexes. That synthetic barriers alter cohesin redistribution demonstrates that the complex translocates processively on chromatin in vivo. The approach provides a relative measure of the maximum size of the DNA binding chamber(s) of cohesin. Together, this study reveals unexplored limitations to cohesin movement on chromosomes.

Significance StatementCohesin is an architectural protein that brings distant chromosomal DNA sites together. The complex links sister chromatids cohesion but it also binds to single pieces of DNA in ways that do not generate cohesion. One class of non-cohesive complexes organizes chromosomal DNA into loops. All cohesin complexes move on DNA but the constraints on such movement are not fully explored. Here, we use size-calibrated obstacles in yeast to interrogate cohesin and the properties of its movement on DNA. We show that both cohesive and non-cohesive complexes translocate processively on chromosomes. In addition, we show that cohesive and non-cohesive complexes are blocked by obstacles of different size. Lastly, we show that stalled cohesive complexes block passage of non-cohesive complexes.


title: The DNAPKcs long-range C-NHEJ complex is required for blunt DNA end joining when XLF is weakened

abstract: Canonical non-homologous end joining (C-NHEJ) factors can assemble into a long-range (LR) complex with DNA ends relatively far apart that contains DNAPKcs, XLF, XRCC4, LIG4, and the KU heterodimer and a short-range (SR) complex lacking DNAPKcs that has the ends positioned for ligation. Since the SR complex can form de novo, the role of the LR complex (i.e., DNAPKcs) for chromosomal EJ is unclear. We have examined EJ of chromosomal blunt DNA double-strand breaks (DSBs), and found that DNAPKcs is significantly less important than XLF and XRCC4 for such EJ. However, weakening XLF via disrupting interaction interfaces (e.g., disrupting the XLF homodimer interface) causes a marked requirement for DNAPKcs, its kinase activity, and its ABCDE-cluster autophosphorylation sites for blunt DSB EJ. In contrast, other aspects of genome maintenance are sensitive to DNAPKcs kinase inhibition in a manner that is not further enhanced by XLF loss (i.e., suppression of homology-directed repair and structural variants, and IR-resistance). We suggest that DNAPKcs is required to position a weakened XLF in an LR complex that can transition into a functional SR complex for blunt DSB EJ, but also has distinct functions for other aspects of genome maintenance.


title: EXOSC10/Rrp6 is essential for the eight-cell embryo/morula transition

abstract: The conserved 3-5 exoribonuclease EXOSC10/Rrp6 is required for gametogenesis, brain development, erythropoiesis and blood cell enhancer function. The human ortholog is essential for mitosis in cultured cancer cells. Little is known, however, about the role of Exosc10 during embryo development and organogenesis. We generated an Exosc10 knockout model and find that Exosc10-/- mice show an embryonic lethal phenotype. We demonstrate that Exosc10 maternal wild type mRNA is present in mutant oocytes and that the gene is expressed during all stages of early embryogenesis. Furthermore, we observe that EXOSC10 early on localizes to the periphery of nucleolus precursor bodies in blastomeres, which is in keeping with the proteins role in rRNA processing and may indicate a function in the establishment of chromatin domains during initial stages of embryogenesis. Finally, we infer from genotyping data for embryonic days e7.5, e6.5 and e4.5 and embryos cultured in vitro that Exosc10-/- mutants arrest at the eight-cell embryo/morula transition. Our results demonstrate a novel essential role for Exosc10 during early embryogenesis, and they are consistent with earlier work showing that impaired ribosome biogenesis causes a developmental arrest at the morula stage.

category: developmental biology


title: The archaeal division protein CdvB1 assembles into polymers that are depolymerized by CdvC

abstract: The Cdv proteins constitute the cell-division system of the Crenarchaea, in a protein machinery that is closely related to the ESCRT system of eukaryotes. The CdvB paralog CdvB1 is believed to play a major role in the constricting ring that is the central actor in cell division in the crenarchaea. Here, we present an in vitro study of purified CdvB1 from the crenarchaeon M. sedula with a combination of TEM imaging and biochemical assays. We show that CdvB1 self-assembles into filamentous polymers that are depolymerized by the action of the Vps4-homolog ATPase CdvC. Using liposome flotation assays, we show that CdvB1 binds to negatively charged lipid membranes and can be detached from the membrane by the action of CdvC. Interestingly, we find that the polymerization and the membrane binding are mutually exclusive properties of the protein. Our findings provide novel insight into one of the main components of the archaeal cell division machinery.


title: Regulation of drought tolerance in Arabidopsis involves PLATZ4-mediated transcriptional suppression of PIP2;8

abstract:
PLATZ transcription factors play important roles in plant growth, development, and biotic and abiotic stress responses. However, how PLATZ regulates plant drought tolerance and ABA sensitivity remains largely unknown. Here, we show that PLATZ4 increases drought tolerance and ABA sensitivity in Arabidopsis thaliana by suppressing the expression of PIP2;8, DEG3, At1g33840 and At3g01345, while upregulating the expression of PR1, ABI3, ABI4 and ABI5. Moreover, PLATZ4 directly binds A/T-rich sequences within the PIP2;8 promoter but not ABI3, ABI4 and ABI5 promoters. Consistent with this, PIP2;8 acts epistatically to PLATZ4. Furthermore, the aquaporin activity of PIP2;8 was confirmed in Xenopus laevis oocytes and yeast cells in response to osmotic stress. Analysis of water loss of seedlings overexpressing PIP2;8 or lacking PIP2;8 function indicated that PIP2;8-mediated water flow is particularly active in response to drought stress in planta. Accordingly, stomatal closure was enhanced in pip2;8 mutants but reduced in PIP2;8-overexpressing plants. In platz4 mutant and PLATZ4-overexpressing plants, water loss and stomatal closure changed oppositely to those in pip2;8 mutants and PIP2;8-overexpressing plants, respectively. In addition, the interaction between PLATZ4 and AITR6 was confirmed by several assays, and the binding of PIP2;8 promoter by PLATZ4 was strengthened by an interaction with AITR6. Collectively, our findings reveal that PLATZ4 interacts with AITR6 to increase ABA sensitivity and drought tolerance in Arabidopsis by upregulating expression of PR1, ABI3, ABI4 and ABI5 while inhibiting the expression of PIP2;8 and associated genes.

One sentence summaryPLATZ4 interacted with AITR6 to increase drought tolerance and ABA sensitivity in Arabidopsis thaliana by directly bind A/T-rich sequences within the plasma membrane aquaporin (PIP)2;8 promoter.

category: plant biology


title: Spatial differences in stoichiometry of EGR phosphatase and Microtubule-Associated Stress Protein 1 control root meristem activity during drought stress.

abstract:
During moderate severity drought and low water potential ({psi}w) stress, poorly understood signaling mechanisms restrict both meristem cell division and subsequent cell expansion. We found that the Clade E Growth-Regulating 2 (EGR2) protein phosphatase and Microtubule Associated Stress Protein 1 (MASP1) differed in their stoichiometry of expression across the root meristem and had opposing effects on root meristem activity at low {psi}w. Ectopic MASP1 or EGR expression increased or decreased, respectively, root meristem size and root elongation during low {psi}w stress. This, along with the ability of phosphomimic MASP1 to overcome EGR suppression of root meristem size and observation that ectopic EGR expression had no effect on unstressed plants, indicated that during low {psi}w EGR activation and attenuation of MASP1 phosphorylation in their overlapping zone of expression determines root meristem size and activity. Ectopic EGR expression also decreased root cell size at low {psi}w. Conversely, both the egr1-1egr2-1 and egr1-1egr2-1masp1-1 mutants had similarly increased root cell size; but, only egr1-1egr2-1 had increased cell division. These observations demonstrated that EGRs affect meristem activity via MASP1 but affect cell expansion via other mechanisms. Interestingly, EGR2 was highly expressed in the root cortex, a cell type important for growth regulation and environmental response.

One Sentence SummarySpatial differences in EGR-MASP1 expression and control of MASP1 phosphorylation adjust root meristem activity to regulate growth during drought stress.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Paul E. Verslues (paulv@gate.sinica.edu.tw).


title: A Diversity Outbred F1 mouse model identifies host-intrinsic genetic regulators of response to immune checkpoint inhibitors

abstract:
Immune checkpoint inhibitors (ICI) have improved outcomes for a variety of malignancies; however, many patients fail to benefit. While tumor-intrinsic mechanisms are likely involved in therapy resistance, it is unclear to what extent host genetic background influences response. To investigate this, we utilized the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. DO mice are an outbred stock generated by crossbreeding 8 inbred founder strains, and CC mice are recombinant inbred mice generated from the same 8 founders. We generated 207 DOB6F1 mice representing 48 DO dams and demonstrated that these mice reliably accept the C57BL/6-syngeneic B16F0 tumor and that host genetic background influences response to ICI. Genetic linkage analysis from 142 mice identified multiple regions including one within chromosome 13 that associated with therapeutic response. We utilized 6 CC strains bearing the positive (NZO) or negative (C57BL/6) driver genotype in this locus. We found that 2/3 of predicted responder CCB6F1 crosses show reproducible ICI response. The chromosome 13 locus contains the murine prolactin family, which is a known immunomodulating cytokine associated with various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Prolactin augmented ICI response against B16F0, with increased CD8 infiltration and 5/8 mice exhibiting slowed tumor growth relative to controls. This study highlights the role of host genetics in ICI response and supports the use of F1 crosses in the DO and CC mouse populations as powerful cancer immunotherapy models.

One Sentence SummaryUsing germline genetic analysis in Diversity Outbred and Collaborative Cross models, we identify prolactin as a host-intrinsic regulator of response to combined immune checkpoint inhibitors.


title: PBP1 of Staphylococcus aureus has multiple essential functions in cell division

abstract: Bacterial cell division is a complex process requiring the coordination of multiple components, to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multi- stage PG architecture that develops during septation. Penicillin binding proteins (PBPs) are essential for the final steps of PG biosynthesis - their transpeptidase activity links together the peptide sidechain of nascent glycan strands together. PBP1 is required for cell division in S. aureus and here we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C-terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thus coordinate the cell division process. The transpeptidase activity of PBP1 is also essential but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1-specific {beta}-lactam, meropenem. Together these results lead to a model for septal PG synthesis where PBP1 enzyme activity is responsible for the characteristic architecture of the septum and PBP1 protein molecules coordinate cell division allowing septal plate formation.


title: Topology and contribution to the pore channel lining of plasma membrane-embedded S. flexneri type 3 secretion translocase IpaB

abstract:
Shigella spp. are human bacterial pathogens that cause bacillary dysentery. Virulence depends on a type 3 secretion system (T3SS), a highly conserved structure present in multiple important human and plant pathogens. Upon host cell contact, the T3SS translocon is delivered to the host membrane, facilitates bacterial docking to the membrane, and enables delivery of effector proteins into the host cytosol. The Shigella translocon is composed of two proteins, IpaB and IpaC, which together form this multimeric structure within host plasma membranes. Upon interaction of IpaC with host intermediate filaments, the translocon undergoes a conformational change that allows for bacterial docking onto the translocon and, together with host actin polymerization, enables subsequent effector translocation through the translocon pore. To generate additional insights into the translocon, we mapped the topology of IpaB in plasma membrane-embedded pores using cysteine substitution mutagenesis coupled with site-directed labeling and proximity-enabled crosslinking by membrane permeant sulfhydryl reactants. We demonstrate that IpaB function is dependent on post translational modification by a plasmid-encoded acyl carrier protein. We show that the first transmembrane domain of IpaB lines the interior of the translocon pore channel such that the IpaB portion of the channel forms a funnel-like shape leading into the host cytosol. In addition, we identify regions of IpaB within its cytosolic domain that protrude into and are closely associated with the pore channel. Taken together, these results provide a framework for how IpaB is arranged within translocons natively delivered by Shigella during infection.

ImportanceType 3 secretion systems are nanomachines employed by many bacteria, including Shigella, which deliver into human cells bacterial virulence proteins that alter cellular function in ways that promote infection. Delivery of Shigella virulence proteins occurs through a pore formed in human cell membranes by the proteins IpaB and IpaC. Here, we define how IpaB contributes to the formation of pores natively delivered into human cell membranes by S. flexneri. We show that a specific domain of IpaB (transmembrane domain 1) lines much of the pore channel and that portions of IpaB that lie in the inside of the human cell loop back into and/or are closely associated with the pore channel. These findings provide new insights into the organization and function of the pore in serving as the conduit for delivery of virulence proteins into human cells during Shigella infection.


title: Genomic and antigenic diversity of carried Klebsiella pneumoniae isolates mirrors that of invasive isolates in Blantyre, Malawi

abstract:
Klebsiella pneumoniae is an antimicrobial resistance (AMR) associated pathogen of global importance, and polyvalent vaccines targeting K. pneumoniae O-antigens are in development. Genomes from sub-Saharan Africa (sSA) are underrepresented in global sequencing efforts. We therefore carried out a genomic analysis of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae complex isolates colonising adults in Blantyre, Malawi, placed these isolates in a global genomic context, and compared colonising to invasive isolates from the main public hospital in Blantyre. 203 isolates from stool and rectal swabs from adults were whole-genome sequenced and compared to a publicly available multicountry collection of 484 K. pneumoniae genomes sampled to cover maximum diversity of the species, 150 previously sequenced Malawian and 66 Kenyan isolates from blood or sterile sites. We inferred phylogenetic relationships and analysed the diversity of genetic loci linked to AMR, virulence, capsule (K-) and LPS O-antigen (O-types). We find that the diversity of Malawian Klebsiella isolates is representative of the species population structure, but with local success and expansion of sequence types (STs) ST14, ST15, ST340 and ST307. Siderophore and hypermucoidy genes were more frequent in invasive versus carriage isolates (present in 13% vs 1%, p < 0.001) but still generally lacking in most invasive isolates. The population structure and distribution of O-antigen types was similar in Malawian invasive and carriage isolates, with O4 being more common in Malawian isolates (14%) than in previously published studies (2-5%). We conclude that host factors, pathogen opportunity or alternate virulence loci not linked to invasive disease elsewhere are likely to be the major determinants of invasive disease in Malawi. Distinct ST and O-type distributions in Malawi highlights the need for geographically aware sampling to robustly define secular trends in Klebsiella diversity. Colonising and invasive isolates in Blantyre are similar and hence O-typing of colonising Klebsiella isolates may be a rapid and cost-effective approach to describe global diversity and guide vaccine development.

Data SummaryAll data and code to replicate this analysis is available as the blantyreESBL v1.0.0 R package (https://doi.org/10.5281/zenodo.5554082) available at https://github.com/joelewis101/blantyreESBL. Reads from all isolates sequenced as part of this study have been deposited in the European Nucleotide Archive, and accession numbers (as well as accession numbers of publicly available genomes used in this analysis) are provided in the R package.


title: Genomic and phenotypic characterization of Pseudomonas hygromyciniae, a novel bacterial species discovered from a commercially purchased antibiotic

abstract:
A purchased lot of the antibiotic hygromycin B was found to be contaminated with a novel bacterial species, which we designate Pseudomonas hygromyciniae. Characteristics of P. hygromyciniae include its ability to use a variety of compounds as carbon sources, its pathogenicity towards lettuce and Galleria mellonella, and its ability to inhibit the growth of an E. coli strain. P. hygromyciniae is unlikely to be a human pathogen, as it did not survive at 37 {degrees}C and was not cytotoxic towards a mammalian cell line. The P. hygromyciniae strain harbors a novel 250 kb megaplasmid which confers resistance to hygromycin B and contains numerous other genes predicted to encode replication and conjugation machinery. These findings indicate that commercially manufactured antibiotics represent another extreme environment that may support the growth of novel bacterial species.

IMPORTANCEMicrobial ecologists have surveyed numerous natural and manmade environments in search of new microbial species. In some instances, these microbes are discovered in harsh conditions, such as deep-sea vents, and their discovery leads to better understanding of how microbes adapt to their environment. Here, we have discovered a new species of bacteria from an extreme manmade environment: a lyophilized, commercially available bottle of the antibiotic hygromycin B.


title: Gut-derived bacterial flagellin induces beta-cell inflammation and dysfunction

abstract:
ObjectiveHyperglycemia and type 2 diabetes (T2D) are caused by failure of pancreatic beta cells. The role of the gut microbiota in T2D has been studied but causal links remain enigmatic.

DesignObese individuals with or without T2D were included from two independent Dutch cohorts. Human data was translated in vitro and in vivo by using pancreatic islets from C57BL6/J mice and by injecting flagellin into obese mice.

ResultsFlagellin is part of the bacterial locomotor appendage flagellum, present on gut bacteria including Enterobacteriaceae, which we show to be more abundant in the gut of individuals with T2D. Subsequently, flagellin induces a pro-inflammatory response in pancreatic islets mediated by the Toll-like receptor (TLR)-5 expressed on resident islet macrophages. This inflammatory response associated with beta-cell dysfunction, characterized by reduced insulin gene expression, impaired proinsulin processing and stress-induced insulin hypersecretion in vitro and in vivo in mice.

ConclusionWe postulate that increased systemically disseminated flagellin in T2D is a contributing factor to beta cell failure in time and represents a novel therapeutic target.

Graphical abstract

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title: Heat Stress Regulates the Expression of TPK1 Gene at Transcriptional and Post-Transcriptional Levels in Saccharomyces cerevisiae

abstract:
In Saccharomyces cerevisiae, cAMP regulates a number of different cellular processes, such as cell growth, metabolism, stress resistance and gene transcription. The intracellular target for this second messenger in yeast cells is the cAMP-dependent protein kinase (PKA). The way in which a broad specificity protein kinase mediates one right physiological response after cAMP increase indicates that specificity is highly regulated in the cAMP / PKA system. Here we address the mechanism through which cAMP-PKA signalling mediates its response to heat shock thermotolerance in Saccharomyces cerevisiae. Yeast PKA is a tetrameric holoenzyme composed of a regulatory (Bcy1) subunit dimer and two catalytic subunits (Tpk1, Tpk2 and Tpk3). PKA subunits are differentially expressed under certain stress conditions. In the present study we show that, although the mRNA levels of TPK1 are upregulated upon heat shock at 37{degrees}C, no change is detected in Tpk1 protein levels. The half-life of TPK1 mRNA increases and this mRNA condensates in cytoplasmic foci upon thermal stress. The resistance of TPK1 mRNA foci to cycloheximide-induced disassembly, together with the polysome profiling analysis suggest that TPK1 mRNA is impaired for entry into translation. TPK1 mRNA foci and TPK1 expression were also evaluated during thermotolerance. The crosstalk of cAMP-PKA pathway and cell wall integrity (CWI) signalling was also studied. Wsc3 sensor and other components of the CWI pathway are necessary for the upregulation of TPK1 mRNA upon heat shock conditions. The assembly in cytoplasmic foci upon thermal stress shows to be dependent of Wsc3. Finally, evidence of an increase in the abundance of Tpk1 in the PKA holoenzyme in response to heat shock is presented, suggesting that a recurrent stress enhanced the fitness for the coming favorable conditions The results indicate the existence of a mechanism that exclusively regulates Tpk1 subunit expression and therefore contributing to the specificity of cAMP-PKA.

SUMMARY STATEMENTPKA subunits are differentially expressed under heat-shock conditions. The mRNA of the TPK1 subunit is upregulated upon heat-shock at 37{degrees}C and thermotolerance, the half-life increases upon heat-stress and also this transcript condensates in cytoplasmic foci upon thermal stress and thermotolerance. The resistance to cycloheximide treatment of TPK1 mRNA foci together with the analysis by polysome profiling suggest that TPK1 mRNA is impaired for entry into translation upon thermal stress. An increase in Tpk1 protein and PKA activity was detected after the heat stress treatments.

Cell Wall Integrity pathway, through Wsc3 sensor, is involved in TPK1 expression. Heat-stress regulates TPK1 expression through this pathway from an intermediate step of the cascade and independently of the upstream elements of the CWI pathway activation. These results demonstrate a new crosstalk between the two signalling pathways.

The increment in Tpk1-dependent PKA activity during cell adaptation to heat stress might contribute to the overall cellular fitness when more favorable environmental conditions are restored.

The results indicate the existence of a mechanism that exclusively regulates TPK1 subunit expression and therefore contributing to the specificity of cAMP-PKA.


title: seRNA PAM-1 regulates skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression synergistically with Ddx5

abstract: Muscle satellite cells (SCs) are responsible for muscle homeostasis and regeneration; and lncRNAs play important roles in regulating SC activities. Here in this study, we identify PAM-1 (Pax7 Associated Muscle lncRNA) that is induced in activated SCs to promote SC activation into myoblast cells upon injury. PAM-1 is generated from a myoblast specific super-enhancer (SE); as a seRNA it binds with a number of target genomic loci predominantly in trans. Further studies demonstrate that it interacts with Ddx5 to tether PAM-1 SE to it inter-chromosomal targets Timp2 and Vim to activate the gene expression. Lastly, we show that PAM-1 expression is increased in aging SCs, which leads to enhanced inter-chromosomal interaction and target genes up-regulation. Altogether, our findings identify PAM-1 as a previously unknown lncRNA that regulates both SC activation and aging through its trans gene regulatory activity.


title: Lysophosphatidylcholine acyltransferase 1 controls the mitochondrial reactive oxygen species generation and survival of the retinal photoreceptor cells

abstract: Due to the high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice with a frameshift mutation of lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration (rd11 mice). In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 decreases saturated PC production and affects the proper cellular fatty acid flux, presumably by altering saturated fatty acyl-CoA availabilities. Furthermore, we demonstrated that Lpcat1 deletion increased mitochondrial reactive oxygen species (ROS) levels in photoreceptor cells, but not in other retinal cells without affecting the OS structure and trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC is critical for metabolic adaptation during photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration-related retinal diseases.


title: Characterization of divalent cation interactions with AASTY nanodiscs

abstract: Amphiphilic copolymers show promise in extracting membrane proteins directly from lipid bilayers into  native nanodiscs. However, many such copolymers are polyanionic and sensitive to divalent cations, limiting their applicability. We characterize the Ca2+ and Mg2+ sensitivity of poly(acrylic acid-co-styrene) (AASTY) copolymers with analytical UV and fluorescent size exclusion chromatography, enabling us to separate signals from nanodiscs, copolymers, and soluble aggregates. We find that divalent cations promote aggregation and precipitation of both free and lipid bound copolymers. We see that excess, free copolymer acts as a  cation sink that protects nanodiscs from Ca2+ induced aggregation. Removal of the free copolymer through dialysis induces aggregation that can be mitigated by KCl. Finally, we find that the nanodisc size is dynamic and dependent on lipid concentration. Our results offer insight to nanodisc behaviour, and can help guide experimental design, aimed at mitigating the shortcomings inherent in negatively charged nanodisc forming copolymers.


title: Characterization of divalent cation interactions with AASTY native nanodiscs


title: Development and characterization of influenza M2 ectodomain and/or HA stalk-based DC-targeting vaccines for different influenza infections

abstract: A universal influenza vaccine is required for broad protection against influenza infection. Here, we revealed the efficacy of novel influenza vaccine candidates based on Ebola glycoprotein (EboGP) DC-targeting domain (E{Delta}M) fusion protein technology. We fused influenza hemagglutinin stalk (HAcs) and extracellular matrix protein (M2e) or four copies of M2e (referred to as tetra M2e (tM2e)) with E{Delta}M to generate E{Delta}M-HM2e or E{Delta}M-tM2e, respectively, and revealed that E{Delta}M facilitates DC/macrophage targeting in vitro. In a mouse study, E{Delta}M-HM2e- or E{Delta}M-tM2e-pseudotyped viral particles (PVPs) induced significantly higher titers of anti-HA and/or anti-M2e antibodies. We also developed recombinant vesicular stomatitis virus (rVSV)-E{Delta}M-HM2e and rVSV-E{Delta}M-tM2e vaccines that resulted in rapid and potent induction of HA and/or M2 antibodies in mouse sera and mucosa. Importantly, vaccination protects mice from influenza H1N1 and H3N2 challenges. Taken together, our study suggests that recombinant rVSV-E{Delta}M-HM2e and rVSV-E{Delta}M-tM2e are efficacious and protective universal vaccines against influenza.


title: Evolutionary sweeps of subviral parasites and their phage host bring unique parasite variants and disappearance of a phage CRISPR-Cas system

abstract: Vibrio cholerae is a significant threat to global public health in part due to its propensity for large-scale evolutionary sweeps where lineages emerge and are replaced. These sweeps may originate from the Bay of Bengal where bacteriophage predation and the evolution of anti-phage counter defenses is a recurring theme. The bacteriophage ICP1 is a key predator of epidemic V. cholerae and is notable for acquiring a CRISPR-Cas system to combat PLE, a defensive subviral parasite encoded by its V. cholerae host. Here we describe the discovery of five previously unknown PLE variants, including one found during recent surveillance of patient samples in Bangladesh. We also observed a lineage sweep of PLE negative V. cholerae occurring within a patient population in under a year which coincided with a loss of ICP1s CRISPR-Cas system. These findings reinforce the importance of surveillance to better understand the selective pressures that drive pandemic cholera.


title: Zika virus impacts extracellular vesicle composition and cellular gene expression in macaque early gestation trophoblasts

abstract: Zika virus (ZIKV) infection at the maternal-placental interface is associated with adverse pregnancy outcomes including fetal demise and pregnancy loss. To determine how infection impacts placental trophoblasts, we utilized rhesus macaque trophoblast stem cells (TSC) that can be differentiated into early gestation syncytiotrophoblasts (ST) and extravillous trophoblasts (EVT). TSCs and STs, but not EVTs, were highly permissive to productive infection with ZIKV strain DAK AR 41524. The impact of ZIKV on the cellular transcriptome showed that infection of TSCs and STs increased expression of immune related genes, including those involved in type I and type III interferon responses. ZIKV exposure altered extracellular vesicle (EV) protein, mRNA, and miRNA cargo, regardless of productive infection. These findings suggest that early gestation macaque TSCs and STs are permissive to ZIKV infection, and that EV analysis may provide a foundation for identifying non-invasive biomarkers of placental infection in a highly translational model.

category: cell biology


title: Sumo-mediated recruitment allows timely function of the Yen1 nuclease in mitotic cells

abstract: The modification of DNA damage response proteins with Sumo is an important mechanism to orchestrate a timely and orderly recruitment of repair factors to damaged sites. After replication stress and double-strand break formation a number of repair factors are Sumoylated and interact with other Sumoylated factors, including the nuclease Yen1. Yen1 plays a critical role to ensure genome stability and unperturbed chromosome segregation by removing covalently linked DNA intermediates that are formed by homologous recombination. Here we show how this important role of Yen1 is dependent on interactions mediated by non-covalent binding to Sumoylated partners. Mutations in the motifs that allow Sumo-mediated recruitment of Yen1 impair its ability to resolve DNA intermediates and result in increased genome instability and chromosome mis-segregation.


title: Role of TLR4 signaling on Porphyromonas gingivalis LPS-induced cardiac dysfunction in mice

abstract: Oral infections, particularly periodontitis, are a well-established risk factor for cardiovascular diseases, although the molecular mechanisms involved remain elusive. The aims of the present study were to investigate the effects of lipopolysaccharide derived from Porphyromonas gingivalis (PG-LPS) on cardiac function in mice, and to elucidate the underlying mechanisms. Mice (C57BL/6) were injected with PG-LPS (0.8 mg/kg/day) with or without an inhibitor of Toll-like receptor 4 (TLR4) signaling (TAK-242, 0.8 mg/kg/day) for 4 weeks. Left ventricular ejection function was significantly decreased at 1 week (from 67 {+/-} 0.5 to 58 {+/-} 1.2 %) and remained low at 4 weeks (57 {+/-} 1.0 %). The number of apoptotic myocytes was increased (approximately 7.4-fold), the area of fibrosis was increased (approximately 3.3-fold) and the number of 8-hydroxydeoxyguanosine-positive myocytes, a sensitive indicator of oxidative DNA damage, was increased (approximately 7.6-fold) at 4 weeks in the heart of PG-LPS treated mice. However, levels of various serum pro-inflammatory cytokines in PG-LPS-treated mice were similar to those in control mice. The impairment of cardiac function in PG-LPS-treated mice appears to involve activation of TLR4-NADPH oxidase (NOX) 4 signaling, leading to abundant production of reactive oxygen species and Ca2+ leakage from sarcoplastic reticulumn induced by calmodulin kinase II (CaMKII)-mediated phosphorylation of phospholamban (at Thr-17) and ryanodine receptor 2 (at Ser-2448). Pharmacological inhibition of TLR4 with TAK-242 attenuated the changes in cardiac function in PG-LPS-treated mice. Our results indicate that TLR4-NOX4 signaling may be a new therapeutic target for treatment of cardiovascular diseases in patients with periodontitis.

category: physiology


title: Impaired trigeminal control of ingestive behavior in the Prrxl1- / - mouse is associated with a lemniscal-biased orosensory deafferentation

abstract: Although peripheral deafferentation studies have demonstrated a critical role for trigeminal afference in modulating the orosensorimotor control of eating and drinking, the central trigeminal pathways mediating that control, as well as the timescale of control, remain to be elucidated. In rodents, three ascending somatosensory pathways process and relay orofacial mechanosensory input: the lemniscal, paralemniscal, and extralemniscal. Two of these pathways (the lemniscal and extralemniscal) exhibit highly structured topographic representations of the orofacial sensory surface, as exemplified by the one-to-one somatotopic mapping between vibrissae on the animals face and barrelettes in brainstem, barreloids in thalamus, and barrels in cortex. Here we use the Prrxl1 knockout mouse model to investigate ingestive behavior deficits associated with disruption of the lemniscal pathway. The Prrxl1 deletion disrupts somatotopic patterning and axonal projections throughout the lemniscal pathway but spares patterning in the extralemniscal nucleus. Our data reveal an imprecise and inefficient ingestive phenotype with deficits that span timescales from milliseconds to months, tightly linking trigeminal input with ingestion, from moment-to-moment consummatory to long term appetitive control. We suggest that ordered assembly of trigeminal sensory information along the lemniscal pathway is critical for the rapid and precise modulation of motor circuits driving eating and drinking action sequences.


title: LysM receptors in Coffea arabica: identification, characterization, and gene expression in response to Hemileia vastatrix

abstract: Pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors (PRRs) localized on the host plant cell wall. These receptors activate a broad-spectrum and durable defense, which are desired characteristics for disease resistance in plant breeding programs. In this study, candidate sequences for PRRs with lysin motifs (LysM) were investigated in the Coffea arabica genome. For this, approaches based on the principle of sequence similarity, conservation of motifs and domains, phylogenetic analysis, and modulation of gene expression in response to Hemileia vastatrix were used. The candidate sequences for PRRs in C. arabica (Ca1-LYP, Ca2-LYP, Ca1-CERK1, Ca2-CERK1, Ca-LYK4, Ca1-LYK5 and Ca2-LYK5) showed high similarity with the reference PRRs used: Os-CEBiP, At-CERK1, At-LYK4 and At-LYK5. Moreover, the ectodomains of these sequences showed high identity or similarity with the reference sequences, indicating structural and functional conservation. The studied sequences are also phylogenetically related to the reference PRRs described in Arabidopsis, rice, and other plant species. All candidates for receptors had their expression induced after the inoculation with H. vastatrix, since the first time of sampling at 6 hours post-inoculation (hpi). At 24 hpi, there was a significant increase in expression, for most of the receptors evaluated, and at 48 hpi, a suppression. The results showed that the candidate sequences for PRRs in the C. arabica genome display high homology with fungal PRRs already described in the literature. Besides, they respond to pathogen inoculation and seem to be involved in the perception or signaling of fungal chitin, acting as receptors or coreceptors of this molecule. These findings represent an advance in the understanding of the basal immunity of this species.


title: Screening coffee genotypes for Cercospora coffeicola resistance in Brazil

abstract: Several efforts have been made by many researchers worldwide to develop coffee plants resistant to different Cercospora species; however, studies concerning C. coffeicola, specifically, are still incipient. In the present study, a blend of strains from this pathogen was inoculated into 18 Brazilian commercial cultivars, a coffee clone of Arabica, as well as into 41 accessions from the Germplasm Collection of Minas Gerais, to evaluate the genetic resistance ability within the population and select superior genotypes for the breeding program. After predicting genotypic values of the evaluated material, the most efficient way to select genotypes based on the data of severity to brown eye spot (BES) was also examined. Moreover, the action of defense mechanisms against C. coffeicola attacks was investigated by assessing the levels of total soluble phenolic compounds and soluble lignin in contrasting genotypes regarding disease susceptibility. Based on the results, the accession MG 1207 Sumatra demonstrated an intrinsic genetic capacity to maintain low levels of severity to brown eye spot. This genotype can thus substantially contribute to the development of new cultivars, which may lead to reduced use of pesticides. This study also evidenced that four evaluations of severity is enough to reach accuracy and efficiency for the severity of BES, thus providing expressive genetic gains. Finally, it is suggested that the levels of lignin and phenolic compounds are not associated with the resistance of coffee genotypes to brown eye spot.


title: Identification of a novel modulator of the actin cytoskeleton, mitochondria, nutrient metabolism and lifespan in yeast

abstract: In yeast, actin cables are F-actin bundles that are essential for cell division through their function as tracks for cargo movement from mother to daughter cell. Actin cables also affect yeast lifespan by promoting transport and inheritance of higher-functioning mitochondria to daughter cells. Here, we report that actin cable stability declines with age. Our genome-wide screen for genes that affect actin cable stability identified the open reading frame YKL075C. Deletion of YKL075C results in increases in actin cable stability and abundance, mitochondrial fitness, and replicative lifespan. Transcriptome analysis revealed a role for YKL075C in regulating branched-chain amino acid (BCAA) metabolism. Consistent with this, modulation of BCAA metabolism or decreasing leucine levels promotes actin cable stability and function in mitochondrial quality control. Our studies support a role for actin stability in yeast lifespan, and demonstrate that this process is controlled by BCAA and a previously uncharacterized ORF YKL075C, which we refer to as actin, aging and nutrient modulator protein 1 (AAN1).


title: The Relative Binding Position of Nck and Grb2 Adaptors Dramatically Impacts Actin-Based Motility of Vaccinia Virus

abstract: Phosphotyrosine (pTyr) motifs in unstructured polypeptides orchestrate important cellular processes by engaging SH2-containing adaptors to nucleate complex signalling networks. The concept of phase separation has recently changed our appreciation of such multivalent networks, however, the role of pTyr motif positioning in their function remains to be explored. We have now explored this parameter in the assembly and operation of the signalling cascade driving actin-based motility and spread of Vaccinia virus. This network involves two pTyr motifs in the viral protein A36 that recruit the adaptors Nck and Grb2 upstream of N-WASP and Arp2/3-mediated actin polymerization. We generated synthetic networks on Vaccinia by manipulating pTyr motifs in A36 and the unrelated p14 from Orthoreovirus. In contrast to predictions, we find that only specific spatial arrangements of Grb2 and Nck binding sites result in robust N-WASP recruitment, Arp2/3 driven actin polymerization and viral spread. Our results suggest that the relative position of pTyr adaptor binding sites is optimised for signal output. This finding may explain why the relative positions of pTyr motifs are usually conserved in proteins from widely different species. It also has important implications for regulation of physiological networks, including those that undergo phase transitions.


title: Evolution of chlorophyll degradation is associated with plant transition to land

abstract: Colonization of land by green plants (Viridiplantae) some 500 million years ago was made possible by large metabolic and biochemical adaptations. Chlorophyll, the central pigment of photosynthesis, is highly photo-active. In order to mitigate deleterious effects of pigment accumulation, some plants have evolved a coordinated pathway to deal with chlorophyll degradation end-products, so-called phyllobilins. This pathway has been so far mostly unravelled in Arabidopsis thaliana. Here, large-scale comparative phylogenomic coupled to an innovative biochemical characterization strategy of phyllobilins allow a better understanding how such a pathway appeared in Viridiplantae. Our analysis reveals a stepwise evolution of the canonical pheophorbide a monooxygenase/phyllobilin pathway. It appears to have evolved gradually, first in chlorophytes chloroplasts, to ensure multicellularity by detoxifying chlorophyll catabolites, and in charophytes outside chloroplasts to allow adaptation of embryophytes to land. At least six out of the eight genes involved in the pathway were already present in the last common ancestor of green plants. This strongly suggests parallel evolution of distinct enzymes catalysing similar reactions in various lineages, particularly for the dephytylation step. Together, our study suggests that chlorophyll degradation accompanied the transition from water to land, and was therefore of great importance for plant diversification.


title: Large-scale exploration of whole-brain structural connectivity in anorexia nervosa: alterations in the connectivity of frontal and subcortical networks

abstract:
BackgroundAnorexia nervosa (AN) is characterized by disturbances in cognition and behavior surrounding eating and weight. The severity of AN combined with the absence of localized brain abnormalities suggests distributed, systemic underpinnings that may be identified using diffusion-weighted MRI (dMRI) and tractography to reconstruct white matter pathways.

MethodsdMRI data acquired from female patients with AN (n = 147) and female healthy controls (HC; n = 119), aged 12-40 years, were combined across five studies. Probabilistic tractography was completed, and full cortex connectomes describing streamline counts between 84 brain regions generated and harmonized. Graph theory methods were used to describe alterations in network organization in AN. The network-based statistic tested between-group differences in brain subnetwork connectivity. The metrics strength and efficiency indexed the connectivity of brain regions (network nodes), and were compared between groups using multiple linear regression.

ResultsIndividuals with AN, relative to HC, had reduced connectivity in a network comprising subcortical regions and greater connectivity between frontal cortical regions (p < 0.05, FWE corrected). Node-based analyses indicated reduced connectivity of the left hippocampus in patients relative to HC (p < 0.05, permutation corrected). Severity of illness, assessed by BMI, was associated with subcortical connectivity (p < 0.05, uncorrected).

ConclusionsAnalyses identified reduced structural connectivity of subcortical networks and regions, and stronger cortical network connectivity, amongst individuals with AN relative to HC. These findings are consistent with alterations in feeding, emotion and executive control circuits in AN, and may direct hypothesis-driven research into mechanisms of persistent restrictive eating behavior.


title: Quantitative study of alpha-synuclein prion-like spreading in fully oriented reconstructed neural networks reveals non-synaptic dissemination of seeding aggregates

abstract: The trans-neuronal spread of protein aggregates in a prion-like manner underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinsons disease. Despite being studied actively, the mechanisms of alpha-synuclein (aSyn) aggregates propagation remain poorly understood. This hinders the development of therapeutic approaches aiming at preventing the spatial progression of intracellular inclusions in neural networks. To assess the role of synaptic structures and neuron characteristics in the transfer efficiency of aggregates with seeding propensity, we developed a novel microfluidic culture system which allows for the first time to reconstruct in vitro fully oriented and synaptically connected neural networks. This is achieved by filtering axonal growth with unidirectional "axon valves" microchannels. We exposed the presynaptic compartment of reconstructed networks to well characterized human aSyn aggregates differing in size: Fibrils and Oligomers. Both aggregates were transferred to postsynaptic neurons through active axonal transport, albeit with poor efficiency. By manipulating network maturity, we compared the transfer rate of aggregates in networks with distinct levels of synaptic connectivity. Surprisingly, we found that transfer efficiency was lower in mature networks with higher synaptic connectivity. We then investigated the seeding efficiency of endogenous aSyn in the postsynaptic population. We found that exposure to Fibrils, and not Oligomers, resulted in low efficiency trans-neuronal seeding which was restricted to postsynaptic axons. Finally, we assessed the impact of neuron characteristics and aSyn expression on the propagation of aSyn aggregates. By reconstructing chimeric networks, we found that neuron characteristics, such as the brain region from which they originate or aSyn expression levels, did not significantly impact aggregates transfer, and observed no trans-neuronal seeding where the presynaptic population did not express aSyn. Overall, we demonstrate that this novel platform uniquely allows the quantitative interrogation of original aspects of the trans-neuronal propagation of seeding pathogenic entities.


title: Low dose pig anti-influenza virus monoclonal antibodies reduce lung pathology but do not prevent virus shedding

abstract: We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies (mAbs). In this study we demonstrated that prophylactic intravenous administration of 15mg/kg of porcine mAb pb18, against the K160-163 site of the haemagglutinin, significantly reduced lung pathology and nasal virus shedding and eliminated virus from the lung of pigs following H1N1pdm09 challenge. When given at 1mg/kg, pb18 significantly reduced lung pathology and lung and BAL virus loads, but not nasal shedding. Similarly, when pb18 was given in combination with pb27, which recognised the K130 site, at 1mg/kg each, lung virus load and pathology were reduced, although without an apparent additive or synergistic effect. No evidence for mAb driven virus evolution was detected. These data indicate that intravenous administration of high doses was required to reduce nasal virus shedding, although this was inconsistent and seldom complete. In contrast the effect on lung pathology and lung virus load is consistent and is also seen at one log lower doses, strongly indicating that a lower dose might be sufficient to reduce severity of disease, but for prevention of transmission other measures would be needed.


title: Reservoir host immunology and life history shape virulence evolution in zoonotic viruses

abstract: Future pandemic risk management requires better understanding of the mechanisms that determine the virulence of emerging zoonotic viruses. Bats host viruses that cause higher case fatality rates upon spillover to humans than those derived from any other mammal, suggesting that reservoir host immunological and life history traits may be important drivers of cross-species virulence. Using a nested population-level and within-host modelling approach, we generate virulence predictions for viral zoonoses derived from diverse mammalian reservoirs, successfully recapturing corresponding virus-induced human mortality rates from the literature. Our work offers a mechanistic explanation for the virulence of bat-borne zoonoses and, more generally, demonstrates how key differences in reservoir host longevity, tolerance, and population density impact the evolution of viral traits that generate severe disease following spillover to humans. We provide a theoretical framework that offers a series of testable questions and hypotheses designed to stimulate future work comparing cross-species virulence evolution in zoonotic viruses derived from diverse mammalian hosts.

category: evolutionary biology


title: Dysfunction of cAMP-PKA-calcium signaling axis in striatal medium spiny neurons: a role in schizophrenia and Huntington's disease pathogenesis

abstract:
BackgroundStriatal medium spiny neurons (MSNs) are preferentially lost in Huntingtons disease. Genomic studies also implicate a direct role for MSNs in schizophrenia, a psychiatric disorder known to involve cortical neuron dysfunction. It remains unknown whether the two diseases share similar MSN pathogenesis or if neuronal deficits can be attributed to cell type-dependent biological pathways. Transcription factor BCL11B, which is expressed by all MSNs and deep layer cortical neurons, was recently proposed to drive selective neurodegeneration in Huntingtons disease and identified as a candidate risk gene in schizophrenia.

MethodsUsing human stem cell-derived neurons lacking BCL11B as a model, we investigated cellular pathology in MSNs and cortical neurons in the context of these disorders. Integrative analyses between differentially expressed transcripts and published GWAS datasets identified cell type-specific disease-related phenotypes.

ResultsWe uncover a role for BCL11B in calcium homeostasis in both neuronal types, while deficits in mitochondrial function and protein kinase A (PKA)-dependent calcium transients are detected only in MSNs. Moreover, BCL11B-deficient MSNs display abnormal responses to glutamate and fail to integrate dopaminergic and glutamatergic stimulation, a key feature of striatal neurons in vivo. Gene enrichment analysis reveals overrepresentation of disorder risk genes among BCL11B-regulated pathways, primarily relating to cAMP-PKA-calcium signaling axis and synaptic signaling.

ConclusionsOur study indicates that Huntingtons disease and schizophrenia are likely to share neuronal pathogenesis where dysregulation of intracellular calcium levels is found in both striatal and cortical neurons. In contrast, reduction in PKA signaling and abnormal dopamine/glutamate receptor signaling is largely specific to MSNs.


title: Time-resolved, integrated analysis of clonal genome evolution in parthenogenetic animals and in cancer

abstract: Clonal genome evolution is a key aspect for parthenogenetic species and cancer. While many studies describe precise landscapes of clonal evolution in cancer, few studies determine the underlying evolutionary parameters from molecular data, and fewer integrate theory with data. We derived theoretical results linking mutation rate, time, expansion dynamics, transition to recurrence, and survival. With this, we inferred time-resolved estimates of evolutionary parameters from mutation accumulation, mutational signatures and selection. Using this framework we traced the speciation of the rapidly emerging and invasive marbled crayfish to a time window between 1947 and 1996, which is consistent with biological records. In glioblastoma samples, we determined tumor expansion patterns, and tumor cell survival ratio at resection. Interestingly, our results suggest that the expansion pattern in the primary tumor is predictive of the progress and time to recurrence. In addition, tumor cell survival was always higher after resection and was associated with the expansion pattern and time to recurrence. We further observed selection events in a subset of tumors, with longer and purifying-only selection phases in recurrent tumors. In conclusion, our framework allowed a time-resolved, integrated analysis of key parameters in clonally evolving genomes, and provided novel insights into the evolutionary age of marbled crayfish and the progression of glioblastoma.


title: Tuning the sensitivity of genetically encoded fluorescent potassium indicators through structure-guided and genome mining strategies

abstract: Genetically encoded potassium indicators lack optimal binding affinity for monitoring intracellular dynamics in mammalian cells. Through structure-guided design and genome mining of potassium binding proteins, we developed green fluorescent potassium indicators with a broad range of binding affinities. KRaION1, based on the insertion of a potassium binding protein (Ec-Kbp) into the fluorescent protein mNeonGreen, exhibits an isotonically measured Kd of 69{+/-}10 (mM; mean {+/-} standard deviation used throughout). We identified Ec-Kbps binding site using NMR spectroscopy to detect protein-thallium scalar couplings and refined the structure of Ec-Kbp in its potassium-bound state. Guided by this structure, we modified KRaION1, yielding KRaION2, which exhibits an isotonically measured Kd of 96{+/-}9 (mM). We identified four Ec-Kbp homologs as potassium binding proteins, which yielded indicators with isotonically measured binding affinities in the 39-112 (mM) range. KRaIONs expressed and functioned in HeLa cells, but exhibited lower Kd values, which were mirrored by lower Kd values measured in vitro when holding sodium constant. Thus, potassium indicator Kd may need to be evaluated in the context of a given experimental goal.

category: bioengineering


title: Simultaneous Mnemonic and Predictive Representations in the Auditory Cortex

abstract: Recent studies have shown that stimulus history can be decoded via the use of broadband sensory impulses to reactivate mnemonic representations. It has also been shown that predictive mechanisms in the auditory system demonstrate similar tonotopic organization of neural activity as that elicited by the perceived stimuli. However, it remains unclear if the mnemonic and predictive information can be decoded from cortical activity simultaneously and from overlapping neural populations. Here, we recorded neural activity using electrocorticography (ECoG) in the auditory cortex of anesthetized rats while exposed to repeated stimulus sequences, where events within the sequence were occasionally replaced with a broadband noise burst or omitted entirely. We show that both stimulus history and predicted stimuli can be decoded from neural responses to broadband impulse at overlapping latencies but linked to largely independent neural populations. We also demonstrate that predictive representations are learned over the course of stimulation at two distinct time scales, reflected in two dissociable time windows of neural activity. These results establish a valuable tool for investigating the neural mechanisms of passive sequence learning, memory encoding, and prediction mechanisms within a single paradigm, and provide novel evidence for learning predictive representations even under anaesthesia.


title: Compositional analysis of Sindbis virus ribonucleoproteins reveals an extensive co-opting of key nuclear RNA-binding proteins

abstract:
The expansion of tropical mosquito habitats and associated arboviruses is a risk for human health, and it thus becomes fundamental to identify new antiviral strategies. In this study we employ a new approach to elucidate the composition of the ribonucleoproteins (RNPs) of a prototypical arbovirus called Sindbis (SINV). SINV RNPs contain 453 cellular and 6 viral proteins, many of these proteins are nuclear in uninfected cells and redistribute to the cytoplasm upon infection. These findings suggest that SINV RNAs act as spiderwebs, capturing host factors required for viral replication and gene expression in the cytoplasm. Functional perturbation of several of these host proteins causes profound effects in virus infection, as illustrated here with the tRNA ligase complex. Moreover, inhibition of viral RNP components with available drugs hampers the infection of a wide range of viruses, opening new avenues for the development of broad-spectrum therapies.

Research highlightsO_LISINV RNA interactome includes 453 cellular and 6 viral proteins.
C_LIO_LINuclear RBPs that interact with SINV RNA are selectively redistributed to the cytoplasm upon infection
C_LIO_LIThe tRNA ligase complex plays major regulatory roles in SINV and SARS-CoV- 2 replication
C_LIO_LIThe SINV RNA interactome is enriched in pan-viral regulators with therapeutic potential.
C_LI


title: Non-invasive Imaging of Sense of Smell by Tracking the Voltage-Gated Sodium Channel NaV1.7

abstract:
BackgroundAnosmia/hyposmia affects 13.3 million people in the U.S. alone according to the recent U.S. National Health and Nutrition Examination Survey (NHANES). Hundreds of thousands more people with persistent olfactory dysfunction will be added to this number due to the COVID-19 pandemic. Patients with loss-of-function mutations in SCN9A, the gene encoding NaV1.7, experience anosmia in addition to congenital insensitivity to pain. Tsp1a is a recently discovered peptide that inhibits NaV1.7 with high potency and selectivity. In this study, we examined whether a fluorescently tagged version of Tsp1a could be used to visualize normal and damaged mouse olfactory nerves.

MethodsAthymic nude mice were intravenously injected with Tsp1a-IR800. As a control, mice were injected with PBS only, and as a blocking control were injected with combination of Tsp1a and Tsp1a-IR800. All mice were imaged in-vivo and epifluorescence images were acquired using an IVIS Spectrum animal imaging system. Semiquantitative analysis of the Tsp1a-IR800 signal was conducted by measuring the average radiant efficiency in the region of the olfactory epithelium/bulb (ROEB). Methimazole was used to chemically ablate the olfactory epithelium. We performed a food buried test to correlate the level of anosmia with the level of radiance efficiency.

ResultsThe area of olfactory epithelium/bulb was clearly visible in epifluorescence in-vivo images of mice receiving the imaging agent. The radiant efficiency was significantly less in both mice injected with PBS and in mice injected with the blocking formulation. The mice after olfactory ablation had a significantly reduced radiant efficiency compared with normal mice. Moreover, there was a statistically significant and inverse correlation between the time required for the mouse to find buried food and the radiant efficiency. We also performed immunohistochemistry using NaV1.7 antibody. Mice after olfactory ablation as well as COVID-19-infected mice had significantly lower expression of NaV1.7 on the level of olfactory epithelium/bulb.

ConclusionWe show that the fluorescent imaging of mouse olfactory epithelium/bulb is possible, suggesting that labeled Tsp1a tracers may serve as the first objective diagnostic tool of smell disorders, including those caused by COVID-19.


title: A bacteria-based assay to study SARS-CoV-2 protein-protein interactions

abstract:
Methods for detecting and dissecting the interactions of virally encoded proteins are essential for probing basic viral biology and providing a foundation for therapeutic advances. The dearth of targeted therapeutics for the treatment of COVID-19, an ongoing global health crisis, underscores the importance of gaining a deeper understanding of the interactions of SARS-CoV-2-encoded proteins. Here we describe the use of a convenient bacteria-based two-hybrid (B2H) system to analyze the SARS-CoV-2 proteome. We identify sixteen distinct intraviral protein-protein interactions (PPIs), involving sixteen proteins. We find that many of the identified proteins interact with more than one partner. We further show how our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified B2H system that permits the detection of disulfide bond-dependent PPIs in the normally reducing Escherichia coli cytoplasm and we use this system to detect the interaction of the SARS-CoV-2 spike protein receptor-binding domain (RBD) with its cognate cell surface receptor ACE2. We then examine how the RBD-ACE2 interaction is perturbed by several RBD amino acid substitutions found in currently circulating SARS-CoV-2 variants. Our findings illustrate the utility of a genetically tractable bacterial system for probing the interactions of viral proteins and investigating the effects of emerging mutations. In principle, the system could also facilitate the identification of potential therapeutics that disrupt specific interactions of virally encoded proteins. More generally, our findings establish the feasibility of using a B2H system to detect and dissect disulfide bond-dependent interactions of eukaryotic proteins.

ImportanceUnderstanding how virally encoded proteins interact with one another is essential in elucidating basic viral biology, providing a foundation for therapeutic discovery. Here we describe the use of a versatile bacteria-based system to investigate the interactions of the protein set encoded by SARS-CoV-2, the virus responsible for the current pandemic. We identify sixteen distinct intraviral protein-protein interactions, involving sixteen proteins, many of which interact with more than one partner. Our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified version of our bacteria-based system that permits detection of the interaction between the SARS-CoV-2 spike protein (specifically its receptor binding domain) and its cognate human cell surface receptor ACE2 and we investigate the effects of spike mutations found in currently circulating SARS-CoV-2 variants. Our findings illustrate the general utility of our system for probing the interactions of virally encoded proteins.


title: Inhibition of SARS-CoV-2 spike protein palmitoylation reduces virus infectivity

abstract: Spike glycoproteins of almost all enveloped viruses are known to undergo post-translational attachment of palmitic acid moieties. The precise role of such palmitoylation of the spike protein in membrane fusion and infection is not completely understood. Here, we report that palmitoylation of the first five cysteine residues of the c-terminal cysteine-rich domain of the SARS-CoV-2 spike are indispensable for infection and palmitoylation deficient spike mutants are defective in trimerization and subsequent membrane fusion. The DHHC9 palmitoyltransferase interacts with and palmitoylates the spike protein in the ER and Golgi, and knockdown of DHHC9 results in reduced fusion and infection of SARS-CoV-2. Two bis-piperazine backbone-based DHHC9 inhibitors inhibit SARS-CoV-2 spike protein palmitoylation and the resulting progeny virion particles released are defective in fusion and infection. This establishes these palmitoyltransferase inhibitors as potential new intervention strategies against SARS-CoV-2.


title: Angiotensin converting enzyme 2 (ACE2) is expressed in murine cutaneous under single-cell transcriptome resolution

abstract: Angiotensin converting enzyme 2 (Ace2) is widely distributed in human organs, which was identified as a functional receptor for severe acute respiratory syndrome (SARS) coronavirus in human beings. It was also confirmed that SARS-CoV-2 uses the same cell entry receptor, ACE2, as SARS-CoV. However, related research still not discover the expression data associated with murine skin under single cell RNA resolution. In this study, we performed single-cell RNA sequencing (scRNA-seq) on unsorted cells from mouse dorsal skin after 7 days post-wounding. 8312 sequenced cells from four skin samples met quality control metrics and were analyzed.

category: genomics


title: Quantitative live-imaging of Aquilegia floral meristems reveals distinct patterns of floral organ initiation and cell-level dynamics of floral meristem termination

abstract: In-depth investigation of any developmental process in plants requires knowledge of both the underpinning molecular networks and how they directly determine patterns of cell division and expansion over time. Floral meristems (FM) produce floral organs, after which they undergo floral meristem termination (FMT), and precise control of organ initiation and FMT is crucial to reproductive success of any flowering plant. Using a live confocal imaging, we characterized developmental dynamics during floral organ primordia initiation and FMT in Aquilegia coerulea (Ranunculaceae). Our results have uncovered distinct patterns of primordium initiation between stamens and staminodes compared to carpels, and provided insight into the process of FMT, which is discernable based on cell division dynamics preceding carpel initiation. To our knowledge, this is the first quantitative live imaging of meristem development in a system with numerous whorls of floral organs as well as an apocarpous gynoecium. This study provides crucial information for our understanding of how the spatial-temporal regulation of floral meristem behavior is achieved in both an evolutionary and developmental context.