title: Metagenomic profiling and transfer dynamics of antibiotic resistance determinants in a full-scale granular sludge wastewater treatment plant

abstract:
In the One Health context, wastewater treatment plants (WWTPs) are central to safeguard water resources. Nonetheless, many questions remain about their effectiveness to prevent the dissemination of antimicrobial resistance (AMR). Most surveillance studies monitor the levels and removal of selected antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in intracellular DNA (iDNA) extracted from WWTP influents and effluents. The role of extracellular free DNA (exDNA) in wastewater is mostly overlooked. In this study, we analyzed the transfer of ARGs and MGEs in a full-scale Nereda(R) reactor removing nutrients with aerobic granular sludge. We tracked the composition and fate of the iDNA and exDNA pools of influent, sludge, and effluent samples. Metagenomics was used to profile the microbiome, resistome, and mobilome signatures of iDNA and exDNA extracts. Selected ARGs and MGEs were analyzed by qPCR. From 2,840 ARGs identified, the genes arr-3 (2%), tetC (1.6%), sul1 (1.5%), oqxB (1.2%), and aph(3")-Ib (1.2%) were the most abundant among all sampling points and bioaggregates. Pseudomonas, Acinetobacter, Aeromonas, Acidovorax, Rhodoferax, and Streptomyces populations were the main hosts of ARGs in the sludge. In the effluent, 478 resistance determinants were detected, of which 89% from exDNA potentially released by cell lysis during aeration in the reactor. MGEs and multiple ARGs were co-localized on the same extracellular genetic contigs. These can pose a risk for AMR dissemination by transformation into microorganisms of receiving water bodies. Total intracellular ARGs decreased 3-42% as a result of wastewater treatment. However, the ermB and sul1 genes increased by 2 and 1 log gene copies mL-1, respectively, in exDNA from influent to effluent. The exDNA fractions need to be considered in AMR surveillance, risk assessment, and mitigation.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/482492v1_ufig1.gif" ALT="Figure 1">
View larger version (39K):
org.highwire.dtl.DTLVardef@1a5eedcorg.highwire.dtl.DTLVardef@e4da78org.highwire.dtl.DTLVardef@c324bforg.highwire.dtl.DTLVardef@1a18138_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIA DNA database from an AGS reactor was constructed to study the system resistome, mobilome, and microbiome.
C_LIO_LIThe genera Pseudomonas and Rhodoferax were the predominant ARG carriers in the system.
C_LIO_LIMGEs and ARGs often co-localize on contigs recovered from the exDNA of the effluent.
C_LIO_LIAGS plants are efficient at reducing ARB.
C_LIO_LIThe exDNA is an underestimated DNA fraction containing ARGs in the effluent.
C_LI


title: Bioinformatics and next generation sequencing data analysis to identify key genes and pathways influencing in Parkinson's disease

abstract: Parkinsons disease (PD) is the most commonly diagnosed neurodegenerative disorder Identification of novel prognostic and pathogenesis biomarkers plays a pivotal role in the management of the PD. Next generation sequencing (NGS) dataset from the GEO (Gene Expression Omnibus) database were used to identify differentially expressed genes (DEGs) in PD. Gene Ontology (GO) and REACTOME pathway enrichmental analyses were performed to elucidate the functional roles of the DEGs. Protein-protein interaction (PPI), modules, miRNA-hub gene regulatory network and TF-hub gene regulatory network were established. The receiver operating characteristic curve (ROC) analysis was used to explore the diagnostic values of hub genes in PD. In total, 957 DEGs were identified, of which 478 were up regulated genes and 479 were down regulated genes. GO and pathway enrichment analysis results revealed that the up regulated genes were mainly enriched in nervous system development, cell junction, transporter activity and neuronal system, whereas down regulated genes were mainly enriched in response to stimulus, cell periphery, identical protein binding and immune system. The top hub genes in the constructed PPI network, modules, miRNA-hub gene regulatory network and TF-hub gene regulatory network were OTUB1, PPP2R1A, AP2M1, PIN1, USP11, CDK2, IQGAP1, NEDD4, VIM and CDK1. Furthermore, ROC analysis showed that hub genes were having good diagnostic values. We identified a series of essential genes along with the pathways that were most closely related with PD initiation and progression. Our results provide a more detailed molecular mechanism for the advancement of PD, shedding light on the potential biomarkers and therapeutic targets.

category: bioinformatics


title: A quantitative method for sizing hyaluronan and other glycosaminoglycans on surfaces

abstract: Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift ({Delta}D) and the negative frequency shift (-{Delta}f) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from [~]2 kDa tetrasaccharides to [~]1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the {Delta}D/-{Delta}f ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.

category: biophysics


title: A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces


title: Transmembrane protein KIRREL1 regulates Hippo signaling via a feedback loop and represents a potential therapeutic target in YAP/TAZ-active cancers

abstract: Dysregulation of the Hippo tumor suppressor pathway and hyperactivation of YAP/TAZ are frequently observed in human cancers and represent promising therapeutic targets. However, strategies targeting the mammalian Hippo pathway are limited due to the lack of a well-established cell surface regulator. By combining protein interactome data and clinical data, we have identified transmembrane protein KIRREL1 as an upstream regulator of the Hippo pathway. KIRREL1 interacts with Hippo pathway components SAV1 and LATS1/2 via its intracellular C-terminal domain and promotes LATS1/2 activation by MST1/2 (Hippo kinases), in turn inhibiting YAP/TAZ activity and target gene expression. Conversely, YAP/TAZ directly induce the expression of KIRREL1 in a TEAD1-4-dependent manner. In mouse liver tumors driven by YAP activation, KIRREL1 protein is robustly induced. Moreover, KIRREL1 expression positively correlates with canonical YAP/TAZ target gene expression in clinical tumor specimens and predicts poor prognosis. Finally, transgenic expression of KIRREL1 effectively blocked tumorigenesis in a mouse intrahepatic cholangiocarcinoma model, suggesting an important role of KIRREL1 in inhibiting cancer development. Together, these findings indicate that KIRREL1 constitutes a negative feedback mechanism regulating the Hippo pathway, and serves as a cell surface marker and potential drug target in cancers with YAP/TAZ dependency.

category: cancer biology


title: STING activation promotes robust immune response and tumor regression in glioblastoma models

abstract:
Immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease. However, the aggressive brain tumor glioblastoma (GBM) is highly immunosuppressive and remains largely refractory to current immunotherapeutic approaches. The cGAS-STING cytoplasmic double stranded DNA (dsDNA) sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties.

Here, we investigated the status of the STING pathway in GBM and the modulation of the brain tumor microenvironment (TME) with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature. We show that human GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils and NK populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100-loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. Responses to treatment were abolished by NK cell depletion. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants the further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, CAR T cells, NK therapies or immune checkpoint blockade.

Significance statementModulation of the immune microenvironment is critical for immunosuppressive and therapy refractory tumors like glioblastoma. Activation of the STING pathway deeply remodels the brain tumor environment and attracts innate immune cells and natural killer cell populations, producing a robust antitumor effect with long-term immune memory. We further show that human glioblastoma tissue can respond to the therapy and lay the foundations for combined intracranial immunotherapies by using crosslinked biodegradable brain implants.


title: STING activation promotes robust immune response and NK cell-mediated tumor regression in glioblastoma models


title: In utero exposure to morphine leads to sex-specific behavioral alterations that persist into adulthood in cross-fostered mice

abstract:
IntroductionThe opioid epidemic has seen an increase in drug use among women of reproductive age. It is well established that Opioid Use Disorder (OUD) can have many negative consequences for the health of mothers and their babies, both during pregnancy and after delivery, but our understanding of the impact of fetal opioid exposure on behavior during adolescence and adulthood is less understood. Preclinical studies have unveiled some of the long-term effects of in utero morphine exposure primarily using injections as the route of drug delivery. Our study utilized a model for oral, voluntary morphine self-administration to investigate neonate, adolescent, and adult offsprings behavioral phenotypes and subsequent ethanol misuse liability.

MethodsWe first validated a paradigm for maternal oral intake of morphine, where female mice became morphine dependent pre-pregnancy, and continued to voluntarily consume morphine in the continuous two-bottle choice (C2BC) paradigm during pregnancy and up to offspring postnatal day 7 (PND 7). Offspring were cross-fostered to a drug-naive dam at PND 7, to model first and second trimester in utero exposure in humans and to mimic the stress associated with NOWS. Bodyweight and ultrasonic vocalizations were assessed to determine alterations in the neonates. Offspring from control and morphine-exposed dams were then tested during adolescence and adulthood in a battery of behavioral tests to assess baseline behavioral phenotypes. We also computed a global behavioral score (GBS) to integrate offsprings multiple behavioral outcomes into a composite score that could be used to identify potential vulnerable and resilient populations in offspring exposed prenatally to morphine. Offspring that were tested during adolescence were also evaluated during adulthood in the ethanol intermittent 2BC to assess ethanol misuse risk.

ResultsUsing an oral maternal morphine C2BC protocol, we demonstrated that morphine dams display signs of dependence, measured by somatic signs during withdrawal, and voluntarily drink morphine throughout gestation. Neonate cross-fostered offspring display changes in spontaneous activity, body weight, and ultrasonic vocalization parameters. During adolescence, offspring display both increased baseline anxiety-like/compulsive-like behavior, while in adulthood they display increased anxiety-like behavior. No changes were found for baseline physical signs, locomotion, and depressive-like behavior during adolescence or adulthood. In addition, a greater percentage of adult male offspring exposed to maternal morphine fell into moderate and high GBS classifications, signaling a more severe behavioral phenotype, compared to male control offspring. These effects were not observed in adult female offspring exposed to morphine in utero. Additionally, male adult offspring exposed to maternal morphine reduced their 2-hour ethanol intake in the intermittent two-bottle choice (I2BC) paradigm, although no changes in 24-hour ethanol intake and preference were found. No changes were observed in female offspring of morphine-exposed dams.

ConclusionOverall, maternal morphine exposure leads to sex-specific changes in neonate, adolescent, and adult behavior, including ethanol intake.

category: pharmacology and toxicology


title: Orthogonal synthetases for polyketide precursors

abstract: The absence of orthogonal aminoacyl-tRNA synthetases that accept non-L--amino acids is the primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers. Here we report PylRS enzymes that accept -hydroxy acids, -thio acids, N-formyl-L--amino acids, and -carboxyl acid monomers (malonic acids) that are formally precursors to polyketide natural products. These monomers are all accommodated and accepted by the translation apparatus in vitro. High-resolution structural analysis of the complex between one such PylRS enzyme and a meta-substituted 2-benzylmalonate derivative reveals an active site that discriminates pro-chiral carboxylates and accommodates the large size and distinct electrostatics of an -carboxyl acid substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose -substituent diverges significantly from the -amine embodied in proteinogenic amino acids. These enzymes could act in synergy with natural or evolved ribosomes to generate diverse sequence-defined non-protein hetero-oligomers.

category: synthetic biology


title: Sleep and circadian rhythm disruption alters the lung transcriptome to predispose to viral infection

abstract: Sleep and circadian rhythm disruption (SCRD), as encountered during shift work, increases the risk of respiratory viral infection including SARS-CoV-2. However, the mechanism(s) underpinning higher rates of respiratory viral infection following SCRD remain poorly characterised. To address this, we investigated the effects of acute sleep deprivation on the mouse lung transcriptome. Here we show that sleep deprivation profoundly alters the transcriptional landscape of the lung, causing the suppression of both innate and adaptive immune systems, disrupting the circadian clock, and activating genes implicated in SARS-CoV-2 replication, thereby generating a lung environment that promotes viral infection and associated disease pathogenesis. Our study provides a mechanistic explanation of how SCRD increases the risk of respiratory viral infections including SARS-CoV-2 and highlights therapeutic avenues for the prevention and treatment of COVID-19.

category: neuroscience


title: A single episode of CMV reactivation initiates long-term expansion and differentiation of the NK cell repertoire

abstract:
NK cells play an important role in suppression of viral replication and are critical for effective control of persistent infections such as herpesviruses. Cytomegalovirus infection is associated with expansion of  adaptive-memory NK cells with a characteristic CD16brightCD56dim NKG2C+ phenotype but the mechanisms by which this population is maintained remain uncertain. We studied NK cell reconstitution in patients undergoing haemopoietic stem cell transplantation and related this to CMV reactivation. NK cells expanded in the early post-transplant period but then remained stable in the absence of viral reactivation. However, a single episode of CMV reactivation led to a rapid and sustained 10-fold increase in NK cell number. NKG2C expression was increased on all NK subsets although the kinetics of expansion peaked at 6 months on immature CD56bright cells whilst continuing to rise on the mature CD56dim pool. Phenotypic maturation was observed by acquisition of CD57 and KIR expression. Transplantation from CMV-seropositive donors was associated with more rapid NK expansion and superior control of viral reactivation. Effective control of viral reactivation was seen when the peripheral NK cell count reached 20,000/ml. These data show that a single episode of CMV reactivation acts to reprogramme hemopoiesis to drive a sustained modulation and expansion of the NK cell pool and reveal further insight into long term regulation of the innate immune repertoire by infectious challenge.

Author Summaryim to highlight where your work fits within a broader context; present the significance or possible implications of your work simply and objectively; and avoid the use of acronyms and complex terminology wherever possible. The goal is to make your findings accessible to a wide audience that includes both scientists and non-scientists

Innate immune cells respond rapidly to infectious challenge and it has previously been thought that they were unable to  learn from specific infecitons but provided only short term control. However, in recent years it has become apparent that some subsets of innate cells, termed adaptive natural killer (NK) cells, are permanently increased in people who have become infected with cytomegalovirus. The mechanism by which this is maintained is not known. We studied the kinetics of adaptive NK cell expansion in patients who had recently undergone stem cell (bone marrow) transplantation as CMV very often  reactivates in this setting and allows study of how this affects the immune system. We find that one short term episode of viral replication led to a large and prolonged expansion of adaptive NK cells. Indeed, their levls increasd 11-fold over 10 months.

category: immunology


title: Isolation of a potently neutralizing and protective human monoclonal antibody targeting yellow fever virus

abstract: Yellow fever virus (YFV) causes sporadic outbreaks of infection in South America and sub-Saharan Africa. While live-attenuated yellow fever virus vaccines based on three substrains of 17D are considered some of the most effective vaccines in use, problems with production and distribution have created large populations of unvaccinated, vulnerable individuals in endemic areas. To date, specific antiviral therapeutics have not been licensed for human use against YFV or any other related flavivirus. Recent advances in monoclonal antibody (mAb) technology have allowed for identification of numerous candidate therapeutics targeting highly pathogenic viruses, including many flaviviruses. Here, we sought to identify a highly neutralizing antibody targeting YFV envelope (E) protein as a therapeutic candidate. We used human B cell hybridoma technology to isolate mAbs from the circulating memory B cells from human YFV vaccine recipients. These antibodies bound to recombinant YFV E protein and recognized at least five major antigenic sites on E. Two mAbs (designated YFV-136 and YFV-121) recognized a shared antigenic site and neutralized the YFV 17D vaccine strain in vitro. YFV-136 also potently inhibited infection by multiple wild-type YFV strains, in part, at a post-attachment step in the virus replication cycle. YFV-136 showed therapeutic protection in two animal models of YFV challenge including hamsters and immunocompromised mice engrafted with human hepatocytes. These studies define features of the antigenic landscape on YFV E protein recognized by the human B cell response and identify a therapeutic antibody candidate that inhibits infection and disease caused by highly virulent strains of YFV.


title: dSeqSb: A systems biology approach to decipher dynamics of 1 host-pathogen interactions using temporal dual RNA-seq data

abstract: Infection triggers a dynamic cascade of reciprocal events between host and pathogen wherein the host activates complex mechanisms to recognise and kill pathogens while the pathogen adjusts its virulence and fitness to avoid eradication by the host. The interaction between the pathogen and the host results in large-scale changes in gene expression in both organisms. Dual RNA-seq, the simultaneous detection of host and pathogen transcripts, has become a leading approach to unravel complex molecular interactions between the host and the pathogen and is particularly informative for intracellular organisms. The amount of in vitro and in vivo dual RNA-seq data is rapidly growing which demands computational pipelines to effectively analyse such data. In particular, holistic, systems-level, and temporal analyses of dual RNA-seq data are essential to enable further insights into the host-pathogen transcriptional dynamics and potential interactions. Here, we developed an integrative network-driven bioinformatics pipeline, dRNASb, a systems biology-based computational pipeline to analyse temporal transcriptional clusters, incorporate molecular interaction networks (e.g., protein-protein interactions), identify topologically and functionally key transcripts in host and pathogen, and associate host and pathogen temporal transcriptome to decipher potential between-species interactions. The pipeline is applicable to various dual RNA-seq data from different species and experimental conditions. As a case study, we applied dRNASb to analyse temporal dual RNA-seq data of Salmonella-infected human cells, which enabled us to uncover genes contributing to the infection process and their potential functions and to identify potential host-pathogen interactions between host and pathogen genes. Overall, dRNASb has the potential to identify key genes involved in bacterial growth or host defence mechanisms for future uses as therapeutic targets.

category: microbiology


title: Neuronal miR-138 represses HSV-2 lytic infection by regulating viral and host genes with mechanistic differences compared to HSV-1

abstract:
Herpes simplex virus 2 (HSV-2) establishes latent infection in dorsal root ganglion (DRG) neurons after productive (lytic) infection in peripheral tissues. A neuron-specific microRNA, miR-138, favors HSV-1 latency by repressing viral ICP0, and host Oct-1 and Foxc1 genes, yet the role of miR-138 in HSV-2 infection was unknown. The ICP0 mRNAs of HSV-1, HSV-2 and chimpanzee herpesvirus each have one to two canonical miR-138 binding sites. The sites are 100% conserved in 308 HSV-1 and 300 HSV-2 published sequences of clinical isolates. In co-transfection assays, miR-138 repressed HSV-2 ICP0 expression through the seed region and surrounding interactions that are different from HSV-1. An HSV-2 mutant with disrupted miR-138 binding sites on ICP0 showed increased ICP0 expression in Neuro-2a cells. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation confirmed miR-138 binding to HSV-2 ICP0, and identified UL19 and UL20 as additional targets, whose expression was repressed by miR-138 during co-transfection. In Neuro-2a cells, transfected miR-138 and its antagomir decreased and increased HSV-2 replication, respectively, and knockout experiment showed that miR-138s host targets OCT-1 and FOXC1 were important for HSV-2 replication. In primary mouse DRG neurons, both ICP0 and FOXC1 positively regulated HSV-2 replication, but both overexpressed and endogenous miR-138 suppressed HSV-2 replication primarily by repressing ICP0 expression. Thus, miR-138 can suppress HSV-2 neuronal replication through multiple viral and host pathways. These results reveal functional similarities and mechanistic differences in how miR-138 regulates HSV-1 and HSV-2 infection and indicate an evolutionary advantage of using miR-138 to repress lytic infection in neurons.

ImportanceHerpes simplex virus 1 (HSV-1) and HSV-2 are closely related viruses with major differences. Both viruses establish latency in neurons from which they reactivate to cause disease. A key aspect of HSV latency is repression of productive infection in neurons. Based on previous work with HSV-1, we investigated the role of a neuron-specific microRNA, miR-138, in HSV-2 infection, and established it as a repressor of HSV-2 productive infection in neuronal cells. This repression is mediated mainly by targeting viral ICP0 and host Foxc1 mRNAs, but other pathways also contribute. Despite functional conservation of the role of miR-138 between HSV-1 and HSV-2, many molecular mechanisms differ including how miR-138 represses ICP0 expression and miR-138 targeting of HSV-2 but not HSV-1 UL19 and UL20. To our knowledge, this study provides the first example of host microRNA regulation of HSV-2 infection.


title: Omicron-specific mRNA vaccine elicits potent immune responses in mice, hamsters, and nonhuman primates

abstract: SARS-CoV-2 has infected more than 400 million people around the globe and caused millions of deaths. Since its identification in November 2021, Omicron, a highly transmissible variant, has become the dominant variant in most countries. Omicrons highly mutated spike protein, the main target of vaccine development, significantly compromises the immune protection from current vaccination. We develop an mRNA vaccine (SOmicron-6P) based on an Omicron-specific sequence. In mice, SOmicron-6P shows superior neutralizing antibodies inducing abilities to a clinically approved inactivated virus vaccine, a clinically approved protein subunit vaccine, and an mRNA vaccine (SWT-2P) with the same sequence of BNT162b2 RNA. Significantly, SOmicron-6P induces a 14.4[~]27.7-fold and a 28.3[~]50.3-fold increase of neutralizing activity against the pseudovirus of Omicron and authentic Omicron compared to SWT-2P, respectively. In addition, two doses SOmicron-6P significantly protects Syrian hamsters against challenge with SARS-CoV-2 Omicron variant and elicits high titers of nAbs in a dose-dependent manner in macaques. Our results suggest that SOmicron-6P offers advantages over current vaccines, and it will be helpful for those with weak immunity.


title: Real-Time Flow Cytometry to assess qualitative and quantitative responses of oral pathobionts during exposure to antiseptics

abstract: Antiseptics are widely used in oral healthcare to prevent or treat oral diseases, such as gingivitis and periodontitis. However, the incidence of bacteria being tolerant to standard antiseptics has sharply increased over the last few years. This stresses the urgency for surveillance against tolerant organisms, as well as the discovery of novel antimicrobials. Traditionally, susceptibility to antimicrobials is assessed by broth micro-dilution or disc diffusion assays, both of which are time-consuming, labor-intensive and provide limited information on the mode of action of the antimicrobials. The above-mentioned limitations highlight the need for the development of new methods to monitor and further understand antimicrobial susceptibility. Here, we used real-time flow cytometry, combined with membrane permeability staining, as a quick and sensitive technology to study the quantitative and qualitative response of two oral pathobionts to different concentrations of chlorhexidine, cetylpyridinium chloride or triclosan. Apart from the real-time monitoring of cell damage, we further applied a phenotypic fingerprint method to differentiate between the bacterial subpopulations that arose due to treatment. We quantified the pathobiont damage rate of different antiseptics at different concentrations within 15 minutes of exposure and identified the conditions under which the bacteria were most susceptible. Moreover, we detected species-specific and treatment-specific phenotypic subpopulations. This proves that real-time flow cytometry can provide information on the susceptibility of different microorganisms in a short time frame while differentiating between antiseptics and thus could be a valuable tool in the discovery of novel antimicrobial compounds while at the same time deciphering their mode of action.


title: Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infection during SARS-CoV-2 Infection

abstract: Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate SARS-CoV-2 infection increased the risk of pneumococcal coinfection in a time-dependent, but sexindependent, manner in the transgenic K18-hACE mouse model of COVID-19. Bacterial coinfection was not established at 3 d post-virus, but increased lethality was observed when the bacteria was initiated at 5 or 7 d post-virus infection (pvi). Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.


title: RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2

abstract: SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy towards them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3, a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented supports the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.


title: CircRNA_33702 promotes renal fibrosis by targeting the miR-29b-3p/WISP1 pathway

abstract: Growing evidence suggest that circular RNAs (circRNAs) are critical mediators in renal diseases. However, there have been very few reports about the role of circRNAs in renal fibrosis. In this study, circRNA_33702 was found to be upregulated, both in UUO mice and in TGF-{beta} 1-treated BUMPT cells. Whilst knockdown of circRNA_33702 was shown to relieve the TGF-{beta} 1-induced expression of collagen I, collagen III and fibronectin; overexpression of circRNA_33702 was found to exert an inhibitory effect on the expression of the same genes. Mechanistically, circRNA_33702 was demonstrated to bind directly with miR-29b-3p and inhibit its expression. Moreover, WISP1 was identified as a target of miR-29b-3p and the expression of WISP1 was observed to be repressed by miR-29b-3p. Notably, knockdown of circRNA_33702 was found to attenuate the expression of collagen I, collagen III and fibronectin by inhibiting the expression of WISP1, and the observed inhibitory effect can be reversed by miR-29b-3p inhibitor. Finally, inhibition of circRNA_33702 was shown to attenuate interstitial fibrosis in UUO mice via the miR-29b-3p/WISP1 axis. In general, our data show that circRNA_33702 may promote renal fibrosis via the miR-29b-3p/WISP1 axis, which may potentially be developed as a new therapeutic target.

category: molecular biology


title: Metabolome and transcriptome analysis reveal components regulating soapberry triterpenoid saponin biosynthesis

abstract:
Soapberry (Sapindus mukorossi Gaertn.) pericarps are rich in valuable bioactive triterpenoid saponins. However, the saponin content dynamics and the molecular regulatory network of saponin biosynthesis in soapberry pericarps remain largely unclear. Here, we performed combined metabolite profiling and transcriptome analysis to identify saponin accumulation kinetic patterns, investigate gene networks, and characterize key candidate genes and transcription factors involved in saponin biosynthesis in soapberry pericarps. A total of 54 saponins were tentatively identified, including 25 that were differentially accumulated. Furthermore, 49 genes putatively involved in sapogenin backbone biosynthesis and some candidate genes assumed to be responsible for the backbone modification, including 41 cytochrome P450s and 45 glycosyltransferases, were identified. Saponin-specific clusters/modules were identified by Mfuzz clustering and weighted gene co-expression network analysis, and one TF-gene regulatory network underlying saponin biosynthesis was proposed. The results of yeast one-hybrid assay and electrophoretic mobility shift assay suggested that SmbHLH2, SmTCP4, and SmWRKY27 may play important roles in the triterpenoid saponin biosynthesis by directly regulating the transcription of SmCYP71D-3 in soapberry pericarp. Overall, these findings provide valuable information for understanding the molecular regulatory mechanism of saponin biosynthesis, enriching the gene resources, and guiding further research on triterpenoid saponin accumulation in soapberry pericarps.

One-sentence summaryCombining metabolome and transcriptome analysis to identify saponin kinetic patterns, gene networks, and key candidate genes and transcription factors involved in saponin biosynthesis of soapberry.


title: HIF1A modulate glycolysis function to governs mouse ovarian microenvironment metabolic plasticity in aging single cell resolution

abstract: The molecular machinery of ovarian aging and female age-related pathway remain unclear. Here, we utilized single-cell RNA-seq to profile over 9815 cells from both young and old female mouse and identified age-related alterations in the female somatic microenvironment. Interestingly, by aging-related signature calculation, we examined HIF1A in mouse ovarian cell aging regulated roles which effect pathways included glycolysis, TCA, OXPHOS and fatty acid metabolism. Additionally, inactivated HIF1A, decreased glycolysis was observed. Comparison analysis reveals the aging related regulon; metabolic and nutrient absorption changes provides a comprehensive understanding of the cell-type-specific mechanisms underlying mouse ovarian aging at single-cell resolution. This study, revealing new potential candidate biomarkers for the diagnosis of aging-associated ovary pathology.


title: Evolutionary analysis of genomes of SARS-CoV-2-related bat viruses suggests old roots, constant effective population size, and possible increase of fitness

abstract: It is of vital practical interest to understand the co-evolution of bat {beta}-coronaviruses with their hosts, since a number of these most likely crossed the species boundaries and infected humans. Complete sequences of 47 consensus genomes are available for bat {beta}-coronaviruses related to the SARS-CoV-2 human virus. We carried out several types of evolutionary analyses using these data. First, using the publicly available BEAST 2 software, we generated phylogenetic trees and skyline plots. The roots of the trees, both for the entire sequences and subsequences coding for the E and S proteins as well as the 5 and 3 UTR regions, are estimated to be located from several decades to more than a thousand years ago, while the effective population sizes remained largely constant. Motivated by this, we developed a simple estimator of the effective population size in a Moran model with constant population, which, under the model is equal to the expected age of the MRCA measured in generations. Comparisons of these estimates to those produced by BEAST 2 shows qualitative agreement. We also compared the site frequency spectra (SFS) of the bat genomes to those provided by the Moran Tug-of-War model. Comparison does not exclude the possibility that overall fitness of the bat {beta}-coronaviruses was increasing over time as a result of directional selection. Stability of interactions of bats and their viruses was considered likely on the basis of specific manner in which bat immunity is tuned, and it seems consistent with our analysis.

category: evolutionary biology


title: Hedgehog signaling is non-cell autonomously activated in the cystic kidney of Arl13b mutant mice

abstract:
BackgroundPolycystic kidney disease (PKD) is a ciliopathy characterized by fluid-filled epithelial cysts in the kidney. Although it is well established that the primary cilium is essential for Hedgehog (HH) signalling and HH signalling is abnormally activated in cystic kidneys of multiple PKD models, the mechanism and function of HH activation in PKD pathogenesis remains incompletely understood.

MethodsWe used a transgenic HH reporter line to identify the target tissue of HH signalling in Arl13f/f;Ksp-Cre mutant kidney, in which the cilia biogenesis gene Arl13b is specifically deleted in epithelial cells of the distal nephron. In addition, we treated Arl13bf/f;Ksp-Cre mice with the GLI inhibitor GANT61 and analyzed its impact on PKD progression in this model.

ResultsIn vivo in the mouse kidney, deletion of Arl13b in epithelial cells led to non-cell autonomous activation of the HH pathway in the interstitium. In addition, whole body inhibition of the HH pathway by GANT61 reduced cyst burden, suppressed fibrosis and reduced kidney function decline in Arl13bf/f;Ksp-Cre mice.

ConclusionsOur results reveal non-cell autonomous activation of HH signalling in the interstitium of the cystic kidney of Arl13bf/f;Ksp-Cre mice and suggest that abnormal activation of the HH pathway contributes to PKD progression.

category: genetics


title: Identification of different classes of genome instability suppressor genes through analysis of DNA damage response markers

abstract: Genetic studies in Saccharomyces cerevisiae have identified 266 genes and predicted an additional 38 genes that suppress the accumulation of spontaneous gross chromosomal rearrangements (GCRs). Here we identified mutations that induce two DNA damage response (DDR) markers, Hug1 expression and Ddc2 foci, and combined these data with other published screens identifying mutations inducing other DDR markers, including Rad52 foci and Rnr3 expression. Together, these data identify four categories of mutations: most mutations were DDR- GCR-, 356 were DDR+ GCR-, 72 were DDR- GCR+, and 157 were DDR+ GCR+. These results indicate that induction of DDR markers alone, while allowing DDR analysis, is not a reliable indicator of increased genome instability. They also guide further analysis of mechanistically distinct groups of GCR-inducing mutations, such as mutations that increase levels of GCR-inducing DNA damage and mutations that result in mis-repair of normal levels of DNA damage resulting in GCRs.


title: Combining genetic constraint with predictions of alternative splicing to prioritize deleterious splicing in rare disease studies

abstract:
BackgroundDespite numerous molecular and computational advances, roughly half of patients with a rare disease remain undiagnosed after exome or genome sequencing. A particularly challenging barrier to diagnosis is identifying variants that cause deleterious alternative splicing at intronic or exonic loci outside of canonical donor or acceptor splice sites.

ResultsSeveral existing tools predict the likelihood that a genetic variant causes alternative splicing. We sought to extend such methods by developing a new metric that aids in discerning whether a genetic variant leads to deleterious alternative splicing. Our metric combines genetic variation in the Genome Aggregate Database with alternative splicing predictions from SpliceAI to compare observed and expected levels of splice-altering genetic variation. We infer genic regions with significantly less splice-altering variation than expected to be constrained. The resulting model of regional splicing constraint captures differential splicing constraint across gene and exon categories, and the most constrained genic regions are enriched for pathogenic splice-altering variants. Building from this model, we developed ConSpliceML. This ensemble machine learning approach combines regional splicing constraint with multiple per-nucleotide alternative splicing scores to guide the prediction of deleterious splicing variants in protein-coding genes. ConSpliceML more accurately distinguishes deleterious and benign splicing variants than state-of-the-art splicing prediction methods, especially in "cryptic" splicing regions beyond canonical donor or acceptor splice sites.

ConclusionIntegrating a model of genetic constraint with annotations from existing alternative splicing tools allows ConSpliceML to prioritize potentially deleterious splice-altering variants in studies of rare human diseases.

category: genomics


title: Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing

abstract: In eukaryotes, the nucleolus is the site of ribosome biosynthesis, an essential process in all cells. While human ribosome assembly is largely evolutionarily conserved, many of the regulatory details underlying its control and function have not yet been well-defined. The nucleolar protein RSL24D1 was originally identified as a factor important for ribosome biogenesis, and as an interactor with the PeBoW complex (PES1, BOP1, WDR12) in high-throughput affinity purifications. The PeBoW complex has been shown to be required for pre-28S rRNA processing. In this study, we show that RSL24D1 depletion impairs both pre-ribosomal RNA (pre-rRNA) transcription and mature 28S rRNA production, leading to decreased protein synthesis and p53 stabilization in mammalian cells. Surprisingly, each of the PeBoW complex members is also required for pre-rRNA transcription. We also demonstrate that RSL24D1 is physically complexed with RNA polymerase I, revealing a connection between large ribosomal subunit biogenesis and rDNA transcription. These results uncover the dual role of RSL24D1 and the PeBoW complex in multiple steps of ribosome biogenesis, and provide evidence implicating large subunit biogenesis factors in pre-rRNA transcription control.

category: biochemistry


title: The periplasmic chaperone Skp prevents misfolding of the secretory lipase A from Pseudomonas aeruginosa

abstract: Pseudomonas aeruginosa is a wide-spread opportunistic human pathogen and a high-risk factor for immunodeficient people and patients with cystic fibrosis. The extracellular lipase A belongs to the virulence factors of P. aeruginosa. The lipase undergoes folding and activation in the periplasm prior the secretion. Here, we demonstrate that the ubiquitous periplasmic chaperone Skp of P. aeruginosa, but not SurA, FkpA, PpiD or YfgM, efficiently prevents misfolding of the aggregation-prone lipase A and facilitates its activation by a specific foldase LipH. Small-angle X-ray scattering visualizes the trimeric architecture of P. aeruginosa Skp and identifies two primary conformations of the chaperone, a compact and a widely open. We describe two binding modes of Skp to the lipase, with affinities of 20 nM and 2 M, which correspond to 1:1 and 1:2 stoichiometry of the lipase:Skp complex. Two Skp trimers are required to stabilize the lipase via the apolar interactions, which are not affected by high salt concentrations typical for the sputum of cystic fibrosis patients. The chaperoning effect of Skp points to its potent role in maturation and secretion of the lipase in Pseudomonas species.


title: Molecular Basis for Variations in the Sensitivity of Pathogenic Rhodopsin Variants to 9-cis-Retinal

abstract: Over 100 mutations in the rhodopsin gene have been linked to a spectrum of retinopathies that include retinitis pigmentosa and congenital stationary night blindness. Though most of these variants exhibit a loss of function, the molecular defects caused by these underlying mutations vary considerably. In this work, we utilize deep mutational scanning to quantitatively compare the plasma membrane expression of 123 known pathogenic rhodopsin variants in the presence and absence of the stabilizing cofactor 9-cis-retinal. We identify 69 retinopathy variants, including 20 previously uncharacterized variants, that exhibit diminished plasma membrane expression in HEK293T cells. 67 of these apparent class II variants exhibit a measurable increase in expression in the presence of 9-cis-retinal. However, the magnitude of the response to this molecule varies considerably across this spectrum of mutations. Evaluation of the observed shifts in relation to thermodynamic estimates for the coupling between binding and folding suggests underlying differences in stability constrains the magnitude of their response to retinal. Nevertheless, estimates from computational modeling suggest many of the least sensitive variants also directly compromise binding. Finally, we evaluate the functional properties of three previous uncharacterized, retinal-sensitive variants ({Delta}N73, S131P, and R135G) and show that two retain residual function in vitro. Together, our results provide a comprehensive experimental characterization of the proteostatic properties of retinopathy variants and their response to retinal.


title: Combinatorial engineering for photoautotrophic production of recombinant products from the green microalga Chlamydomonas reinhardtii

abstract: Chlamydomonas reinhardtii has emerged as a powerful green cell factory for metabolic engineering of sustainable products created from the photosynthetic lifestyle of this microalga. Advances in nuclear genome and transgene expression engineering are allowing robust engineering strategies to be demonstrated in this host. However, commonly used lab strains are not equipped with features to enable their broader implementation in non-sterile conditions and high-cell density concepts. Here, we use combinatorial chloroplast and nuclear genome engineering to complement the C. reinhardtii strain UVM4 with publicly available genetic tools to enable the use of inorganic phosphite and nitrate as a sole the source phosphorous and nitrogen, respectively. We present recipes to create phosphite-buffered media solutions that enable high cell density algal cultivation. We then combine previously reported engineering strategies to produce the heterologous sesquiterpenoid patchoulol to high titers from our engineered green cell factories and show these products are possible to produce in under non-sterile conditions. Our work presents a straightforward means to generate C. reinhardtii strains for broader application in bio-processes for the sustainable generation of products.

category: bioengineering


title: IntegronFinder 2.0: identification and analysis of integrons across Bacteria, with a focus on antibiotic resistance in Klebsiella

abstract: Integrons are mobile genetic elements that contain multiple cassettes encoding accessory genes whose order is shuffled by a specific integrase. Integrons within mobile genetic elements often contain multiple antibiotic resistance genes that they spread among nosocomial pathogens and contribute to the current antibiotic resistance crisis. However, most integrons are presumably sedentary and encode a much broader diversity of functions. IntegronFinder is a widely used software to identify novel integrons in bacterial genomes, but has aged and lacks some useful functionalities to handle very large datasets of draft genomes or metagenomes. Here, we present IntegronFinder version 2. We have updated the code, improved its efficiency and usability, adapted the output to incomplete genome data, and added a few novel functions. We describe these changes and illustrate the relevance of the program by analyzing the distribution of integrons across more than 20,000 fully sequenced genomes. We also take full advantage of its novel capabilities to analyze close to 4 thousand Klebsiella pneumoniae genomes for the presence of integrons and antibiotic resistance genes within them. Our data shows that K. pneumoniae has a large diversity of integrons and the largest mobile integron in our database of plasmids. The pangenome of these integrons contains a total of 165 different gene families with most of the largest families being related with resistance to numerous types of antibiotics. IntegronFinder is a free and open-source software available at https://github.com/gem-pasteur/Integron_Finder.


title: C21orf2 mutations found in ALS disrupt primary cilia function

abstract: Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease that affects 1 in 400 people. Almost 40 genes have been associated with ALS, currently explaining about 15% of the ALS risk. These genes tend to cluster in certain disease pathways such as protein quality control, RNA metabolism and axonal function. Despite these advances, adequate treatments for ALS patients are still missing. In this study, we investigate the role of a newly discovered ALS gene, C21orf2, in ALS pathology. We show that C21orf2 is localized to the basal body of the primary cilium and plays an important role in ciliogenesis in vitro and in vivo. Knock down of C21orf2 also lowers cilia frequency and length in human iPSC-derived spinal motor neurons (sMNs). Furthermore, we show that intraflagellar transport is impaired, causing primary cilia to fail in transducing extracellular signals essential in the sonic hedgehog pathway. ALS-associated mutations in C21orf2 lead to loss of binding to centrosomal proteins, loss of proper localization at the basal body and hereby prevent C21orf2 from carrying out its normal function in cilia by loss-of-function. Finally, we confirm that sMNs derived from iPSCs from ALS patients with C21orf2 mutations display similar cilia dysfunction and have disturbed sonic hedgehog signaling. Collectively, our data reveal impaired cilia homeostasis as a novel disease mechanism at play in ALS, opening new avenues for further research.


title: Synaptotagmin 1 mediates toxicity of botulinum neurotoxin type A

abstract:
The extreme neurotoxicity of botulinum neurotoxins (BoNTs), broadly used as a therapeutics, is mediated by their direct binding to two plasma membrane receptors: polysialogangliosides (PSGs) and serotype-dependently either synaptotagmin 1/2 (Syt1/2) or synaptic vesicle glycoprotein 2 (SV2). Here, we demonstrate that although BoNT/A serotype binds directly to PSG and SV2, its neurotoxicity depends on Syt1. Using single-molecule superresolution microscopy of pathological concentrations of BoNT/A holotoxins, we demonstrate that the toxin binds to a preassembled PSG-Syt1 complex that forms nanoclusters with SV2 on the plasma membrane of hippocampal neurons. This coincidental interaction controls the selective endocytic targeting of BoNT/A into synaptic vesicles, leading to incapacitation of neuronal communication. Our results suggest that Syt1-SV2 plasma membrane nanoclusters may acts as a common target for various BoNT serotypes.

One-Sentence SummaryBoNT/A hijacks an intrinsic tripartite PSG-Syt1-SV2 endocytic sorting mechanism to incapacitate neuronal communication


title: The nuclear receptor NR4A1 restrains neutrophil granulocyte mediated brain damage in cerebral ischemia

abstract: Immigration and activation of immune cells play a significant role in damage progression after ischemic stroke. It has been shown that the nuclear receptor NR4A1 exerts a crucial role within the inflammatory response of various immune diseases via regulating immune cell activation. In this study, we investigated the role of NR4A1 on the activation and recruitment of brain resident and peripheral immune cells after cerebral ischemia. Here, we show that NR4A1 mediates an anti-inflammatory and damage limiting effect after ischemic stroke through immigrating neutrophil granulocytes. Importantly, NR4A1-activation with its ligand Cytosporone-B improves functional outcome and diminishes brain damage. Therefore, modulation of NR4A1 is a promising therapeutic target in the treatment of stroke.


title: A BALB/c IGHV Reference Set, defined by haplotype analysis of long-read VDJ-C sequences from F1 (BALB/c / C57BL/6) mice

abstract: The immunoglobulin genes of inbred mouse strains that are commonly used in models of antibody-mediated human diseases are poorly characterized. This compromises data analysis. To infer the immunoglobulin genes of BALB/c mice, we used long-read SMRT sequencing to amplify VDJ-C sequences from F1 (BALB/c x C57BL/6) hybrid animals. Previously unreported strain variations were identified in the Ighm and Ighg2b genes, and analysis of VDJ rearrangements led to the inference of 278 germline IGHV alleles. 169 alleles are not present in the C57BL/6 genome reference sequence. To establish a set of expressed BALB/c IGHV germline gene sequences, we computationally retrieved IGHV haplotypes from the IgM dataset. Haplotyping led to the confirmation of 162 BALB/c IGHV gene sequences. A musIGHV398 pseudogene variant also appears to be present in the BALB/cByJ substrain, while a functional musIGHV398 gene is highly expressed in the BALB/cJ substrain. Only four of the BALB/c alleles were also observed in the C57BL/6 haplotype. The full set of inferred BALB/c sequences has been used to establish a BALB/c IGHV reference set, hosted at https://ogrdb.airr-community.org. We assessed whether assemblies from the Mouse Genome Project (MGP) are suitable for the determination of the genes of the IGH loci. Only 37 (43.5%) of the 85 confirmed IMGT-named BALB/c IGHV and 33 (42.9%) of the 77 confirmed non-IMGT IGHV were found in a search of the MGP BALB/cJ genome assembly. This suggests that Adaptive Immune Receptor Repertoire sequencing (AIRR-Seq) data, but not currently-available genome assemblies, are suited to the documentation of germline IGHV genes.


title: The CTP-binding domain is disengaged from the DNA-binding domain in a co-crystal structure of Bacillus subtilis Noc-DNA complex

abstract: In Bacillus subtilis, a ParB-like nucleoid occlusion protein (Noc) binds specifically to Noc-binding sites (NBS) around the chromosome to help coordinate chromosome segregation and cell division. Noc does so by binding to cytidine triphosphate (CTP) to form large membrane-associated nucleoprotein complexes to physically inhibit the assembly of the cell division machinery. The site-specific binding of Noc to NBS DNA is a prerequisite for CTP-binding and the subsequent formation of a membrane-active DNA-entrapped protein complex. Here, we solve the structure of a truncated B. subtilis Noc bound to NBS DNA to reveal the conformation of Noc at this crucial step. Our structure reveals the disengagement between the N-terminal CTP-binding domain and the NBS-binding domain of each DNA-bound Noc subunit, this is driven, in part, by the swapping of helices 4 and 5 at the interface of the two domains. Site-specific crosslinking data suggest that this conformation of Noc-NBS exists in solution. Overall, our results lend support to the recent proposal that parS/NBS-binding catalyzes CTP-binding and DNA-entrapment by preventing the re-engagement of the NTD and DBD from the same ParB/Noc subunit.


title: Stable nebulization and muco-trapping properties of Regdanvimab/IN-006 support its development as a potent, dose-saving inhaled therapy for COVID-19

abstract: The respiratory tract represents the key target for antiviral delivery in early interventions to prevent severe COVID-19. While neutralizing monoclonal antibodies (mAb) possess considerable efficacy, their current reliance on parenteral dosing necessitates very large doses and places a substantial burden on the healthcare system. In contrast, direct inhaled delivery of mAb therapeutics offers the convenience of self-dosing at home, as well as much more efficient mAb delivery to the respiratory tract. Here, building on our previous discovery of Fc-mucin interactions crosslinking viruses to mucins, we showed that regdanvimab, a potent neutralizing mAb already approved for COVID-19 in several countries around the world, can effectively trap SARS-CoV-2 virus-like-particles in fresh human airway mucus. IN-006, a reformulation of Regdanvimab, was stably nebulized across a wide range of concentrations, with no loss of activity and no formation of aggregates. Finally, nebulized delivery of IN-006 resulted in 100-fold greater mAb levels in the lungs of rats compared to serum, in marked contrast to intravenously dosed mAbs. These results not only support our current efforts to evaluate the safety and efficacy of IN-006 in clinical trials, but more broadly substantiate nebulized delivery of human antiviral mAbs as a new paradigm in treating SARS-CoV-2 and other respiratory pathologies.


title: Reduced gene templates for supervisedanalysis of scale-limited CRISPR-Cas9 fitnessscreens

abstract:
Pooled genome-wide CRISPR-Cas9 screens are furthering our mechanistic understanding of human biology and have allowed us to identify new oncology therapeutic targets. Scale-limited CRISPR-Cas9 screens - typically employing guide RNA libraries targeting subsets of functionally related genes, individual biological pathways, or portions of the druggable genome - constitute an optimal setting for investigating narrow hypotheses and they are easier to execute on complex models, such as organoids and in-vivo models. Different supervised methods are used for the computational analysis of genome-wide CRISPR-Cas9 screens; most are not well suited for scale-limited screens as they require large sets of positive/negative control genes (gene templates) to be included among the screened ones. We have developed a computational framework identifying optimal subsets of known essential and nonessential genes (at different subsampling percentages) that can be used as templates for supervised analyses of scale-limited CRISPR-Cas9 screens, while having a reduced impact on the size of the employed library.

HighlightsO_LIScale-limited CRISPR-Cas9 screens are experimentally easier than genome-wide screens
C_LIO_LIReference gene templates (RTs) are used for supervised analyses of genome-wide screens
C_LIO_LIReduced RTs allow supervised analyses of scale-limited CRISPR-Cas9 screens
C_LIO_LIWe present optimal reduced RTs and a computational method to assemble them
C_LI

Graphical abstract

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title: RdRp-scan: A Bioinformatic Resource to Identify and Annotate Divergent RNA Viruses in Metagenomic Sequence Data

abstract: Despite a rapid expansion in the number of known RNA viruses following the advent of metagenomic sequencing, the identification and annotation of highly divergent RNA viruses remains challenging, particularly from poorly characterized hosts and environmental samples. Protein structures are more conserved than primary sequence data, such that structure-based comparisons provide an opportunity to reveal the viral "dusk matter": viral sequences with low, but detectable, levels of sequence identity to known viruses with available protein structures. Here, we present a new open computational and resource - RdRp-scan - that contains a standardized bioinformatic toolkit to identify and annotate divergent RNA viruses in metagenomic sequence data based on the detection of RNA dependent RNA polymerase (RdRp) sequences. By combining RdRp-specific Hidden Markov models (HMM) and structural comparisons we show that RdRp-scan can efficiently detect RdRp sequences with identity levels as low as 10% to those from known viruses and not identifiable using standard sequence-to-sequence comparisons. In addition, to facilitate the annotation and placement of newly detected and divergent virus-like sequences into the known diversity of RNA viruses, RdRp-scan provides new custom and curated databases of viral RdRp sequences and core motif, as well as pre-built RdRp alignments. In parallel, our analysis of the sequence diversity detected by RdRp-scan revealed that while most of the taxonomically unassigned RdRps fell into pre-established clusters, some sequences cluster into potential new orders of RNA viruses related to the Wolframvirales and Tolivirales. Finally, a survey of the conserved A, B and C RdRp motifs within the RdRp-scan sequence database revealed additional variations of both sequence and position, which might provide new insights into the structure, function and evolution of viral RdRps.


title: Rule-based habitat suitability modelling for the reintroduction of the grey wolf (Canis lupus) in Scotland

abstract: Though native to Scotland, the grey wolf (Canis lupus) was extirpated c.250 years ago as part of a global eradication drive. The global population has recently expanded, now occupying 67% of its former range. Evidence is growing that apex predators provide a range of ecological benefits, most stemming from the reduction of overgrazing by deer - something from which Scotland suffers. In this study, we build a rule-based habitat suitability model for wolves on the Scottish mainland. From existing literature, we identify the most important variables as land cover, prey density, road density and human density, and establish thresholds of suitability for each. Fuzzy membership functions are used to assign suitability values to each variable, followed by fuzzy overlay to combine all four: a novel approach to habitat suitability modelling for terrestrial mammals. Model sensitivity is tested for land cover and prey density, as these variables constitute a knowledge gap and an incomplete dataset, respectively. The Highlands and Grampian mountains emerge strongly and consistently as the most suitable areas, largely due to high negative covariance between prey density and road/human density. Sensitivity testing reveals the models are fairly robust to changes in prey density, but less robust to changes in the scoring of land cover, with the latter altering the distribution of land mainly through the 70 - 100% suitability range. However, in statistical significance tests, only the least and most generous versions of the model emerge as giving significantly different results. Depending on the version of the model, a contiguous area of between 10,139km2 and 18,857km2 is shown to be 80 to 100% suitable. This could be sufficient to support between 50 and 94 packs of four wolves, if the average pack range size is taken to be 200km2. We conclude that in terms of habitat availability, reintroduction should be feasible.

category: ecology


title: Individually unique, fixed body patterns of Octopus chierchiae allow for photo-identification in long-term studies

abstract: The Lesser Pacific Striped Octopus, Octopus chierchiae, is a small iteroparous octopus known to inhabit intertidal regions of the Pacific coast of Central America. Many details about the species life history in the wild remain unknown. For apparently rare and delicate animals such as O. chierchiae, non-extractive sampling methods are necessary to study individuals and populations over time. After photographically documenting the physical development of 25 octopuses from hatching, we have concluded that individual O. chierchiae have individually unique body patterns that remain constant throughout their lifetimes. Furthermore, using photographs taken of animals in captivity on different dates over many months, we show that untrained volunteers can accurately identify whether or not a pair of images depicts the same individual octopus. These results demonstrate that wild individuals could be recognized and observed for in situ longitudinal studies. In addition, our results imply potential for photoidentification and community science to be used as non-extractive, non-intrusive sampling methods for future studies of wild O. chierchiae and other octopuses.

category: zoology


title: Rate-distortion theory of neural coding and its implications for working memory

abstract: Rate-distortion theory provides a powerful framework for understanding the nature of human memory by formalizing the relationship between information rate (the average number of bits per stimulus transmitted across the memory channel) and distortion (the cost of memory errors). Here we show how this abstract computational-level framework can be realized by a model of neural population coding. The model reproduces key regularities of visual working memory, including some that were not previously explained by population coding models. We verify a novel prediction of the model by reanalyzing recordings of monkey prefrontal neurons during an oculomotor delayed response task.


title: Integrating temporal single-cell gene expression modalities for trajectory inference and disease prediction

abstract: Current methods for analyzing single-cell datasets have relied primarily on static gene expression measurements to characterize the molecular state of individual cells. However, capturing temporal changes in cell state is crucial for the interpretation of dynamic phenotypes such as the cell cycle, development, or disease progression. RNA velocity infers the direction and speed of transcriptional changes in individual cells, yet it is unclear how these temporal gene expression modalities may be leveraged for predictive modeling of cellular dynamics. Here, we present the first task-oriented benchmarking study that investigates integration of temporal sequencing modalities for dynamic cell state prediction. We benchmark eight integration approaches on eight datasets spanning different biological contexts, sequencing technologies, and species. We find that integrated data more accurately infers biological trajectories and achieves increased performance on classifying cells according to perturbation and disease states. Furthermore, we show that simple concatenation of spliced and unspliced molecules performs consistently well on classification tasks and can be used over more memory intensive and computationally expensive methods. This work provides users with practical recommendations for task-specific integration of single-cell gene expression modalities.


title: Self-organizing Single-Rosette Brain Organoids from Human Pluripotent Stem Cells

abstract:
The field of brain organoid research is complicated by morphological variability with multiple neural rosette structures per organoid. We have developed a new human brain organoid technique that generates self-organizing, single-rosette spheroids (SOSRS) with reproducible size, cortical-like lamination, and cell diversity. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells (hPSCs) that is patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D transition from neural plate to neural tube that occurs during neurodevelopment. The vast majority of monolayer fragments form spheres with a single central lumen and consistent growth rates. Over time, the SOSRS develop appropriately ordered lamination consistent with six cortical layers by immunocytochemistry and single cell RNA-sequencing. The reproducibility of this method has allowed us to demonstrate robust structural phenotypes arising from chemical teratogen exposure or when modeling a genetic neurodevelopmental epileptic disorder. This platform should advance studies of human cortical development, brain disorder mechanisms, and precision therapies.

SUMMARY STATEMENTSimple procedure for generating reproducible single rosette cortical brain organoids used to identify robust structural phenotypes with neuroteratogen exposure and in a genetic neurodevelopmental disease model.


title: Redundant PLETHORA activity promotes development of early embryonic cell lineages in Arabidopsis

abstract: The BABY BOOM/PLETHORA4 (BBM/PLT4) transcription factor has received much attention due to its ability to induce somatic and zygotic embryogenesis, two processes of pivotal importance in plant breeding. Loss of additional AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) genes, encoding members of the APETALA2 transcription factor family, causes embryo arrest and abortion, but whether BBM/PLT4 provides specific information for embryo development has remained unknown. Here, we reveal that AIL/PLT members are expressed in partially overlapping domains from their first appearance in the apical cell daughter of the zygote. Redundant early embryonic activity of BABY BOOM/PLT4 and PLT2 triggers development of the apical cell lineage and is required to initiate embryonic primordia. Furthermore, promoter swap experiments show that PLT1 and PLT3 expression in the expression domains of PLT2 and BABY BOOM/PLT4 is sufficient to rescue plt2 bbm double mutants. Our data indicate that generic AIL/PLT factors, involved in maintenance of stem cells, promotion of cell division and suppression of cell differentiation, provide the necessary information to initiate embryogenesis in Arabidopsis.

category: plant biology


title: Separability of Human Motor Memories during Reaching Adaptation with Force Cues

abstract:
Judging by the breadth of our motor repertoire during daily activities, it is clear that acquiring multiple motor skills is a hallmark of the human motor system. However, for reaching movements the different planning conditions under which this is possible in laboratory settings have remained a challenging question. Considering the sensitivity of limb afferent feedback relative to externally applied forces, we hypothesised that independent cues delivered by means of background loads could support simultaneous formation of different motor memories of various velocity-dependent force fields. We demonstrate in a series of experiments that indeed healthy adults can form internal priors about opposite force fields, independently of the direction of the background force cue. However, when the cue and force field were only related by their magnitude, a separation was still observed but the associated mechanism was subject to interference. Finally, we highlight that this paradigm allows dissociating trial-by-trial acquisition of internal representations from within-trial feedback adaptation, as these two adaptation mechanisms are associated with different time scales that can be measured reliably.

Significance StatementThe conditions under which humans can adapt to different contexts in parallel remain the subject of active debates. Mounting evidence highlights that contextual factors linked to movement planning are necessary to form different motor memories. Here we show that background forces indicative of the direction of a force field could play the role of contextual factors, and enable dual adaptation to different force fields. However, we uncovered that when the cue and the force field were in the same direction but of different magnitudes, the motor memories were still distinct but subject to interference. We further show that different timescales of adaptation observed can be explained by offline and online mechanisms in a model of reaching adaptation and control.


title: Rac1 controls cell turnover and mammary gland reversibility in post-partum involution

abstract: Cell turnover in adult tissues is essential for maintaining tissue homeostasis over a lifespan and for inducing the morphological changes associated with the reproductive cycle. However, the underlying mechanisms that coordinate the balance of cell death and proliferation remain unsolved. Using the mammary gland we have discovered that Rac1 acts as a nexus to control cell turnover. Post-lactational tissue regression is characterized by the death of milk secreting alveoli, but the process is reversible within the first 48h if feeding recommences. In mice lacking epithelial Rac1, alveolar regression was delayed. This defect did not result from failed cell death but rather increased cell turnover. Fitter progenitor cells inappropriately divided, regenerating the alveoli but cell death also concomitantly accelerated. We discovered that progenitor cell hyperproliferation was linked to non-autonomous effects of Rac1 deletion on the macrophageal niche with heightened inflammation. Moreover, loss of Rac1 impaired cell death with autophagy but switched the cell death route to apoptosis. Finally, mammary gland reversibility failed in the absence of Rac1 as the regenerated alveoli failed to recommence lactation upon re-suckling.

category: developmental biology


title: Maximize crop production and environmental sustainability: insights from an ecophysiological model of plant-pest interactions and multi-criteria decision analysis

abstract: Satisfying the demand for agricultural products while also protecting the environment from negative impacts of agriculture is a major challenge for crop management. We used an ecophysiological model of plant-pest interaction and multi-criteria decision analysis to optimize crop management when considering two contrasting objectives: (1) maximizing crop production and (2) minimizing environmental impact related to fertilization, irrigation and pesticide deployment. The model provides an indicator of crop production for 27 management scenarios, obtained combining three levels of fertilization, irrigation and pesticide use, respectively. We computed the environmental impact relevant to each management scenario by means of a weighted sum of costs assigned to fertilization, irrigation and pesticide use. We identified the optimal scenarios with respect to the considered objectives analysing the Pareto front. These scenarios were mostly characterized by high fertilization and no pesticide use. We evaluated the multi-functionality of the optimal scenarios by mean of the Gini coefficient: the scenario better assuring the equality between the two objectives was characterized by high fertilization, intermediate irrigation and no pesticide. Although our results remain qualitative and not immediately transferable to agronomic practices, our analytical framework provides a useful tool to evidence trade-offs among two contrasting objectives and provide solutions to act in an efficient way by leaving a certain degree of freedom to the political decision maker.


title: Belowground plant responses to root herbivory depend on the composition and structure of their root-colonising arbuscular mycorrhizal fungi

abstract:
O_LIThere is growing interest in managing arbuscular mycorrhizal (AM) fungi in agriculture to support plant production. These fungi can support crop growth and nutrient uptake but also affect plant-herbivore interactions. Our knowledge of how native AM fungal diversity and community composition influence these interactions is limited, while our understanding of this in relation to root-herbivory is lacking altogether.
C_LIO_LITo begin to address these knowledge gaps, plants were grown with no AM fungi or were inoculated with native fungal communities sourced from either a crop field (field community), a sclerophyll forest (forest community), or a crop field in fallow (fallow community). We then explored how the composition and structure (species richness and relative abundance) of root-colonising AM fungal communities was associated with the growth and belowground nutrient responses of a major crop (Sorghum bicolor) to attack from a root-feeding insect (Dermolepida albohirtum).
C_LIO_LIDNA metabarcoding revealed plants associated with three distinct root-colonising AM fungal communities. Fungal taxon richness in roots was highest in the field community and lowest in the fallow community. Both the field and fallow communities were dominated by the putatively ruderal genera Glomus and Claroideoglomus, while the forest-derived community contained greater proportions of Paraglomus and Ambispora.
C_LIO_LIIn response to root herbivory, plants without AM fungi and plants colonised by the forest community exhibited root biomass losses of 61% and 44%, respectively. Similarly, these two groups also had reductions of 59% and 65% in their root phosphorus content, respectively, when subjected to the root herbivore. In contrast, plants associating with communities harbouring greater proportions of Glomus and Claroideoglomus (the field and fallow communities) did not exhibit reductions in root biomass or nutrient content.
C_LIO_LIOur results show that plant responses to root-herbivory vary with root-colonising AM fungal community composition and structure. In a community context, our findings suggest that stronger associations with the genera Claroideoglomus and Glomus may potentially support crop tolerance-associated responses belowground. There is an urgent need for more exploration of how natural assemblages of AM fungi differentially mediate plant-herbivore interactions if we are to effectively manage soil fungi in sustainable agricultural systems.
C_LI


title: In Silico Evaluation of Plant Nitrification Suppression Effects on Agroecosystem Nitrogen Loss

abstract: AO_SCPLOWBSTRACTC_SCPLOWNitrification regulates potential for nitrogen (N) loss from ecosystems because it converts ammonium to nitrate, which is susceptible to leaching and gaseous emissions. Crops can suppress the microbes that perform nitrification by exuding nitrification-inhibiting compounds from their roots and taking up available ammonium, the substrate for nitrification. However, the effect of nitrification suppression on agroecosystem N losses remains poorly characterized, and a lack of temporal synchrony between nitrification, N losses, and nitrification suppression by plants could limit the effect of nitrification suppression. We used the DayCent-CABBI model to evaluate the effectiveness of the suppression of nitrification by sorghum to reduce N2O emissions and nitrate leaching in an energy sorghum/soybean rotation at the Energy Farm in Urbana-Champaign, IL. We simulated nitrification suppression at the measured levels (measNS) and at the maximum measured level applied to the entire growing season (maxNS), and we also explored ways to better utilize nitrification suppression by altering the timing of UAN fertilizer applications. Model experiments showed that most nitrification occurred immediately after fertilizer was applied, whereas nitrification suppression begins to ramp up more than a month after planting. On an annual basis, measNS experiments showed a 1-2% reduction in annual N2O emissions relative to no nitrification suppression (noNS), and maxNS experiments showed a 4-9% reduction in annual N2O emissions relative to noNS. Both nitrification suppression levels showed < 1% reduction in nitrate leaching. Altering the timing of fertilizer applications to better synchronize nitrification suppression with high soil ammonium levels had mixed effects on annual N2O emissions and nitrate leaching and sometimes resulted in increased N losses. The timing of simulated N2O emissions shifted with the timing of fertilization, and N2O emissions from denitrification increased when N2O emissions from nitrification decreased. Increasing N retention during the non-growing season may be more effective and growing-season nitrification suppression for reducing annual N losses in the rainfed Midwest, particularly for NO3- leaching in the early spring. Optimizing the timing of nitrification suppression alongside off-season N retention strategies would best improve the N sustainability of agroecosystems.


title: Genetic structure analysis on leopard cat reveals the effects of habitat fragmentation on subpopulation differentiation in Beijing

abstract: In the face of habitat shrinkage and segregation, the survival of wild cats looks bleak. Interpreting the population genetic structure during habitat fragmentation is critical to planning effective management strategies. To reveal the segregation effects of road construction and human settlement on population genetic structure, we noninvasively analyzed fecal DNA samples from leopard cats (Prionailurus bengalensis) from five nature reserves in mountainous areas around Beijing. We focused on molecular microsatellite markers and the mitochondrial control region. A total of 112 individual leopard cats were identified among 601 samples of scat, and generally moderate population genetic diversity was detected. Microsatellite marker-based gene flow (Nm) and STRUCTURE analyses showed no obvious discrepancies among the subpopulations; however, the calculation of genetic distance and construction of a phylogenetic tree of the mitochondrial control region revealed a trend toward differentiation from the other subpopulations in the Songshan subpopulation, which indicates habitat fragmentation effects. Because the isolated Songshan subpopulation may evolve independently, we suggest that its genetic structure be monitored every 5 years to detect any changes and that individuals be introduced as needed to maintain the viability of this subpopulation.


title: Fine-mapping and identification of candidate causal genes for tail length in the Merinolandschaf breed

abstract:
BackgroundDocking the tails of young lambs in long-tailed sheep breeds is a common practice worldwide. This practice is associated with pain, suffering and damage to the affected animals. Breeding for a shorter tail in long-tailed sheep breeds could offer one of the alternatives. This study aimed to analyze the natural tail length variation in the most common German Merino variety, and to identify possible causal alleles for the short tail phenotype segregating within a typical long-tailed breed.

ResultsHaplotype-based mapping in 362 genotyped (Illumina OvineSNP50) and phenotyped Merinolandschaf lambs resulted in a genome-wide significant mapping at position 37,111,462 bp on sheep chromosome 11 and on chromosome 2 at position 94,538,115 bp (Oar_v4.0). Targeted capture sequencing of these regions in 48 selected sheep and comparative analyses of WGS data of various long and short-tailed sheep breeds as well as wild sheep subspecies identified a SNP and a SINE element as the promising candidates. The PCR genotyping of these candidates revealed complete linkage of both the candidate variants. The SINE element is located in the promotor region of HOXB13, while the SNP was located in the first exon of HOXB13 and predicted to result in a nonsynonymous mutation.

ConclusionsOur approach successfully identified HOXB13 as candidate genes and the likely causal variants for tail length segregating within a typical long-tailed Merino breed. This would enable more precise breeding towards shorter tails, improve animal welfare by amplification of ancestral alleles and contribute to a better understanding of differential embryonic development.


title: The Root of the Red Maple Paradox: Two distinct regeneration mechanisms determine leaf morphology, physiology, and competitive success in Acer rubrum.

abstract: Red maple is one of the most abundant and wide-ranging tree species in eastern North America and has rapidly increased in abundance over the past century following forest disturbances. This species is both a site- and light-generalist and has the unique ability to regenerate in forms that represent multiple stages of forest succession. However, red maple has modest physiological traits compared to its competitors, including low maximum photosynthetic rate, low photosynthetic nitrogen-use efficiency, and low foliar nutrient content. Red maples unremarkable physiology contradicts its competitive success. To untangle this paradox, we examine red maples two distinct regeneration mechanisms: seedlings and vegetative sprouts. Red maple can regenerate from seed, but can also sprout clonally from a stump following forest disturbance. We compare the morphology, physiology, and plasticity of these two regeneration mechanisms over 24 years of forest succession using a chronosequence of regenerating forest stands. We found that sprout-origin maples grow on average 6.5x taller and 5.5x faster than seed-origin maples. Sprout-origin trees display greater leaf spectral reflectance in the near-infrared range and greater stomatal density than seed-origin trees, demonstrating vegetative sprouts low water-use efficiency. Sprout-origin trees have more robust light-capture traits, including thicker palisade mesophyll than seed-origin trees. Sprouts also have a high plasticity between upper and lower leaves in many morphological and physiological traits, while seed-origin trees exhibited much less plasticity. The two distinct regeneration mechanisms of seed-origin and sprout-origin give rise to red maple trees with dramatically different leaf traits, allowing red maples to regenerate and thrive in a range of ecological conditions. Seed-origin maples are slow-growing, late successional, and shade-tolerant. Sprout-origin maples are fast-growing, early successional, and shade intolerant. This unique bimodal regeneration strategy may explain the red maple paradox and help predict forest composition and structure following disturbance.


title: Structure of cyanobacterial photosystem I complexed with Cytochrome c6 and Ferredoxin at 1.97 A resolution

abstract: Photosystem I (PSI) is a light driven electron pump transferring electrons from Cytochrome c6 (Cyt c6) to Ferredoxin (Fd). An understanding of this electron transfer process is hampered by a paucity of structural detail concerning the binding of Fd and the bound Cyt c6. Here we describe the high resolution cryo-EM structure of Thermosynechococcus elongatus BP-1 PSI in complex with Fd and Cyt c6. In our structure Cyt c6 is loosely bound in a non-productive site on the periphery of the complex. Side chain interactions at the PSI:Fd interface are visualized in detail. The structure explains the properties of mutants of PsaE and PsaC and suggests a molecular switch for the dissociation of Fd upon reduction. Calorimetry-based thermodynamic analyses confirms a single binding site for Fd and demonstrates that PSI:Fd complexation is purely driven by entropy. A reaction cycle for the efficient transfer of electrons from Cyt c6 to Fd via PSI is proposed.


title: Multiscale structural control of hydraulic bending in the sensitive plant Mimosa pudica

abstract: Mechanistic studies of animal and plant motion often focus on the fundamental units of biological actuation: individual motor cells. Recent advances spanning the fields of muscle physiology and biomimetic actuation, however, reveal that important mechanical behaviors also arise at higher levels of organization, driven by the interplay of motor cells with connective tissue networks that guide and control en masse deformation. Here we illustrate how a paradigm of cell-driven actuation augmented by a mechanically crucial extracellular matrix is equally applicable to plant pulvini; motor organs evolutionarily distinct from but mechanically analogous to animal muscles. Using pulvini from the sensitive plant Mimosa pudica, we visualized anatomical sources of mechanical anisotropy at three hierarchical scales of pulvinus organization, built hydraulic physical models of observed morphologies, and analyzed 3D changes in the shapes of osmotically pressurized pulvini. We find that extracellular guidance controls the direction and extent of turgor-induced pulvinus deformation, an effect that ultimately influences the magnitude, speed, and energetic cost of pulvinus-driven motion. We discuss the relevance of these findings to ongoing work in the fields of muscle physiology and soft robotics, and we situate pulvini within a conceptual framework for understanding the design of biological motor organs generally.


title: ColorBayes: Improved color correction of high-throughput plant phenotyping images to account for local illumination differences

abstract:
BackgroundColor distortion is an inherent problem in image-based phenotyping systems that are illuminated by artificial light. This distortion is problematic when examining plants because it can cause data to be incorrectly interpreted. One of the leading causes of color distortion is the non-uniform spectral and spatial distribution of artificial light. However, color correction algorithms currently used in plant phenotyping assume that a single and uniform illuminant causes color distortion. These algorithms are consequently inadequate to correct the local color distortion caused by multiple illuminants common in plant phenotyping systems, such as fluorescent tubes and LED light arrays. We describe here a color constancy algorithm, ColorBayes, based on Bayesian inference that corrects local color distortions. The algorithm estimates the local illuminants using the Bayes rule, the maximum a posteriori, the observed image data, and prior illuminant information. The prior is obtained from light measurements and Macbeth ColorChecker charts located on the scene.

ResultsThe ColorBayes algorithm improved the accuracy of plant color on images taken by an indoor plant phenotyping system. Compared with existing approaches, it gave the most accurate metric results when correcting images from a dataset of Arabidopsis thaliana images.

The software is available at https://github.com/diloc/Color_correction.git.


title: Novel QTLs for cucumber resistance to two Colletotrichum orbiculare strains of different pathogenic races

abstract:
Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber (Cucumis sativus L.) and other cucurbit crops. The cucumber accession Ban Kyuri (G100) has a high level of resistance to anthracnose and is considered to be promising breeding material. Here, we report genetic mapping of loci from this cultivar that confer resistance to 104-T and CcM-1 of C. orbiculare that belong to pathogenic races 0 and 1, respectively. Quantitative trait locus (QTL) analysis based on phenotypic data from 196 F2:3 families detected one major QTL, An5, and one minor QTL, An6.2, for resistance to race 0, and one major QTL, An2, and three minor QTLs (An1.1, An1.2 and An6.1) for resistance to race 1. We identified lysM domain receptor-like kinase 3 (CsaV3_5G036150) and wall-associated receptor kinase-like (CsaV3_6G048820) as candidate genes for An5 and An6.2, respectively. Multiple genes encoding pattern recognition receptors were located in the regions of the QTLs conferring resistance to race 1. Thus, we identified potential sources of genetic resistance to different pathogenic races of C. orbiculare in the Ban Kyuri cultivar of cucumber.

Key messageQuantitative trait locus analysis identified independent novel loci in cucumber responsible for resistance to races 0 and 1 of the anthracnose fungal pathogen Colletotrichum orbiculare.


title: Structure-informed microbial population genetics elucidate selective pressures that shape protein evolution

abstract:
Comprehensive sampling of natural genetic diversity with metagenomics enables highly resolved insights into the interplay between ecology and evolution. However, intra-population genomic variation represents the outcome of both stochastic and selective forces, making it difficult to identify whether variants are maintained by adaptive, neutral, or purifying processes. This is partly due to the reliance on gene sequences to interpret variants, which disregards the physical properties of three-dimensional gene products that define the functional landscape on which selection acts. Here we describe an approach to analyze genetic variation in the context of predicted protein structures, and apply it to study a marine microbial population within the SAR11 subclade 1a.3.V, which dominates low-latitude surface oceans. Our analyses reveal a tight association between the patterns of nonsynonymous polymorphism, selective pressures, and structural properties of proteins such as per-site relative solvent accessibility and distance to ligands, which explain up to 59% of genetic variance in some genes. In glutamine synthetase, a central gene in nitrogen metabolism, we observe decreased occurrence of nonsynonymous variants from ligand binding sites as a function of nitrate concentrations in the environment, revealing genetic targets of distinct evolutionary pressures maintained by nutrient availability. Our data also reveals that rare codons are purified from ligand binding sites when genes are under high selection, demonstrating the utility of structure-aware analyses to study the variants that likely impact translational processes. Overall, our work yields insights into the governing principles of evolution that shape the genetic diversity landscape within a globally abundant population, and makes available a software framework for structure-aware investigations of microbial population genetics.

SignificanceIncreasing availability of metagenomes offers new opportunities to study evolution, but the equal treatment of all variants limits insights into drivers of sequence diversity. By capitalizing on recent advances in protein structure prediction capabilities, our study examines subtle evolutionary dynamics of a microbial population that dominates surface oceans through the lens of structural biology. We demonstrate the utility of structure-informed metrics to understand the distribution of nonsynonymous polymorphism, establish insights into the impact of changing nutrient availability on protein evolution, and show that even synonymous variants are scrutinized strictly to maximize translational efficiency when selection is high. Overall, our work illustrates new opportunities for discovery at the intersection between metagenomics and structural bioinformatics, and offers an interactive and scalable software platform to visualize and analyze genetic variants in the context of predicted protein structures and ligand-binding sites.


title: Classifying interactions in a synthetic bacterial community is hindered by inhibitory growth medium

abstract: Predicting the fate of a microbial community and its member species relies on understanding the nature of their interactions. However, designing simple assays that distinguish between interaction types can be challenging. Here, we performed spent media assays based on the predictions of a mathematical model to decipher the interactions between four bacterial species: Agrobacterium tumefaciens (At), Comamonas testosteroni (Ct), Microbacterium saperdae (Ms) and Ochrobactrum anthropi (Oa). While most experimental results matched model predictions, the behavior of Ct did not: its lag phase was reduced in the pure spent media of At and Ms, but prolonged again when we replenished with our growth medium. Further experiments showed that the growth medium actually delayed the growth of Ct, leading us to suspect that At and Ms could alleviate this inhibitory effect. There was, however, no evidence supporting such "cross-detoxification" and instead, we identified metabolites secreted by At and Ms that were then consumed or "crossfed" by Ct, shortening its lag phase. Our results highlight that even simple, defined growth media can have inhibitory effects on some species and that such negative effects need to be included in our models. Based on this, we present new guidelines to correctly distinguish between different interaction types, such as cross-detoxification and cross-feeding.