title: GOLPH3 and GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi transport vesicles

abstract: Glycosylation is a diverse and abundant modification of proteins, lipids and RNA. The fidelity of glycosylation is, in part, assured by the correct compartmentalisation of Golgi-resident glycosylation enzymes within the Golgi stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention in the Golgi. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterisation of the clientele of GOLPH3 and its paralogue GOLPH3L has been lacking. The role of GOLPH3 is of particular interest as it is frequently amplified in several solid tumour types. Here, we combine two orthogonal proteomic analyses to identify a diverse range of GOLPH3+3L clients and find that they act in a wide spectrum of glycosylation pathways, or have other roles in the Golgi. Using binding studies, bioinformatics and an in vivo Golgi retention assay, we show that GOLPH3+3L interact with the cytoplasmic tails of their clients through membrane-proximal positively-charged residues. Furthermore, deletion of GOLPH3+3L causes diverse defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.

category: cell biology


title: The Caenorhabditis elegans TDRD5/7-like protein, LOTR-1, interacts with the helicase ZNFX-1 to balance epigenetic signals in the germline

abstract: LOTUS and Tudor domain containing proteins have critical roles in the germline. Proteins that contain these domains, such as Tejas/Tapas in Drosophila, help localize Vasa to the germ granules and facilitate piRNA-mediated transposon silencing. The homologous proteins in mammals, TDRD5 and TDRD7, are required during spermiogenesis. Until now, proteins containing both LOTUS and Tudor domains in Caenorhabditis elegans have remained elusive. Here we describe LOTR-1 (D1081.7), which derives its name from its LOTUS and Tudor domains. Interestingly, LOTR-1 docks next to P granules to colocalize with the broadly conserved Z-granule helicase, ZNFX-1. LOTR-1s Z-granule association requires its Tudor domain, but both LOTUS and Tudor deletions affect brood size when coupled with a knockdown of the Vasa homolog glh-1. In addition to interacting with the germ-granule components WAGO-1, PRG-1 and DEPS-1, we identified a Tudor-dependent association with ZNFX-1. Like znfx-1 mutants, lotr-1 mutants lose small RNAs from the 3 ends of WAGO and Mutator targets, reminiscent of the loss of piRNAs from the 3 ends of piRNA precursor transcripts in mouse Tdrd5 mutants. Our work suggests that LOTR-1 acts in a conserved mechanism that brings small RNA generating mechanisms towards the 3 ends of small RNA templates or precursors.

category: developmental biology


title: Single-molecule, full-length transcript isoform sequencing reveals disease mutation-associated RNA isoforms in cardiomyocytes

abstract: Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we established an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generated a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms (http://steinmetzlab.embl.de/iBrowser/). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identified 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by novel IMMT isoforms. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms as the functional unit, instead of genes or exons, providing more immediate biological interpretation and higher resolution transcriptome comparisons.

category: genetics


title: Extensive protein dosage compensation in aneuploid human cancers

abstract: Aneuploidy is a hallmark of human cancers, but the effects of aneuploidy on protein expression remain poorly understood. To uncover how chromosome copy number changes influence the cancer proteome, we have conducted an analysis of hundreds of human cancer cell lines with matched copy number, RNA expression, and protein expression data. We found that a majority of proteins exhibit dosage compensation and fail to change by the degree expected based on chromosome copy number alone. We uncovered a variety of gene groups that were recurrently buffered upon both chromosome gain and loss, including protein complex subunits and cell cycle genes. Several genetic and biophysical factors were predictive of protein buffering, highlighting complex post-translational regulatory mechanisms that maintain appropriate gene product dosage. Finally, we established that chromosomal aneuploidy has an unexpectedly moderate effect on the expression of oncogenes and tumor suppressors, demonstrating that these key cancer drivers can be subject to dosage compensation as well. In total, our comprehensive analysis of aneuploidy and dosage compensation across cancers will help identify the key driver genes encoded on altered chromosomes and will shed light on the overall consequences of aneuploidy during tumor development.

category: genomics


title: Higher order phosphatase-substrate contacts terminate the Integrated Stress Response

abstract: Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits are known to engage PP1cs surface, locally affecting flexible phosphopeptides access to the active site. However, catalytic efficiency of holophosphatases towards their natively-folded phosphoprotein substrates is largely unexplained. Here we present a Cryo-EM structure of the tripartite PP1c/PPP1R15A/G-actin holophosphatase that terminates signalling in the Integrated Stress Response (ISR) in pre-dephosphorylation complex with its substrate, translation initiation factor 2 (eIF2). G-actins role in eIF2 dephosphorylation is supported crystallographically by the structure of the binary PPP1R15A-G-actin complex, and by biochemical and genetic confirmation of the essential role of PPP1R15A-G-actin contacts to eIF2P dephosphorylation. In the pre-dephosphorylation CryoEM complex, G-actin aligns the catalytic and regulatory subunits, creating a composite surface that engages eIF2s N-terminal domain to position the distant phosphoserine-51 at the active site. eIF2 residues specifying affinity for the holophosphatase are confirmed here to make critical contacts with the eIF2 kinase PERK. Thus, a convergent process of higher-order substrate recognition specifies functionally-antagonistic phosphorylation and dephosphorylation in the ISR.

category: biochemistry


title: Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2

abstract: The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 is an attractive target for COVID-19 vaccine developments, which naturally exists in a trimeric form. Here, guided by structural and computational analyses, we present a mutation-integrated trimeric form of RBD (mutI tri-RBD) as a broadly protective vaccine candidate, in which three RBDs were individually grafted from three different circulating SARS-CoV-2 strains including the prototype, Beta (B.1.351) and Kappa (B.1.617). The three RBDs were then connected end-to-end and co-assembled to possibly mimic the native trimeric arrangements in the natural S protein trimer. The recombinant expression of the mutI tri-RBD, as well as the homo-tri-RBD where the three RBDs were all truncated from the prototype strain, by mammalian cell exhibited correct folding, strong bio-activities, and high stability. The immunization of both the mutI tri-RBD and homo-tri-RBD plus aluminum adjuvant induced high levels of specific IgG and neutralizing antibodies against the SARS-CoV-2 prototype strain in mice. Notably, regarding to the "immune-escape" Beta (B.1.351) variant, mutI tri-RBD elicited significantly higher neutralizing antibody titers than homo-tri-RBD. Furthermore, due to harboring the immune-resistant mutations as well as the evolutionarily convergent hotspots, the designed mutI tri-RBD also induced strong broadly neutralizing activities against various SARS-CoV-2 variants, especially the variants partially resistant to homo-tri-RBD. Homo-tri-RBD has been approved by the China National Medical Products Administration to enter clinical trial (No. NCT04869592), and the superior broad neutralization performances against SARS-CoV-2 support the mutI tri-RBD as a more promising vaccine candidate for further clinical developments.

category: immunology


title: Exploration of molecular mechanism underlying the self-flocculation of Zymomonas mobilis through comparative omics analyses and experimental validations for developing robust production systems

abstract: Zymomonas mobilis metabolizes sugar through the Entner-Doudoroff pathway with less ATP generated for lower biomass accumulation and more substrate to product formation with improved yield, since ATP is dissipated predominately through growth for intracellular energy homeostasis, making it a platform to be engineered as microbial cell factories, particularly for producing bulk commodities with major cost from feedstock consumption. ZM401, a self-flocculating mutant, presents advantages for production including cost-effective biomass recovery through gravity sedimentation, self-immobilization within bioreactors for high cell density to improve productivity and enhanced tolerance to environmental stresses for high product titers, but molecular mechanism underlying this phenotype is largely unknown. In this work, we sequenced and assembled the genome of ZM401 to explore genetic basis for the self-flocculation of the bacterial cells through comparative genomic and transcriptomic analyses, molecular docking simulations for enzymes encoded by functional genes and their substrates/activators, and experimental validations. Our results demonstrated that the single nucleotide deletion in ZMO1082 disrupted its stop codon for the putative gene being fused with ZMO1083, which created an exciting gene encoding the subunit A of the bacterial cellulose synthase with unique function for synthesizing cellulose microfibrils to flocculate the bacterial cells, and the single nucleotide mutation in ZMO1055 compromised the function of bifunctional diguanylate cyclase/phosphodiesterase encoded by the gene on the degradation of c-di-GMP for its intracellular accumulation to activate the cellulose biosynthesis. These discoveries are significant not only for optimizing the self-flocculation of Z. mobilis, but also engineering other bacteria with the self-flocculating phenotype for robust production.

category: microbiology


title: RPAP2 regulates a transcription initiation checkpoint by prohibiting assembly of preinitiation complex

abstract: RNA polymerase II (Pol II)-mediated transcription in metazoan requires precise regulation. RNA polymerase II-associated protein 2 (RPAP2) was previously identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD). We found that RPAP2 binds hypo/hyper-phosphorylated Pol II with undetectable phosphatase activity. Structure of RPAP2-Pol II shows mutually exclusive assembly of RPAP2-Pol II and pre-initiation complex (PIC) due to three steric clashes. RPAP2 prevents/disrupts Pol II-TFIIF interaction and impairs in vitro transcription initiation, suggesting a function in prohibiting PIC assembly. Loss of RPAP2 in cells leads to global accumulation of TFIIF and Pol II at promoters, indicating critical role of RPAP2 in inhibiting PIC assembly independent of its putative phosphatase activity. Our study indicates that RPAP2 functions as a gatekeeper to prohibit PIC assembly and transcription initiation and suggests a novel transcription checkpoint.

category: molecular biology


title: Arabidopsis stomatal polarity protein BASL mediates distinct processes before and after cell division to coordinate cell size and fate asymmetries

abstract: In many land plants, asymmetric cell divisions (ACDs) create and pattern differentiated cell types on the leaf surface. In the Arabidopsis stomatal lineage, BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) regulates multiple aspects of ACD including division plane placement and cell fate enforcement. Polarized subcellular localization of BASL is initiated before the ACD and persists for many hours after the division in one of the two daughters. Untangling the respective contributions of polarized BASL before and after division is essential to gain a better understanding of its roles in regulating stomatal lineage ACDs and to uncover the rules that guide leaf pattern. Here we combine quantitative imaging and lineage tracking with genetic tools that provide temporally-restricted BASL expression. We find that pre-division BASL is required for division orientation, whereas BASL polarity post-division ensures proper cell fate commitment. These genetic manipulations allowed us to uncouple daughter-cell size asymmetry from polarity crescent inheritance, revealing independent effects of these two asymmetries on subsequent cell behavior. Finally, we show that there is coordination between the division frequencies of sister cells produced by ACDs, and this coupling requires BASL as an effector of peptide signaling.

category: plant biology


title: Introgression of type-IV glandular trichomes from Solanum galapagense to cultivated tomato reveals genetic complexity for the development of acylsugar-based insect resistance

abstract:
Glandular trichomes are involved in the production of food- and medicine-relevant chemicals in plants, besides being associated with pest resistance. In some wild Solanum species closely related to the cultivated tomato (S. lycopersicum), the presence of type-IV glandular trichomes leads to the production of high levels of insecticide acylsugars (AS). Conversely, low AS production observed in the cultivated tomato is attributed to its incapacity to develop type-IV trichomes in adult organs. Therefore, we hypothesized that cultivated tomatoes engineered to harbor type-IV trichomes on the leaves of mature plants can be pest resistant. We introgressed into the tomato cultivar Micro-Tom (MT) the capability of S. galapagense to maintain the development of type-IV trichomes throughout all plant stages, thus creating a line named "Galapagos enhanced trichomes" (MT-Get). Mapping-by-sequencing of MT-Get revealed that five chromosomal regions of S. galapagense were present in MT-Get. Further mapping reveled that S. galapagense alleles on chromosomes 1, 2 and 3 are sufficient for the presence of type-IV trichomes, but in lower densities. GC-MS, LC-MS, and gene expression analyses demonstrated that the increased density of type-IV trichomes was not accompanied by high AS production and exudation in MT-Get. Moreover, MT-Get did not differ from MT in its susceptibility to whitefly (Bemisia tabaci). Our findings demonstrates that type-IV glandular trichome development along with AS production and exudation are partially uncoupled at the genetic level. The MT-Get genotype represents a valuable resource for further studies involving the biochemical manipulation of type-IV trichome content through either genetic introgression or transgenic approaches.

Significance StatementThis work identified loci in the tomato genome that control the heterochronic development of type-IV glandular trichomes and uncoupled the genetic control of this type of trichome ontogeny from acylsugar biosynthesis and accumulation, revealing a higher than anticipated genetic complexity of acylsugar-based insect resistance. The findings reported herein will contribute to further dissect the genetics of trichome development in tomato as well as to introgress broad and durable insect resistance in tomato and other Solanaceae.


title: Single domain antibodies against enteric pathogen virulence factors are active as curli fiber fusions on probiotic E. coli Nissle 1917

abstract:
Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic Escherichia.coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eucaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.

Author SummaryEnteric pathogens are the causative agents of diarrheal disease - a leading cause of infant morbidity and mortality worldwide. While treatment and prevention options such as drugs or vaccines exist for some pathogens, their efficacy and availability are often limited. New therapeutic strategies are therefore needed, especially inexpensive agents in low-income countries where enteric disease burdens are highest. One promising avenue for novel treatments uses VHHs - highly stable, well-expressed, antibody domains derived from camelid species such as llamas and alpacas. The small size, high stability and simple structure of these antibody fragments enables their streamlined production by bacteria such as E. coli, potentially reducing cost and improving scalability. In this work, we describe the development of VHHs targeting multiple virulence factor proteins of pathogenic E. coli and other leading causes of diarrheal disease. These VHHs provide new tools for the research community and may serve as promising components of agents that prevent or treat pathogen infections. Towards that goal, we engineered a novel system in which the probiotic, mucus-establishing bacterial strain E. coli Nissle 1917 (EcN) is used to express and display VHHs at high density on its surface. By demonstrating the ability of these engineered EcN to bind to pathogens, we provide a first step toward using such probiotics as a cheap, simple, and effective treatment for enteric pathogen infections.


title: FMO rewires metabolism to promote longevity throughtryptophan and one carbon metabolism

abstract: Flavin containing monooxygenases (FMOs) are promiscuous enzymes known for metabolizing a wide range of exogenous compounds. In C. elegans, fmo-2 expression increases lifespan and healthspan downstream of multiple longevity-promoting pathways through an unknown mechanism. Here, we report that, contrary to its classification as a xenobiotic enzyme, fmo-2 expression leads to rewiring of endogenous metabolism principally through changes in one carbon metabolism (OCM). Using computer modeling, we identify decreased methylation as the major OCM flux modified by FMO-2 that is sufficient to recapitulate its longevity benefits. We further find that tryptophan is decreased in multiple mammalian FMO overexpression models and is a validated substrate for FMO enzymes. Our resulting model connects a single enzyme to two previously unconnected key metabolic pathways and provides a framework for the metabolic interconnectivity of longevity-promoting pathways such as dietary restriction. FMOs are well-conserved enzymes that are also induced by lifespan-extending interventions in mice, supporting a conserved and critical role in promoting health and longevity through metabolic remodeling.


title: Splice Factor Polypyrimidine tract-binding protein 1 (Ptbp1) is Required for Immune Priming of the Endothelium in Atherogenic Disturbed Flow Conditions

abstract:
NF{kappa}B mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through upregulation of adhesion molecules Icam1 and Vcam. The endothelium is "primed" for cytokine activation of NF{kappa}B by exposure to low and disturbed blood flow (LDF) in vivo and by LDF or static conditions in cultured cells. While priming leads to an exaggerated expression of Icam1 and Vcam following cytokine stimulation, the molecular underpinnings are not fully understood. We showed that alternative splicing of genes regulating NF{kappa}B signaling occurs during priming, but the functional implications of this are not known. We hypothesize that the regulation of splicing by RNA-binding splice factors is critical for priming. Here, we perform a CRISPR screen in cultured aortic endothelial cells to determine whether splice factors active in the response to LDF participate in endothelial cell priming. Using Icam1 and Vcam induction by TNF stimulation as a marker of priming, we identify polypyrimidine tract binding protein (Ptbp1) as a required splice factor. Ptbp1 expression is increased and its motifs are enriched nearby alternatively spliced exons in endothelial cells exposed to LDF in vivo in a platelet dependent manner, indicating its induction by early innate immune cell recruitment. At a mechanistic level, deletion of Ptbp1 inhibited NF{kappa}B nuclear translocation and transcriptional activation. These changes coincided with altered splicing of key components of the NF{kappa}B signaling pathway that were similarly altered in the LDF response. However, these splicing and transcriptional changes could be restored by expression of human PTBP1 cDNA in Ptbp1 deleted cells. In vivo, endothelial specific deletion of Ptbp1 reduced myeloid cell infiltration at regions of LDF in atherosclerotic mice. In human coronary arteries, PTBP1 expression correlates with expression of TNF pathway genes and amount of plaque. Together, our data suggest that Ptbp1, which is activated in the endothelium by innate immune cell recruitment in regions of LDF, is required for priming of the endothelium for subsequent NF{kappa}B activation and myeloid cell recruitment in vascular inflammation.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Plaque forms in low and disturbed flow regions of the vasculature, where endothelial cells are "primed" to respond to cytokines (e.g. TNF) with elevated levels of cell adhesion molecules via the NF{kappa}B signaling pathway. We show that the splice factor Ptbp1 (purple) mediates priming. Ptbp1 is induced in endothelial cells by platelet recruitment, promoting priming and subsequent myeloid cell infiltration into plaque. Mechanistically, Ptbp1 regulates splicing of genes involved in the NF{kappa}B signaling pathway and is required for efficient nuclear translocation of NF{kappa}B in endothelial cells. This provides new insight into the molecular mechanisms underlying an endothelial priming process that reinforces vascular inflammatory responses.

C_FIG_DISPLAY


title: Persistent DNA Repair Signaling and DNA Polymerase Theta Promote Broken Chromosome Segregation

abstract:
Cycling cells must respond to double-strand breaks (DSBs) to avoid genome instability. Mis-segregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophila papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early-acting repair machinery (Mre11 and RPA3) to DSBs. This machinery persists as foci on DSBs as cells enter mitosis. Repair foci are resolved in a step-wise manner during mitosis. Repair signaling kinetics at DSBs depends on both monoubiquitination of the Fanconi Anemia (FA) protein Fancd2 and the alternative end-joining protein DNA Polymerase Theta. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.

SummaryClay et. al. show that cells with DNA breaks that persist into mitosis activate sustained DNA repair signaling, regulated by Fanconi Anemia proteins and the alternative end-joining repair protein DNA Polymerase Theta. This signaling enables broken chromosome segregation and prevents micronuclei.


title: Stem cell-free therapy for glaucoma to preserve vision

abstract:
Glaucoma is the leading cause of irreversible blindness with trabecular meshwork (TM) dysfunction resulting in elevated intraocular pressure and retinal ganglion cell (RGC) damage leading to vision loss. In this study, we discovered that secretome, derived from human TM stem cells, via minimal invasive periocular injection, can reduce intraocular pressure, restore TM homeostasis, protect RGC, and restore RGC function in both steroid-induced and genetic myocilin mutant mouse models of glaucoma. The secretome upregulated the COX2-PGE2 axis via mitochondrial TMEM177 and led to activation of endogenous stem cells and TM regeneration. Inhibition of COX2 abolished the protective effect of secretome on TM cells. Secretome treatment also enhanced RGC survival and function. Proteomic analysis revealed that the secretome is enriched with proteins involved in extracellular matrix modulation leading to the remodeling of TM to restore homeostasis. This study highlights the feasibility of stem cell-free therapy for glaucoma with minimal invasive administration and the involvement of multiple novel pathways for a cumulative regenerative effect on the TM to protect RGC.

Brief summaryThis study describes a cell-free treatment using stem cell secretome in two animal models of glaucoma and explores the potential mechanisms


title: Lamellipodia-like actin networks in cells lacking WAVE Regulatory Complex

abstract: Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases mediating activation of WAVE Regulatory Complex (WRC) driving Arp2/3 complex-dependent actin assembly. Previous genome editing studies solidified the view of an essential, linear pathway employing aforementioned components. Using disruption of the WRC subunit Nap1 and its paralogue Hem1 followed by serum and growth factor stimulation or expression of active GTPases now revealed a pathway to formation of Arp2/3 complex-dependent, lamellipodia-like structures (LLS) that require both Rac and Cdc42, but not WRC. These observations were independent of WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from the latter, induced LLS contained all common lamellipodial regulators tested, including cortactin, the Ena/VASP ligand lamellipodin or FMNL subfamily formins. Our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery operating without WRC and Ena/VASP.


title: School yard biodiversity determines short-term recovery of disturbed skin microbiota in children

abstract: The planting and conservation of biodiverse habitat in urban contexts has been proposed as a public health intervention aimed at reducing the prevalence of non-communicable diseases via microbiome rewilding (Mills et al. 2017; Mills et al. 2019). However, our understanding of the effect of urban biodiversity interventions on the human microbiota remains limited, especially on the skin (Hui et al. 2019; Roslund et al. 2020) and in the context of permanent green spaces (Lehtimaki et al. 2018; Selway et al. 2020). Here, we test the short-term response of experimentally disturbed bacterial communities on the skin of healthy children exposed to different school environments - either a  classroom, a  sports field, or a biodiverse  forest - to understand how exposures to different types of biodiversity may influence skin microbiota. Children exposed to the  forest had significantly increased skin microbiota diversity when compared to pre-exposure, an effect that increased over three days suggesting long-term effects. The microbiota on children exposed to the  forest had the largest structural and compositional community change compared to children exposed to  sports fields, which in turn was larger than those who remained in  classrooms. Children exposed to  sports fields and  forests also acquired new core bacteria after exposure to green spaces, potentially buffering against disturbances to the skin microbiotas diversity, while individuals who remained in the  classroom lost microbes throughout the experiment. Overall, we conclude that urban green spaces can have an enriching influence on the diversity of skin microbiota, including core members shared between all children. These findings have important implications for the design and construction of new school yards and public spaces with respect to biodiversity, health, and human microbiota.

category: ecology


title: Stability of Polygenic Scores Across Discovery Genome-Wide Association Studies

abstract: Polygenic scores (PGS) are commonly evaluated in terms of their predictive accuracy at the population level by the proportion of phenotypic variance they explain. To be useful for precision medicine applications, they also need to be evaluated at the individual patient level when phenotypes are not necessarily already known. Hence, we investigated the stability of PGS in European-American (EUR)- and African-American (AFR)-ancestry individuals from the Philadelphia Neurodevelopmental Cohort (PNC) and the Adolescent Brain Cognitive Development (ABCD) cohort using different discovery GWAS for post-traumatic stress disorder (PTSD), type-2 diabetes (T2D), and height. We found that pairs of EUR-ancestry GWAS for the same trait had genetic correlations > 0.92. However, PGS calculated from pairs of sameancestry and different-ancestry GWAS had correlations that ranged from <0.01 to 0.74. PGS stability was higher for GWAS that explained more of the trait variance, with height PGS being more stable than PTSD or T2D PGS. Focusing on the upper end of the PGS distribution, different discovery GWAS do not consistently identify the same individuals in the upper quantiles, with the best case being 60% of individuals above the 80th percentile of PGS overlapping from one height GWAS to another. The degree of overlap decreases sharply as higher quantiles, less heritable traits, and different-ancestry GWAS are considered. PGS computed from different discovery GWAS have only modest correlation at the level of the individual patient, underscoring the need to proceed cautiously with integrating PGS into precision medicine applications.


title: The non-canonical interaction between calmodulin and calcineurin contributes to the differential regulation of plant-derived calmodulins on calcineurin.

abstract: Calmodulin (CaM) is an important Ca2+ signaling hub that regulates many protein signaling pathways. In recent years, several CaM homologs expressed in plants have been shown to regulate mammalian targets and they are attractive for gene therapy. However, the molecular basis of how the CaM homologs mutations impact target activation is unclear, which limits efforts to engineer their functional properties. To understand these mechanisms, we examined two CaM isoforms found in soybean plants that differentially regulate a mammalian target, calcineurin (CaN). These CaM isofroms, sCaM-1 and sCaM-4 share >90% and ~ 78% identity with the mammalian CaM (mCaM), respectively, activate CaN with comparable or reduced activity relative to mCaM. We used molecular simulations and experimental assays to probe whether calcium and protein-protein binding interactions are altered in plant CaMs relative to mCaM as a basis for differential CaN regulations. We found that the two sCaMs Ca2+-binding properties such as coordination and affinity are comparable to mCaM. Further, the binding of CaM to the CaM binding region (CaMBR) in CaN is also comparable among the three CaMs, as evidenced by calculated binding free energies and experimental measured EC50 [CaM]. However, mCaM and sCaM-1 exhibited stronger binding with a secondary region of CaNs regulatory domain that is weakened for sCaM-4. This secondary interaction is likely to affect the turnover rate (kcat) of CaN based on our modeling of enzyme activity and is consistent with our experimental data. Together, our data show how plant-derived CaM variants can alter target activation through interactions beyond Ca2+-binding and canonical CaMBR binding, which may extend beyond the mammalian CaN target.


title: The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation

abstract: Yeast Cadmium Factor-1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4[A] and 4.0[A] in distinct inward-facing open conformations capturing a previously unobserved ordered state of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the lasso domain. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide long-sought after insights into how R-domains control ABCC transporter activity.


title: 2.5D Tractions in monocytes reveal mesoscale mechanics of podosomes during substrate indenting cell protrusion

abstract: Degradation and protrusion are key to cellular barrier breaching in cancer metastasis and leukocyte extravasation. Cancerous invadopodia and myelomonocytic podosomes are widely considered as structural tools facilitating these processes and are thus summarized under the term invadosomes. Despite similar behaviour on the individual scale, substantial differences have been reported to arise on the collective scale. They are considered to be a result of podosome mesoscale-connectivity. In this study, we investigated global in-plane and out-of-plane mechanical forces of podosome clusters in ER-Hoxb8 cell derived monocytes. We are able to correlate these forces with the interpodosomal connectivity. The observed traction and protrusion patterns fail to be explained by summation of single podosome mechanics. Instead, they appear to originate from superimposed mesoscale effects. Based on mechanistic and morphological similarities with epithelial monolayer mechanics, we propose a spatiotemporal model of podosome cluster mechanics capable of relating single to collective podosome mechanical behaviour. Our results suggest that network contraction-driven (in-plane) tractions lead to a buckling instability that contributes to the out-of-plane indentation into the substrate. First assigning an active mechanical role to the dorsal podosome actomyosin network, we aim at translating actomyosin hierarchy into scale dependency of podosome mechanics.

category: biophysics


title: Tumor PD-L1 selectively suppresses type I interferon in myeloid cells to suppress CTL recruitment to promote lung metastasis

abstract: The mechanism underlying tumor cell PD-L1 (tPD-L1) induction of immune suppression through T cell PD-1 is well-known, but the mechanism underlying tPD-L1 induction of immune suppression via an intermediate cell is incompletely understood. We report here that tPD-L1 does not suppress cytotoxic T lymphocyte (CTL) activation and lytic function when only tumor cells and CTLs are present. Strikingly, knocking out PD-L1 in tumor cells has no effect on primary tumor growth, but significantly decreases lung metastasis in a CTL-dependent manner. Depletion of myeloid cells impaired tPD-L1 promotion of lung metastasis. Single-cell RNA sequencing revealed that tPD-L1 engages myeloid PD-1 (mPD-1) to antagonize type I interferon (IFN-I) and STAT1 signaling to repress Cxcl9 and Cxcl10 expression to impair CTL recruitment to lung metastases. Human patient response to PD-1 blockade immunotherapy correlates with IFN-I response in myeloid cells. Our data determines that the tPD-L1/mPD-1/IFN-I/STAT1/Cxcl9/10 axis controls CTL tumor infiltration in lung metastasis.

category: cancer biology


title: Telomere transcription in MLL-rearranged acute leukemia: increased levels of TERRA associate with lymphoid lineage and are independent of telomere length and ploidy

abstract: Telomere transcription into telomeric repeat-containing RNA (TERRA) is an integral component of all aspects of chromosome end protection consisting of telomerase- or recombination-dependent telomere elongation, telomere capping, and preservation of (sub)telomeric heterochromatin structure. The chromatin modifier and transcriptional regulator MLL associates with telomeres and regulates TERRA transcription in telomere length homeostasis and response to telomere dysfunction. MLL fusion proteins (MLL-FPs), the product of MLL rearrangements in leukemia, are also associated with telomeric chromatin. However, an effect on telomere transcription in MLL-rearranged (MLL-r) leukemia has not yet been evaluated. Here, we show increased UUAGGG repeat-containing RNA levels in MLL-r acute lymphoblastic leukemia (ALL) when compared to myeloid and non-MLL-r ALL leukemia. MLL rearrangements do not affect telomere length and increased levels of UUAGGG repeatcontaining RNA correlate with mean telomere length and reflect increased levels of TERRA. Also, increased TERRA levels in MLL-r ALL occur in the presence of telomerase activity and are independent of ploidy, an underestimated source of variation on the overall transcriptome size in a cell. This MLL rearrangement-dependent and lymphoid lineage-associated increase in levels of TERRA supports a sustained telomere transcription by MLL-FPs that correlates with marked genomic stability previously reported in pediatric MLL-r ALL.


title: Changes in splicing and neuromodulatory gene expression programs in sensory neurons with pheromone signaling and social experience

abstract: Social experience and pheromone signaling in ORNs affect pheromone responses and male courtship behaviors in Drosophila, however, the molecular mechanisms underlying this circuit-level neuromodulation remain less clear. Previous studies identified social experience and pheromone signaling-dependent modulation of chromatin around behavioral switch gene fruitless, which encodes a transcription factor necessary and sufficient for male behaviors. To identify the molecular mechanisms driving social experience-dependent neuromodulation, we performed RNA-seq from antennal samples of mutant fruit flies in pheromone receptors and fruitless, as well as grouped or isolated wild-type males. We found that loss of pheromone detection differentially alters the levels of fruitless exons suggesting changes in splicing patterns. In addition, many Fruitless target neuromodulatory genes, such as neurotransmitter receptors, ion channels, and ion transporters, are differentially regulated by social context and pheromone signaling. Our results suggest that modulation of circuit activity and behaviors in response to social experience and pheromone signaling arise due to changes in transcriptional programs for neuromodulators downstream of behavioral switch gene function.

category: neuroscience


title: Endothelial Resolution of Inflammation is Delayed Following JAK-driven but not NFκB-dependent Activation

abstract: Blood endothelial cells actively regulate egress of leukocytes into peripheral tissues in response to inflammatory insult. The resolution of inflammation is critical for healing and return to homeostasis, but the timing and mechanisms involved in return to a non-inflamed state are not well-understood. We examined vascular endothelial activation comparing NF{kappa}B-driven TNF and JAK/STAT-mediated IFN{gamma}. Pro-adhesive gene expression, phenotype and secretome of human endothelial cells from 6 vascular beds were measured under chronic cytokine stimulation, and after short-term cytokine priming followed by withdrawal. The majority of inducible TNF effectors require continuous exposure for reinforcement of the altered phenotype. NF{kappa}B and target genes are quickly down-regulated in the absence of cytokine. In contrast, the consequences of even short exposure to IFN{gamma} are long-lasting and broad, with sustained elevation of adhesion molecules and chemokines up to 48hr later. JAK/STAT and interferon response factor expression are likewise durable, dependent on new transcription and autonomous of continuous IFN{gamma}. Finally, intact persistent STAT expression and JAK signaling in the endothelium is required to maintain a pro-adhesive phenotype after IFN{gamma} withdrawal, which could be prevented by the JAK1/2 inhibitor ruxolitinib. Our results reveal a sustained JAK-dependent perturbation of endothelial function after exposure to IFN{gamma}, but not after NF{kappa}B-driven inflammation.


title: Cryo-EM reveals new species-specific proteins and symmetry elements in the Legionella pneumophila Dot/Icm T4SS

abstract: Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires Disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 [A] resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure. We hypothesize this conformational flexibility is likely to facilitate the Dot/Icm T4SSs ability to translocate a remarkably large set of [~]300 putative substrates across the inner and outer membranes of the bacterial cell.


title: Preferential lipolysis of DGAT1 over DGAT2 generated triacylglycerol in Huh7 hepatocytes

abstract: Hepatic steatosis is defined by accumulation of neutral lipids in lipid droplets (LDs) including triacylglycerol (TG) and steryl esters. Two distinct diacylglycerol acyltransferases (DGAT1 and DGAT2) catalyze synthesis of TG in hepatocytes. TG formed through either DGAT1 or DGAT2 appears to be preferentially directed to distinct intercellular fates, such as fatty acid production for oxidation or very-low density lipoprotein assembly, respectively. Because of the preferential use of TG generated by DGAT1 and DGAT2, we hypothesized that targeting/association of lipolytic machinery to LDs would differ depending on whether the TG stores were generated through DGAT1 or DGAT2 activities. Inhibition of DGAT1 or DGAT2 in human hepatoma cells (Huh7) incubated with oleic acid resulted in only a small change in TG accretion suggesting that the two DGATs can compensate for each other in fatty acid esterification. This compensation was not accompanied by changes in DGAT1 or DGAT2 mRNA expression. DGAT1 inhibition (TG synthesized by DGAT2) resulted in large LDs, whereas DGAT2 inhibition (TG synthesized by DGAT1) caused the accumulation of numerous small LDs. Oleic acid treatment increased mRNA and protein expression of the LD-associated protein PLIN2 but not PLIN5 or the lipase ATGL and its activator ABHD5/CGI-58. Inactivation of DGAT1 or DGAT2 did not alter expression (mRNA or protein) of ATGL, ABHD5/CGI-58, PLIN2 or PLIN5, but inactivation of both DGATs increased PLIN2 abundance despite a dramatic reduction in the number of LDs. ATGL localized preferentially to DGAT1-made LDs rather than to DGAT2-made LDs, and TG in these LDs was preferentially used for fatty acid (FA) oxidation. A combination of DGAT2 inhibitor and the pan lipase inhibitor E600 resulted in large LDs, suggesting that the small size of DGAT1-made LDs is due to a lipolytic process.


title: Single nucleus pituitary transcriptomic and epigenetic landscape reveals human stem cell heterogeneity with diverse regulatory mechanisms

abstract: Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus (sn) RNAseq and ATACseq resource from pediatric, adult, and aged pituitaries (snpituitaryatlas.princeton.edu) and characterize cell type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors (TFs) that are significantly associated with gene expression in PSCs compared to other cell types and within PSCs. Modeling the heterogeneous expression of two markers for committing cell lineages among PSCs shows significant correlation with regulatory domain accessibility for GATA3, but with TF expression for POMC. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes.


title: The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters

abstract:
The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene activation in specific biological contexts is not well understood. Here we analyzed the role of the non-enzymatic core module during Drosophila oogenesis. We show that depletion of several SAGA-specific subunits belonging to the core module blocked egg chamber development during mid-oogenesis stages, resulting in stronger phenotypes than those obtained after depletion of SAGAs histone acetyltransferase module or deubiquitination module. These results, as well as additional genetic analyses pointing to an interaction with TBP, suggested a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments. Thus, the SAGA complex appears to occupy promoters in its entirety, consistent with the strong biochemical integrity of the complex. The high-resolution genomic binding profiles are congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. The stronger genetic requirement of the core module during oogenesis may therefore be explained through its interaction with TBP or its role in recruiting the enzymatic modules to the promoter. We propose the handyman principle, which posits that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present.

Author SummaryEmbryonic development critically relies on the differential expression of genes in different tissues. This involves the dynamic interplay between DNA, sequence-specific transcription factors, coactivators and chromatin remodelers which guide the transcription machinery to the appropriate promoters for productive transcription. To understand how this happens at the molecular level, we need to understand when and how coactivator complexes such as SAGA function. SAGA consists of multiple modules with well characterized enzymatic functions. This study shows that the non-enzymatic core module of SAGA is required for Drosophila oogenesis, while the enzymatic functions are largely dispensable. Despite this differential requirement, SAGA subunits appear to be broadly recruited to all promoter types, consistent with the biochemical integrity of the complex. These results suggest that genetic requirements and physical organization do not always go hand in hand.


title: GRAND: A database of gene regulatory network models across human conditions

abstract:
Gene regulation plays a fundamental role in shaping tissue identity, function, and response to perturbation. Regulatory processes are controlled by complex networks of interacting elements, including transcription factors, miRNAs and their target genes. The structure of these networks helps to determine phenotypes and can ultimately influence the development of disease or response to therapy. We developed GRAND (https://grand.networkmedicine.org) as a database for gene regulatory network models that can be compared between biological states, or used to predict which drugs produce changes in regulatory network structure. The database includes 12,468 genome-scale networks covering 36 human tissues, 28 cancers, 1,378 unperturbed cell lines, as well as 173,013 TF and gene targeting scores for 2,858 small molecule-induced cell line perturbation paired with phenotypic information. GRAND allows the networks to be queried using phenotypic information and visualized using a variety of interactive tools. In addition, it includes a web application that matches disease states to potentially therapeutic small molecule drugs using regulatory network properties.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Modeling gene regulation across human conditions integrates cancer tissues and cell lines, small molecules, and normal tissue networks.

C_FIG_DISPLAY


title: LD-CNV: rapid and simple discovery of chromosomal translocations using linkage disequilibrium between copy number variable loci

abstract: Large scale structural variations, such as chromosomal translocations, can have profound effects on fitness and phenotype, but are difficult to identify and characterize. Here, we describe a simple and effective method aimed at identifying translocations using only the dosage of sequence reads mapped on the reference genome. We binned reads on genomic segments sized according to sequencing coverage and identified instances when copy number segregated in populations. For each dosage-polymorphic 1Mb bin, we tested linkage disequilibrium with other variable bins. In nine potato (Solanum tuberosum) dihaploid families translocations affecting pericentromeric regions were common and in two cases were due to genomic misassembly. In two populations, we found evidence for translocation affecting euchromatic arms. In cv. PI 310467, a non-reciprocal translocation between chromosome 7 and 8 resulted in a 5-3 copy number change affecting several Mb at the respective chromosome tips. In cv. "Alca Tarma", the terminal arm of chromosome 4 translocated to the tip of chromosome 1. Using oligonucleotide-based fluorescent in situ hybridization painting probes (oligo-FISH), we tested and confirmed the predicted arrangement in PI 310467. In 192 natural accessions of Arabidopsis thaliana, dosage haplotypes tended to vary continuously and resulted in higher noise, but we identified pericentromeric LD suggesting the effect of repeats. This method should be useful in species where translocations are suspected because it tests linkage without the need for genotyping.


title: Fast alignment and preprocessing of chromatin profiles with Chromap

abstract: We present Chromap, an ultrafast method for aligning and preprocessing high throughput chromatin profiles. Chromap is comparable to BWA-MEM and Bowtie2 in alignment accuracy and is over 10 times faster than traditional workflows on bulk ChIP-seq / Hi-C profiles and than 10x Genomics CellRanger v2.0.0 pipeline on single-cell ATAC-seq profiles.

category: bioinformatics


title: Mechanism of CFTR correction by type I folding correctors

abstract: Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the {Delta}F508 mutant and are widely used to treat patients. However, their mechanism of action is unknown. Here we present cryo-electron microscopy structures of CFTR in complex with two FDA-approved correctors: lumacaftor and tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered {Delta}F508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its pre-mature degradation, and thereby allosterically rescue a large number of disease-causing mutations.


title: Synergistic information in the frontal cortex-striatal pathway

abstract: Across the cortico-basal ganglia circuit, the medial frontal cortex (MFC) communicates with the dorsal striatum (DS) during learning and planning. How these two brain regions communicate with each other is, however, not fully understood. Here we report the presence of synergistic information during information transfer across the frontal cortex-striatal pathway. Synergistic information emerges from the positive interaction of DS and MFC neurons and provides the DS with additional cortical information. This information is held latent in neuronal signals. To reveal it, we simultaneously record neuronal activities from the MFC and DS of rats trained on an outcome-based decision-making task and determined whether past neuronal activities of the DS positively influence communication rates. We detect a neuronal synergy that enables the MFC to boost its communication rate to the DS. Our results suggest that past neuronal activities of the DS help decode MFC signals. This ability is not attributed to the inherent autocorrelation of DS spiking activities.


title: Functional characterization of odor responses and gene expression changes in olfactory co-receptor mutants in Drosophila

abstract:
Two large families of olfactory receptors, the Odorant Receptors (ORs) and the Ionotropic Receptors (IRs), mediate responses to most odors in the insect olfactory system. Individual odor binding "tuning" OR receptors are expressed by olfactory neurons in basiconic and trichoid sensilla and require the co-receptor Orco to function. The situation for IRs is more complex. Different tuning IR receptors are expressed by olfactory neurons in coeloconic sensilla and rely on either the Ir25a or Ir8a co-receptors; some evidence suggests that Ir76b may also act as a coreceptor, but its function has not been systematically examined. This is particularly important as recent data indicate that nearly all coeloconic olfactory neurons co-express Ir25a, Ir8a, and Ir76b. Here, we report the effects of Drosophila olfactory co-receptor mutants on odor detection by coeloconic olfactory neurons and determine their broader impact on gene expression through RNASeq analysis. We demonstrate that Ir76b and Ir25a function together in all amine-sensing olfactory receptor neurons. In most neurons, loss of either co-receptor abolishes amine responses, whereas in ac1 sensilla, amine responses persist in the absence of Ir76b or Ir25a, but are lost in a double-mutant. Such responses do not require Ir8a. Conversely, acid-sensing ORNs require Ir8a, but not Ir76b or Ir25a. Using antennal transcriptional profiling, we find that the expression of acid-sensing IR receptors is significantly reduced in Ir8a mutants, but is unaffected by the loss of Ir25a or Ir76b. Similarly, select OR tuning receptors are also downregulated in Orco2 mutants. In contrast, expression of amine-sensing IR receptors is mostly unchanged in Ir25a and Ir76b mutants. Together, our data reveal new aspects of co-receptor function in the olfactory system.

SummaryInsect vectors of human disease rely on their sense of smell to locate humans for blood meals. A critical first step in olfaction is the odorant-induced activation of receptors on olfactory neurons. There are two major olfactory receptor families in insects, with each species having dozens of different odorant-binding "tuning" receptors. The receptor complexes also contain non-tuning co-receptors, which are highly conserved across insect species and are required for function. Here we characterize co-receptor mutants with electrophysiological recordings and transcriptome analysis in Drosophila. Our findings resolve the differential co-receptor dependence of olfactory neuron responses to volatile amines and acids. We also report changes in antennal gene expression that result from the absence of these co-receptors. Most notably, the absence of some co-receptors leads to a selective loss of transcript expression for the tuning olfactory receptors whose function depends on the missing co-receptors. Together our data provide new insight into the roles of co-receptors in insect olfaction.


title: Genomic islands targeting dusA in Vibrio species are distantly related to Salmonella Genomic Island 1 and mobilizable by IncC conjugative plasmids

abstract:
Salmonella Genomic Island 1 (SGI1) and its variants are significant contributors to the spread of antibiotic resistance among Gammaproteobacteria. All known SGI1 variants integrate at the 3 end of trmE, a gene coding for a tRNA modification enzyme. SGI1 variants are mobilized specifically by conjugative plasmids of the incompatibility groups A and C (IncA and IncC). Using a comparative genomics approach based on genes conserved among members of the SGI1 group, we identified diverse integrative elements distantly related to SGI1 in several species of Vibrio, Aeromonas, Salmonella, Pokkaliibacter, and Escherichia. Unlike SGI1, these elements target two alternative chromosomal loci, the 5 end of dusA and the 3 end of yicC. Although they share many features with SGI1, they lack antibiotic resistance genes and carry alternative integration/excision modules. Functional characterization of IMEVchUSA3, a dusA-specific integrative element, revealed promoters that respond to AcaCD, the master activator of IncC plasmid transfer genes. Quantitative PCR and mating assays confirmed that IMEVchUSA3 excises from the chromosome and is mobilized by an IncC helper plasmid from Vibrio cholerae to Escherichia coli. IMEVchUSA3 encodes the AcaC homolog SgaC that associates with AcaD to form a hybrid activator complex AcaD/SgaC essential for its excision and mobilization. We identified the dusA-specific recombination directionality factor RdfN required for the integrase-mediated excision of dusA-specific elements from the chromosome. Like xis in SGI1, rdfN is under the control of an AcaCD-responsive promoter. Although the integration of IMEVchUSA3 disrupts dusA, it provides a new promoter sequence and restores the reading frame of dusA for proper expression of the tRNA-dihydrouridine synthase A. Phylogenetic analysis of the conserved proteins encoded by SGI1-like elements targeting dusA, yicC, and trmE gives a fresh perspective on the possible origin of SGI1 and its variants.

Author summaryWe identified integrative elements distantly related to the Salmonella Genomic Island 1 (SGI1), a key vector of antibiotic resistance genes in Gammaproteobacteria. SGI1 and its variants reside at the 3 end of trmE, share a large, highly conserved core of genes, and carry a complex integron that confers multidrug resistance phenotypes to their hosts. Unlike members of the SGI1 group, these novel genomic islands target the 5 end dusA or the 3 end of yicC, lack multidrug resistance genes, and seem much more diverse. We showed here that, like SGI1, these elements are mobilized by conjugative plasmids of the IncC group. Based on comparative genomics and functional analyses, we propose a hypothetical model of the evolution of SGI1 and its siblings from the progenitor of IncA and IncC conjugative plasmids via an intermediate dusA-specific integrative element through gene losses and gain of alternative integration/excision modules.


title: Properties of Two-Locus Genealogies and Linkage Disequilibrium in Temporally Structured Samples

abstract: Archaeogenetics has been revolutionary, revealing insights into demographic history and recent positive selection in many organisms. However, most studies to date have ignored the non-random association of genetic variants at different loci (i.e., linkage disequilibrium, LD). This may be in part because basic properties of LD in samples from different times are still not well understood. Here, we derive several results for summary statistics of haplotypic variation under a model with time-stratified sampling: 1) The correlation between the number of pairwise differences observed between time-staggered samples ({pi}{Delta}t) in models with and without strict population continuity; 2) The product of the LD coefficient, D, between ancient and modern samples, which is a measure of haplotypic similarity between modern and ancient samples; and 3) The expected switch rate in the Li and Stephens haplotype copying model. The latter has implications for genotype imputation and phasing in ancient samples with modern reference panels. Overall, these results provide a characterization of how haplotype patterns are affected by sample age, recombination rates, and population sizes. We expect these results will help guide the interpretation and analysis of haplotype data from ancient and modern samples.


title: SLIMP: Supervised learning of metabolite-protein interactions from co-fractionation mass spectrometry data

abstract: Metabolite-protein interactions affect and shape diverse cellular processes. Yet, despite advances, approaches for identifying metabolite-protein interactions at a genome-wide scale are lacking. Here we present an approach termed SLIMP that predicts metabolite-protein interactions using supervised machine learning on features engineered from metabolic and proteomic profiles from a co-fractionation mass spectrometry-based technique. By applying SLIMP with gold standards, assembled from public databases, along with metabolic and proteomic data sets from multiple conditions and growth stages we predicted over 9,000 and 20,000 metabolite-protein interactions for Saccharomyces cerevisiae and Arabidopsis thaliana, respectively. Extensive comparative analyses corroborated the quality of the predictions from SLIMP with respect to widely-used performance measures (e.g. F1-score exceeding 0.8). SLIMP predicted novel targets of 2, 3 cyclic nucleotides and dipeptides, which we analysed comparatively between the two organisms. Finally, predicted interactions for the dipeptide Tyr-Asp in Arabidopsis and the dipeptide Ser-Leu in yeast were independently validated, opening the possibility for future applications of supervised machine learning approaches in this area of systems biology.

category: systems biology


title: Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software

abstract: Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously in a single experiment. Variant libraries targeting full-length ORFs typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a variant differs from the template ORF by a single codon variation, variant quantification presents the most profound challenge to this technology. Efforts such as dividing a library into sub-libraries for direct sequencing, or tag-directed subassembly are practical only for small ORFs. Our approach, however, features an enhanced variant-calling tool that processes massive shotgun sequencing data, allowing one to screen a single variant library for ORFs sized up to [~]3600 bases. The variant-calling tool detects variants reliably, and also presents variant annotations in datafiles enabling analyses that have reshaped our strategies governing library design, screen deconvolution, sequencing and sequencing data analysis.


title: Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software ASMv1.0


title: Claustral Projections to Anterior Cingulate Cortex Modulate Engagement with the External World

abstract: Engagement is a major determinant of performance. Hyper-engagement risks impulsivity and is fatiguing over time, while hypo-engagement could lead to missed opportunities. Even in sleep, when engagement levels are minimal, sensory responsiveness varies. Thus, maintaining an optimal engagement level with the environment is a fundamental cognitive ability. The claustrum, and in particular its reciprocal connectivity with executive regions in the frontal cortex, has been associated with salience, attention and sleep. These apparently disparate roles can be consolidated within the context of engagement. Here we describe the activity of claustro-frontal circuits in a task imposing a tradeoff between response inhibition and sensory acuity ( ENGAGE). Recording calcium fiber photometry during >80,000 trials, we characterize claustrum recruitment during salient behavioral events, and find that a moderate level of activity in claustro-cingulate projections defines optimal engagement. Low activity of this pathway is associated with impulsive actions, while high activity is associated with behavioral lapses. Chemogenetic activation of cingulate-projecting claustrum neurons suppressed impulsive behavior and reduced the engagement of mice in the task. This relationship became even clearer upon addressing individual variability in the strategy mice employed during the ENGAGE task. Furthermore, this association of claustrum activity and engagement extends into sleep. Using simultaneous EEG and photometry recordings in the claustrum, we find that cingulate projecting claustrum neurons are most active during deep unresponsive slow-wave sleep, when mice are less prone to awakening by sensory stimuli.


title: Cortical neuroprosthesis-mediated functional ipsilateral control of locomotion in rats with incomplete spinal cord injury

abstract: Control of voluntary limb movement is predominantly attributed to the contralateral motor cortex. Nevertheless, increasing evidence suggests the involvement of ipsilateral cortical networks in this process. Ipsilateral control particularly emerges in motor tasks requiring bilateral coordination, which is an essential characteristic of locomotion. Here, we combined a unilateral thoracic spinal cord injury (SCI) with a cortical neuroprosthetic intervention that uncovered a functional role of the ipsilateral cortex in rat movement. In all rats, after SCI, ipsilesional cortex excitation promoted a bilateral synergy, whereby the elevation of the contralateral foot was complemented by ipsilateral hindlimb extension. In two animals, we found that stimulation of a medial cortical sub-region modulated ipsilateral hindlimb flexion. Ipsilateral cortical stimulation delivered after SCI alleviated multiple locomotor and postural deficits. These results establish a causal link between cortical activation and a remarkably fine and proportional ipsilateral control of hindlimb movement, a potential target for future neuroprosthetic technology.


title: Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1

abstract: The actin motor protein myosin VI is a multivalent protein with diverse functions. Here, we identified and characterised a myosin VI ubiquitous interactor, the oral-facial-digital syndrome 1 (OFD1) protein, whose mutations cause malformations of the face, oral cavity, digits, and polycystic kidney disease. We found that myosin VI regulates the localisation of OFD1 at the centrioles and, as a consequence, the recruitment of the distal appendage protein cep164. Myosin VI depletion in non-tumoural cell lines causes an aberrant localisation of OFD1 along the centriolar walls, which is due to a reduction in the OFD1 mobile fraction. Finally, loss of myosin VI triggers a severe defect in ciliogenesis that could be causally linked to an impairment in the autophagic removal of OFD1 from satellites. Altogether, our results highlight an unprecedent layer of regulation of OFD1 and a pivotal role of myosin VI in coordinating the formation of the distal appendages and primary cilium with important implications for the genetic disorders known as ciliopathies.


title: Biogenesis of hepatitis B virus e antigen is driven by translocon-associated protein complex and regulated by conserved cysteines in signal peptide

abstract: Hepatitis B virus (HBV) uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. P22 can be retro-translocated back to the cytosol or enter the secretory pathway and undergo a second cleavage event, resulting in secreted p17 (HBe). Here, we report that translocation of p25 to the ER is promoted by translocon-associated protein complex (TRAP). We found that p25 is not completely translocated into the ER; a fraction of p25 is phosphorylated and remains in the cytoplasm and nucleus. Within the p25 sp sequence, we identified three cysteine residues that control the efficiency of sp cleavage and contribute to proper subcellular distribution of the precore pool.


title: Ovipositor and mouthparts in a fossil insect support a novel ecological role for early orthopterans in Pennsylvanian forests

abstract: Lobeattid insects represented a high portion of the earliest known, Pennsylvanian insect faunas. However, their systematic affinities and their role as foliage feeders which severely influenced their ecosystems remain debated. We investigated hundreds of samples of a new lobeattid species from the Xiaheyan locality using Reflectance Transforming Imaging combined with geometric morphometrics in order to assess its morphology, infer its ecological role, and phylogenetic position. Ctenoptilus frequens sp. nov. possessed a sword-shaped ovipositor whose valves interlocked by two ball-and-socket mechanisms. This unambiguously supports lobeattids as stem-relatives of all Orthoptera (crickets, grasshoppers, katydids). Given the herein presented and other remains, it follows that this group experienced an early diversification coupled with high levels of abundance. The ovipositor shape additionally indicates that ground was the preferred substrate for eggs. Visible mouthparts made it possible to assess the efficiency of the mandibular food uptake system in comparison to a wide array of recent species. The new species was omnivorous which explains the paucity of external damage on contemporaneous plant foliage.

category: evolutionary biology


title: Mendelian randomization suggests a bidirectional, causal relationship between physical inactivity and obesity

abstract: Physical inactivity is associated with excess weight gain in observational studies. However, some longitudinal studies indicate reverse causality where weight gain leads to physical inactivity. As observational studies suffer from reverse causality, it is challenging to assess the true causal directions. Here, we assess the bidirectional causality between physical inactivity and obesity by bidirectional Mendelian randomization analysis. We used results from genome-wide association studies for accelerometer-based physical activity and sedentary time in 91,105 individuals and for body mass index (BMI) in 806,834 individuals. We implemented Mendelian randomization using CAUSE method that accounts for pleiotropy and sample overlap using full genome-wide data. We also applied inverse variance-weighted, MR-Egger, weighted median, and weighted mode methods using genome-wide significant variants only. We found evidence of bidirectional causality between sedentary time and BMI: longer sedentary time was causally associated with higher BMI [beta (95%CI) from CAUSE method: 0.11 (0.02, 0.2), P=0.02], and higher BMI was causally associated with longer sedentary time (0.13 (0.08, 0.17), P=6.3.x10-4). Our analyses suggest that higher moderate and vigorous physical activity are causally associated with lower BMI (moderate: -0.18 (-0.3,-0.05), P=0.006; vigorous: -0.16 (-0.24,-0.08), P=3.8x10-4), but indicate that the association between higher BMI and lower levels of physical activity is due to horizontal pleiotropy. The bidirectional, causal relationship between sedentary time and BMI suggests that decreasing sedentary time is beneficial for weight management, but also that targeting obesity may lead to additional health benefits by reducing sedentary time.


title: Identification of essential genes in Caenorhabditis elegans through whole genome sequencing of legacy mutant collections

abstract: It has been estimated that 15-30% of the [~]20,000 genes in C. elegans are essential, yet many of these genes remain to be identified or characterized. With the goal of identifying unknown essential genes, we performed whole genome sequencing on complementation pairs from legacy collections of maternal-effect lethal and sterile mutants. This approach uncovered maternal genes required for embryonic development and genes with putative sperm-specific functions. In total, 58 essential genes were identified on chromosomes III, IV, and V, of which 49 genes are represented by novel alleles in this collection. Of these 49 genes, 19 (40 alleles) were selected for further functional characterization. The terminal phenotypes of embryos were examined, revealing defects in cell division, morphogenesis, and osmotic integrity of the eggshell. Mating assays with wild-type males revealed previously unknown male-expressed genes required for fertilization and embryonic development. The result of this study is a catalogue of mutant alleles in essential genes that will serve as a resource to guide further study toward a more complete understanding of this important model organism. As many genes and developmental pathways in C. elegans are conserved and essential genes are often linked to human disease, uncovering the function of these genes may also provide insight to further our understanding of human biology.


title: A dose-sensitive OGT-TET3 complex is necessary for normal Xist RNA distribution and function

abstract: Female (XX) mouse embryonic stem cells (mESCs) differ from their male (XY) counterparts because they have lower levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). This difference in DNA modifications is a result of having two X chromosomes (Xs), both of which are active at this developmental stage. We identified an X-linked gene, Ogt, that controls levels of 5mC and 5hmC in mESCs. OGT is a post-translational modification enzyme and we identified the 5-methylcytosine dioxygenase TET3 as an OGT target that is differentially modified in XX and XY mESCs. In addition to influencing 5mC and 5hmC abundance, OGT dose also controls TET3 and OGT distribution. OGT and TET3 are predominantly nuclear in XX mESCs and cytoplasmic in XY mESCs. Furthermore, these proteins are present in different complexes in XX and XY mESCs. Mutational analysis revealed that TET3 determines the XX-specific abundance of 5mC and 5hmC in mESCs. While TET3 null XX mESCs exhibited modest changes in gene expression, there were substantial alterations upon differentiation into epiblast-like cells (mEpiLCs). In addition, these TET3 null XX mESCs did not undergo X-chromosome inactivation (XCI) when differentiated. These data suggest that an X-dose sensitive complex containing OGT and TET3 regulates cytosine modifications and XCI.


title: Understanding the mechanism of monoADP ribosylation in OsSRT1 and its linkage to the DNA repair system under stress conditions.

abstract:
The role of sirtuins in plants are slowly unraveling. There are only reports of H3K9Ac deacetylation by OsSRT1. Here our studies shade light on its dual enzyme capability with preference for mono ADP ribosylation over deacetylation. OsSRT1 can specifically transfer the single ADP ribose group on its substrates in an enzymatic manner. This mono ADPr effect is not well known in plants, more so for deacetylases. The products of this reaction (NAM and ADP ribose) have immense negative effect on this enzyme suggesting a tighter regulation. Resveratrol, a natural plant polyphenol proves to be a strong activator of this enzyme at 150 M concentration. Under different abiotic stress conditions, we could link this ADP ribosylase activity to the DNA repair pathway by activating the enzyme PARP1. Metal stress in plants also influences these enzyme activities.

HighlightsO_LIOsSRT1 can transfer a single moiety of ADP-ribose on itself as well as other nuclear proteins like histones H3 and H2A.
C_LIO_LINAM, ADP-ribose and certain metal ions negatively regulate this ADP-ribose transfer.
C_LIO_LIADPr of OsPARP1 and OsPARP2 links OsSRT1 to DNA damage repair pathways.
C_LIO_LIOsSRT1 positively regulates the activity of OsPARP1 by ADP ribosylating it.
C_LIO_LIOn plants exposure to H2O2 (oxidative stress) and Arsenic toxicity, there is a link between the increased activity of the players of DNA damage repair system and overexpression of OsSRT1.
C_LI


title: Understanding the mechanism of monoADP ribosylation in OsSRT1 and its linkage to the DNA repair system under stress conditions


title: Supervised Capacity Preserving Mapping: A Clustering Guided Visualization Method for scRNAseq data

abstract: The rapid development of scRNA-seq technologies enables us to explore the transcriptome at the cell level in a large scale. Recently, various computational methods have been developed to analyze the scR-NAseq data such as clustering and visualization. However, current visualization methods including t-SNE and UMAP are challenged by the limited accuracy of rendering the geometic relationship of populations with distinct functional states. Most visualization methods are unsupervised, leaving out information from the clustering results or given labels. This leads to the inaccurate depiction of the distances between the bona fide functional states and the variance of clusters. We present supCPM, a robust supervised visualization method, which separates different clusters, preserves global structure, and tracks the cluster variance. Compared with six visualization methods using synthetic and real data sets, supCPM shows improved performance than other methods in preserving the global geometric structure and data variance. Overall, supCPM provides an enhanced visualization pipeline to assist the interpretation of functional transition and accurately depict population segregation.


title: Recovery of conformational continuum from single-particle cryo-EM data: Optimization of ManifoldEM informed by ground-truth studies

abstract: This work is based on the manifold-embedding approach to study biological molecules exhibiting continuous conformational changes. Previous work established a method capable of reconstructing 3D movies and accompanying energetics of atomic-level structures from single-particle cryo-EM images of macromolecules displaying multiple conformational degrees of freedom. Here, we introduce an unsupervised geometric machine learning approach that is informed by detailed heuristic analysis of manifolds formed by simulated heterogeneous cryo-EM datasets generated from an atomic structure. These simulated data were generated with increasing complexity to account for multiple conformational motions, state occupancies and typical microscope parameters in a wide range of signal-to-noise ratios. Using these datasets as ground-truth, we provide detailed exposition of our findings using several conformational motions while exploring the available parameter space. Guided by these insights, we build a framework to leverage the high-dimensional geometric information obtained towards reconstituting a quasi-continuum of conformational states in the form of a free-energy landscape and respective 3D density maps for all states therein. As shown by a direct comparison of results, this framework offers substantial improvements relative to the previous work.


title: Geometric machine learning informed by ground truth: Recovery of conformational continuum from single-particle cryo-EM data of biomolecules


title: Pathway-based approach reveals differential sensitivity of glioblastoma to E2F1 inhibition

abstract: Targeting glioblastoma (GBM) based on molecular subtyping have not yet translated into successful therapies. Here, we used gene set enrichment analysis (GSEA) to conduct an unsupervised clustering analysis to condense the gene expression data from bulk patient samples and patient-derived gliomasphere lines into new gene lists. We then identified key molecular pathways differentially regulated between tumors. These gene lists associated not only with cell cycle and stemness signatures, but also with cell-type specific markers and different cellular states of GBM. We identified the transcription factor E2F1 as a key regulator of tumor cell proliferation and self-renewal in only the subset of proliferating gliomasphere cultures predicted to be E2F1-activated and validated its functional significance in tumor formation capacity. E2F1 inhibition also sensitized E2F1-activated gliomasphere cultures to radiation treatment. Our findings indicate that a pathway-based approach can be leveraged to deconstruct inter-tumoral heterogeneity and uncover key therapeutic vulnerabilities for targeting GBM.


title: Immune-evasion of KRAS-mutant lung adenocarcinoma mediated by cAMP response element-binding protein

abstract: cAMP response element-binding protein (CREB) mediates proliferative and inflammatory gene transcription in neurodegeneration and cancer, but its role in malignant immune-evasion of lung adenocarcinoma (LUAD) is unknown. We show that human LUAD of smokers are frequently altered along the CREB pathway and we employ mouse models to discover that KRAS-mutant LUAD co- opt CREB to evade immune rejection by tumoricidal neutrophils. For this, KRAS- driven CREB activation suppresses CXC-chemokine expression and prevents recruitment of CXCR1+ neutrophils. CREB1 is shown to be pro-tumorigenic in five different LUAD models, a function that is dependent on host CXCR1. Pharmacologic CREB blockade prevents tumor growth and restores neutrophil recruitment only when initiated before immune-evasion of KRAS-mutant LUAD. CREB and CXCR1 expression in human LUAD are compartmentalized to tumor and stromal cells, respectively, while CREB-regulated genes and neutrophils impact survival. In summary, CREB-mediated immune evasion of KRAS-mutant LUAD relies on signaling to neutrophil CXCR1 and is actionable.


title: IKZF3 deficiency potentiates chimeric antigen receptor T cells targeting solid tumors

abstract: Chimeric antigen receptor (CAR) T cell therapy has been successful in treating hematological malignancy, but solid tumors remain refractory. Here, we demonstrated that knocking out transcription factor IKZF3 in HER2-specific CAR T cells targeting breast cancer cells did not affect proliferation or differentiation of the CAR T cells in the absence of tumors, but markedly enhanced killing of the cancer cells in vitro and in a xenograft model. Furthermore, IKZF3 KO had similar effects on the CD133-specific CAR T cells targeting glioblastoma cells. AlphaLISA and RNA-seq analyses indicate that IKZF3 KO increased the expression of genes involved in cytokine signaling, chemotaxis and cytotoxicity. Our results suggest a general strategy for enhancing CAR T efficacy on solid tumors.


title: Requirement for an Otopetrin-Like protein for acid taste in Drosophila

abstract: Many of the Drosophila receptors required for bitter, sugar and other tastes have been identified. However, the receptor required for the taste of acid has been elusive. In Drosophila, the major families of taste receptors, such as "Gustatory Receptors" and "Ionotropic Receptors" are unrelated to taste receptors in mammals. Previous work indicated that members of these major families do not appear to be broadly required acid sensors. Here, to identify the enigmatic acid taste receptor, we interrogated three genes encoding proteins distantly related the mammalian Otopertrin1 proton channel. We found that RNAi knockdown or mutation of Otopetrin-Like A (OtopLA) by CRISPR/Cas9, severely impairs the behavioral rejection of sugary foods laced with HCl or carboxylic acids. Mutation of OtopLA also greatly reduces acid-induced action potentials. We identified an isoform of OtopLA that was expressed in the proboscis and was sufficient to restore acid sensitivity to OtopLA mutant flies. OtopLA functioned in acid taste in a subset of bitter-activated gustatory receptor neurons that senses protons. This work highlights an unusual functional conservation of a receptor required for a taste modality in flies and mammals.


title: Communication between the mediodorsal thalamus and prelimbic cortex regulates timing performance in rats

abstract: Predicting when future events will occur and adjusting behavior accordingly is critical to adaptive behavior. Despite this, little is known about the brain networks that encode time and how this ultimately impacts decision-making. One established finding is that the prefrontal cortex (PFC) and its non-human analogues (e.g., the rodent prelimbic cortex; PL) mediate timing. This provides a starting point for exploring the networks that support temporal processing by identifying areas that interact with the PFC during timing tasks. For example, substantial work has explored the role of frontostriatal circuits in timing. However, other areas are undoubtedly involved. The mediodorsal nucleus of the thalamus (MD) is an excellent candidate region. It shares dense, reciprocal connections with PFC-areas in both humans and non-human species and is implicated in cognition. However, causal data implicating MD-PFC interactions in cognition broadly is still sparse, and their role in timing specifically is currently unknown. To address this, we trained male rats on a time-based, decision-making task referred to as the  peak-inter- val procedure. During the task, presentation of a cue instructed the rats to respond after a specific interval of time elapsed (e.g., tone-8 seconds). We incorporated two cues; each requiring a response after a distinct time-interval (e.g., tone-8 seconds / light-16 seconds). We tested the effects of either reversibly inactivating the MD or PL individually or functionally disconnecting them on performance. All manipulations caused a comparable timing deficit. Specifically, responses showed little organization in time, as if primarily guided by motivational systems. These data expand our understanding of the networks that support timing and suggest that MD-PL interactions specifically are a core component. More broadly, our results suggest that timing tasks provide a reliable assay for characterizing the role of MD-PL interactions in cognition using rodents, which has been difficult to establish in the past.


title: SARS-CoV-2 mRNA Vaccine Induces Robust Specific and Cross-reactive IgG and Unequal Strain-specific Neutralizing Antibodies in Naïve and Previously Infected Recipients

abstract: With the advance of SARS-CoV-2 vaccines, the outlook for overcoming the global COVID-19 pandemic has improved. However, understanding of immunity and protection offered by the SARS-CoV-2 vaccines against circulating variants of concern (VOC) is rapidly evolving. We investigated the mRNA vaccine-induced antibody responses against the referent WIV04 (Wuhan) strain, circulating variants, and human endemic coronaviruses in 168 naive and previously infected people at three-time points. Samples were collected prior to vaccination, after the first and after the second doses of one of the two available mRNA-based vaccines. After full vaccination, both naive and previously infected participants developed comparable robust SARS-CoV-2 specific spike IgG levels, modest IgM and IgA binding antibodies, and varying degrees of HCoV cross-reactive antibodies. However, the strength and frequency of neutralizing antibodies produced in naive people were significantly lower than in the previously infected group. We also found that 1/3rd of previously infected people had undetectable neutralizing antibodies after the first vaccine dose; 40% of this group developed neutralizing antibodies after the second dose. In all subjects neutralizing antibodies produced against the B.1.351 and P.1 variants were weaker than those produced against the reference and B.1.1.7 strains. Our findings provide support for future booster vaccinations modified to be active against the circulating variants.


title: Measles fusion complexes from central nervous system clinical isolates: decreased interaction between hemagglutinin and fusion proteins

abstract:
Measles virus (MeV) viral entry is mediated by a fusion complex comprised of a receptor binding protein (hemagglutinin, H) and a fusion protein (F). The wild type H/F complex requires interaction with specific proteinaceous receptors (CD150/SLAM and nectin-4) in order to be activated. In contrast the H/F complexes isolated from viruses infecting the central nervous system (CNS) do not require a specific receptor. A single amino acid change in the F protein (L454W) was previously identified in two patients with lethal sequelae of MeV CNS infection, and the F bearing this mutation mediates fusion even without the H protein. We show here that viruses bearing the L454W fusion complex are less efficient than wt virus at targeting receptor expressing cells and that this defect is associated with a decreased interaction between the H and the F proteins.

ImportanceMeasles (Mev) infection can cause serious complications including measles inclusion body encephalitis (MIBE) and subacute sclerosing panencephalitis (SSPE). MIBE and SSPE are relatively rare but lethal. We have shown that the fusion complex of CNS adapted clinical samples can spread in the absence of known receptor. We now provide evidence that HRC mutations leading to CNS adaptation come at a cost to the efficiency of viral entry.

One Sentence SummaryMeasles CNS adapted fusion complexes have altered H/F interaction.


title: Validation of selective agars for detection and quantification of Escherichia coli resistant to critically important antimicrobials

abstract:
Success in the global fight against antimicrobial resistance (AMR) is likely to improve if surveillance can be performed more rapidly, affordably and on a larger scale. An approach based on robotics and agars incorporated with antimicrobials has enormous potential to achieve this. However, there is a need to identify the combinations of selective agars and key antimicrobials yielding the most accurate counts of susceptible and resistant organisms. A series of designed experiments involving 1,202 plates identified the best candidate-combinations from six commercially available agars and five antimicrobials using 18 Escherichia coli strains as either pure cultures or inoculums within faeces. The effect of various design factors on colony counts were analysed in generalised linear models. Without antimicrobials, Brilliance E. coli (Brilliance) and CHROMagar ECC (CHROMagar) agars yielded 28.9% and 23.5% more colonies than MacConkey agar. The order of superiority of agars remained unchanged when faecal samples with and without spiking of resistant E. coli were inoculated onto agars with or without specific antimicrobials. When incorporating antimicrobials at varying concentrations, it was revealed that ampicillin, tetracycline and ciprofloxacin are suitable for incorporation into Brilliance and CHROMagar agars at all defined concentrations. Gentamicin was only suitable for incorporation at 8 and 16 g/mL while ceftiofur was only suitable at 1 g/mL. CHROMagar ESBL agar supported growth of a wider diversity of extended-spectrum cephalosporin-resistant E. coli. The findings demonstrate the potential for combining robotics with agars to deliver AMR surveillance on a vast scale with greater sensitivity of detection and strategic relevance.

IMPORTANCEEstablished models of surveillance for AMR in livestock typically have a low sampling intensity which creates a tremendous barrier to understanding the variation of resistance amongst animal and food enterprises. However, developments in laboratory robotics now make it possible to rapidly and affordably process high volumes of samples. Combined with modern selective agars incorporating antimicrobials, this forms the basis of a novel surveillance process for identifying resistant bacteria by chromogenic reaction including accurately detecting and quantifying their presence even when present at low concentration. As Escherichia coli is a widely preferred indicator bacterium for AMR surveillance, this study identifies the optimal selective agar for quantifying resistant E. coli by assessing the growth performance on agars with antimicrobials. The findings are the first step towards exploiting laboratory robotics in an up-scaled approach to AMR surveillance in livestock with wider adaptations in food, clinical microbiology and public health.


title: Interplay between bacterial clone and plasmid in the spread of antibiotic resistance genes in the gut: lessons from a temporal study in veal calves

abstract: Intestinal carriage of extended spectrum {beta}-lactamase (ESBL)-producing Escherichia coli is a frequent, increasing and worrying phenomenon, but little is known about the molecular scenario and the evolutionary forces at play. We screened 45 veal calves, known to have high prevalence of carriage, for ESBL-producing E. coli on 514 rectal swabs (one randomly selected colony per sample) collected over six months. We characterized the bacterial clones and plasmids carrying blaESBL genes with a combination of genotyping methods, whole genome sequencing and conjugation assays. One hundred and seventy-three ESBL-producing E. coli isolates [blaCTX-M-1 (64.7%), blaCTX-M-14 (33.5%) or blaCTX-M-15 (1.8%)] were detected, belonging to 32 bacterial clones, mostly of phylogroup A. Calves were colonized successively by different clones with a trend in decreasing carriage. The persistence of a clone in a farm was significantly associated with the number of calves colonized. Despite a high diversity of E. coli clones and blaCTX-M-carrying plasmids, few blaCTX-M gene/plasmid/chromosomal background combinations dominated, due to (i) efficient colonization of bacterial clones and/or (ii) successful plasmid spread in various bacterial clones. The scenario  clone vs. plasmid spread depended on the farm. Thus, epistatic interactions between resistance genes, plasmids and bacterial clones contribute to optimize fitness in specific environments.