title: Antimicrobial susceptibility testing using MYCO test-system and MIC distribution of 8 drugs against clinical isolates from Shanghai of Nontuberculous Mycobacteria

abstract: Given the increased incidence and prevalence of nontuberculous mycobacteria (NTM) diseases and the natural resistance of NTM to multiple antibiotics, in vitro susceptibility testing of different NTM species against drugs from the MYCO test system and new applied drugs are required. 241 NTM clinically isolates were under analyzed, including 181 slowly growing mycobacterium (SGM) and 60 rapidly growing mycobacterium (RGM). The Sensititre SLOMYCO and RAPMYCO panels were used for the drug susceptibility testing to commonly used anti-NTM antibiotics. Furthermore, Minimum inhibitory concentration (MIC) distributions were determined against 8 potential anti-NTM drugs, including vancomycin (VA), bedaquiline (BDQ), delamanid (DLM), faropenem (FAR), meropenem (MPM), clofazimine (CFZ), avibactam (CAZ), and Cefoxitin (FOX) and epidemiological cut-off values (ECOFFs) were analyzed using ECOFFinder. The results showed that most of the SGM strains were susceptible to clarithromycin (CLA), rifampicin (RFB) from the SLOMYCO panels and BDQ, CFZ from the 8 applied drugs, while, RGM strains were susceptible to tigecycline (TGC) from the RAPMYCO panels and also BDQ, CFZ. The ECOFF values of CFZ were 0.25g/ml, 0.25g/ml, 0.5g/ml, and 1g/ml for M. kansasii, M. avium, M. intracellulare, and M. abscessus, respectively, and BDQ was 0.5g/ml for the same four prevalent NTM species. Due to the weak activity of the other 6 drugs, no ECOFF was determined. This study on the susceptibility of NTM includes 8 potential anti-NTM drugs and a large sample size of Shanghai clinical isolates. and demonstrated that BDQ and CFZ had efficient activities against different NTM species in vitro, which can be applied for the treatment of NTM diseases.


title: Subtyping evaluation of Salmonella Enteritidis using SNP and core genome MLST with nanopore reads

abstract: Whole genome sequencing (WGS) for public health surveillance and epidemiological investigation of foodborne pathogens predominantly relies on sequencing platforms that generate short reads. Continuous improvement of long-read nanopore sequencing such as Oxford Nanopore Technologies (ONT) presents a potential for leveraging multiple advantages of the technology in public health and food industry settings, including rapid turnaround and onsite applicability in addition to superior read length. However, evaluation, standardization and implementation of the ONT approach to WGS-based, strain-level subtyping is challenging, in part due to its relatively high base-calling error rates and frequent iterations of sequencing chemistry and bioinformatic analytics. Using an established cohort of Salmonella Enteritidis isolates for subtyping evaluation, we assessed the technical readiness of ONT for single nucleotide polymorphism (SNP) analysis and core-genome multilocus sequence typing (cgMLST) of a major foodborne pathogen. By multiplexing three isolates per flow cell, we generated sufficient sequencing depths under seven hours of sequencing for robust subtyping. SNP calls by ONT and Illumina reads were highly concordant despite homopolymer errors in ONT reads (R9.4.1 chemistry). In silico correction of such errors allowed accurate allelic calling for cgMLST and allelic difference measurements to facilitate heuristic detection of outbreak isolates. Our study established a baseline for the continuously evolving nanopore technology as a viable solution to high quality subtyping of Salmonella, delivering comparable subtyping performance when used standalone or together with short-read platforms.

category: microbiology


title: Large-Scale, Multi-Year Microbial Community Survey of a Freshwater Trout Aquaculture Facility

abstract:
Aquaculture is an important tool for solving growing worldwide food demand, but infectious diseases of the farmed animals represent a serious roadblock to continued industry growth. Therefore, it is essential to understand the microbial communities that reside within the built environments of aquaculture facilities to identify reservoirs of bacterial pathogens and potential correlations between commensal species and specific disease agents. Here, we present the results from three years of sampling a commercial rainbow trout aquaculture facility. The sampling was focused on the early-life stage hatchery building and included sampling of the facility source water and outdoor production raceways. We observed that the microbial communities residing on the abiotic surfaces within the hatchery were distinct from those residing on the surfaces of the facility water source as well as the production raceways, despite similar communities in the water column at each location. Within the hatchery building, most of the microbial classes and families within surface biofilms were also present within the water column, suggesting that these biofilms are seeded by a unique subgroup of microbial taxa from the water. Lastly, we detected a common fish pathogen, Flavobacterium columnare, within the hatchery, including at the source water inlet. Importantly, the relative abundance of this pathogen was correlated with clinical disease. Our results characterized the microbial communities in an aquaculture facility, established that the hatchery environment contains a unique community composition, and demonstrated that a specific fish pathogen resides within abiotic surface biofilms and is seeded from the natural source water.

ImportanceThe complex microbial consortium residing in the built environment of aquaculture facilities is poorly understood. In this study, we provide a multi-year profile of the surface- and water-associated microbial communities of this biome. The results demonstrated that distinct community structures exist in the water and on surfaces. Furthermore, it was shown that a common and economically impactful bacterial pathogen, F. columnare, is continually introduced via the source water, is widespread within surface biofilms in the hatchery environment, and is likely amplified within these raceways but does not always cause disease despite being present. These results advance our understanding of pathogen localization at fish farms, show the interplay between host and environmental microbiomes, and reveal the importance of microbial community sequencing in aquaculture for identifying potential beneficial and harmful microbes. This study adds to the aquaculture microecology dataset and enhances our ability to understand this environment from a "One Health" perspective.


title: Cyclic nucleotide-induced superhelical structure activates a bacterial TIR immune effector

abstract: Cyclic nucleotide signalling is a key component of anti-viral defence in all domains of life, from bacteria to humans. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway 1, which originated in bacteria 2. These defence systems require a sensor domain such as STING or SAVED to bind the cyclic nucleotide, coupled with an effector domain that causes cell death when activated by destroying essential biomolecules 3. One example is the TIR (Toll/interleukin-1 receptor) domain, which degrades the essential cofactor NAD+ when activated in response to pathogen invasion in plants and bacteria 2,4,5 or during nerve cell programmed death 6. Here, we show that a bacterial anti-viral defence system generates a cyclic tri-adenylate (cA3) signal which binds to a TIR-SAVED effector, acting as the "glue" to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organise and complete a composite active site, allowing NAD+ degradation. Our study illuminates a striking example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.

category: biochemistry


title: In search of the universal method: a comparative survey of bottom-up proteomics sample preparation methods

abstract: Robust, efficient and reproducible protein extraction and sample processing is a key step for bottom-up proteomics analyses. While many sample preparation protocols for mass spectrometry have been described, selecting an appropriate method remains challenging, since some protein classes may require specialized solubilization, precipitation, and digestion procedures. Here we present a comprehensive comparison of 16 most widely used sample preparation methods, covering in-solution digests, device-based methods, as well as commercially available kits. We find a remarkably good performance of the majority of the protocols with high reproducibility, little method dependencies and low levels of artifact formation. However, we revealed method-dependent differences in the recovery of specific protein features, which we summarized in a descriptive guide-matrix. Our work thereby provides a solid basis for the selection of MS sample preparation strategies for a given proteomics project.


title: Huntingtin turnover: Modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550

abstract: Huntingtons disease (HD) is a neurodegerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phospho-sites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein, by proteomic and phospho-proteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phospho-sites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together these findings highlight categories of phospho-sites that merit further study and provide a phospho-site kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells.


title: Combination therapy targeting inflammasome and fibrogenesis alleviates inflammation and fibrosis in a zebrafish model of silicosis

abstract: Silicosis is a long-term lung disease caused by the inhalation of large amounts of crystalline silica dust. As there is no effective treatment available, patients are provided with supportive care, and some may be considered for lung transplantation. There is therefore an evident need for a better understanding of the diseases biology and for identifying new therapeutic targets and therapies. In this context, our group has developed a larval zebrafish model of silicosis by injecting silica crystals into the hindbrain ventricle, a cavity into which immune cells can be recruited and that mimics the alveolar environment of the human lung. The injection of silica crystals into this cavity led to the initiation of local and systemic immune responses driven through both TLR- and inflammasome-dependent signaling pathways, followed by fibrosis, as happens in human patients. The combination of the inflammasome inhibitor VX-765 and the antifibrotic agent pirfenidone was found to be the best therapy to alleviate both inflammation and fibrosis. The zebrafish model of silicosis developed here is a unique tool that will shed light onto the molecular mechanisms involved in the progression of this devastating disease and for identifying novel drugs that improve the quality of life of silicosis patients.

category: immunology


title: Dual mode of PGRP-LE-dependent NF-κB pathway activation in bacteria infected guts

abstract: Some of the interactions between prokaryotes and eukaryotes are mediated by a molecular dialogue between microbial MAMPs and host PRRs. Bacteria-derived peptidoglycan, which possesses all the characteristics of a MAMP, is detected by membrane-bound or cytosolic PRRs belonging to various families of proteins in both animals and plants (PGRP, Nod, Lys-M...). If the identity and the epistatic relationship between the downstream components of the signaling cascades activated upon PGN/PRR interactions are well characterized, little is known about the subcellular events requires to translate these early sensing steps into downstream target gene transcription. Using a model of Drosophila enteric infection, we show that gut-associated bacteria can induce PGRP-LE intracellular aggregation. Observed in both enterocytes and entero-endocrine cells, these aggregates were found to co-localize with the early endosome marker Rab5. In vivo functional analysis further demonstrates that, whereas some PGRP-LE target genes, such as antimicrobial peptides, can be activated independently of Rab5, other such as the PGRP-SC1 amidase, need the combine action of PGRP-LE and Rab5 to be transcribed. These results demonstrate how by using different intracellular signaling routes, the same ligand/receptor complex can activate different target genes in the same cell.


title: Mapping the T cell repertoire to a complex gut bacterial community

abstract: Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with a complex defined community (97 or 112 bacterial strains) and profile T cell responses to each strain individually. Unexpectedly, the pattern of T cell responses suggests that many T cells in the gut repertoire recognize multiple bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all of the bacteria-specific TCRs exhibit a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that are polyspecific for 18 Firmicutes in the community. By screening three pooled bacterial genomic libraries against 13 pooled hybridomas, we discover that they share a single target: a conserved substrate-binding protein (SBP) from an ABC transport system. Treg and Th17 cells specific for an epitope from this protein are abundant in community-colonized and specific-pathogen-free mice. Our work reveals that T cell recognition of Firmicutes is focused on a widely conserved cell-surface antigen, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.


title: Role of High Mobility Group B protein HmbA, orthologue of yeast Nhp6p, in Aspergillus nidulans

abstract: The mammalian HMGB1 protein belongs to the high-mobility-group B (HMG-B) family, which is not only architectural but also functional element of the chromatin. The fungal counterpart of HMGB1 was identified in Saccharomyces cerevisiae as Nhp6p and the pleiotropic physiological functions of this protein were thoroughly studied during the last decades. Although filamentous Ascomycete fungi also possess the orthologues of Nhp6p, their physiological functions, apart from their role in the sexual development, have not been investigated, yet. Here we study the physiological functions of the Nhp6p orthologue HmbA from Aspergillus nidulans in the primary and secondary metabolism, stress tolerance, hypha elongation and maintenance of polarized growth through the analysis of hmbA deletion mutant. We also revealed that the endochitinase ChiA acts in the cell wall remodelling and contributes to polar growth. Additionally, by conducting heterologous expression studies, we further demonstrated that HmbA and Nhp6p is interchangeable for several functions. We hypothesized that the fully complemented functions might predominantly depend on the DNA binding ability of the HmbA and Nhp6p proteins rather than on the interaction of these HMG-B proteins with other functional protein components of the chromatin.


title: Efficient and accurate prime editing strategy to correct genetic alterations in hiPSC using single EF-1alpha driven all-in-one plasmids

abstract: Prime editing (PE) is currently the most effective and versatile technology to make targeted alterations in the genome. Several improvements to the PE machinery have recently been made, and have been tested in a range of model systems, including immortalized cell lines, stem-cells and animal models. While nick RNA (ncRNA)-dependent PE systems like PE3 and PE5 are currently considered to be the most effective, they come with undesired indels or SNVs at the edit locus. Here, we aimed to improve ncRNA-independent systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids, driven by a tissue-broad EF-1alpha promoter, that is especially suitable for human iPSC models, and linked to a GFP tag for fluorescent based sorting. These novel pAIO systems effectively corrected mutations observed in patients suffering from epileptic encephalopathy, including a truncating SCN1A R612* variant in HEK293T cells and a gain-of-function KCNQ2 R201C variant in patient-derived hiPSC, with edit efficiency up to 50%. We also show that introducing additional silent PAM-removing mutations can negatively influence edit efficiency. Finally, we observed an absence of genome-wide PE off-target effects at pegRNA homology sites. Taken together, our study shows an improved efficacy and accuracy of EF-1alpha driven ncRNA-independent pAIO PE plasmids in hiPSC.

category: molecular biology


title: Nrf1 is an indispensable redox-determining factor for mitochondrial homeostasis by integrating multi-hierarchical regulatory networks

abstract: To defend a vast variety of challenges in the oxygenated environments, all life forms have been evolutionally established a set of antioxidant, detoxification and cytoprotective systems during natural selection and adaptive survival, in order to maintain cell redox homeostasis and organ integrity in the healthy development and growth. Such antioxidant defense systems are predominantly regulated by two key transcription factors Nrf1 and Nrf2, but the underlying mechanism(s) for their coordinated redox control remains elusive. Here, we found that loss of full-length Nrf1 led to a dramatic increase in reactive oxygen species (ROS) and oxidative damages in Nrf1-/- cells, and this increase was not eliminated by drastic elevation of Nrf2, even though the antioxidant systems were also substantially enhanced by hyperactive Nrf2. Further studies revealed that the increased ROS production in Nrf1-/- resulted from a striking impairment in the mitochondrial oxidative respiratory chain and its gene expression regulated by nuclear respiratory factors, called PalNRF1 and GABPNRF2. In addition to antioxidant capacity of cells, glycolysis was greatly augmented by aberrantly-elevated Nrf2, so to partially relieve the cellular energy demands, but aggravate its mitochondrial stress. The generation of ROS was also differentially regulated by Nrf1 and Nrf2 through miR-195 and/or mIR-497-mediated UCP2 pathway. Consequently, the epithelial-mesenchymal transformation (EMT) of Nrf1-/- cells was activated by putative ROS-stimulated signaling via MAPK, HIF1, NF-[Formula]B, PI3K and AKT, all players involved in cancer development and progression. Taken together, it is inferable that Nrf1 acts as a potent integrator of redox regulation by multi-hierarchical networks.


title: Structural basis for activation of Arf1 at the Golgi complex

abstract: The Golgi complex is the central sorting station of the eukaryotic secretory pathway. Traffic through the Golgi requires activation of Arf GTPases that orchestrate cargo sorting and vesicle formation by recruiting an array of effector proteins. Arf activation and Golgi membrane association is controlled by large guanine nucleotide exchange factors (GEFs) possessing multiple conserved regulatory domains. Here we present cryoEM structures of full-length Gea2, the yeast paralog of the human Arf-GEF GBF1, that reveal the organization of these regulatory domains and explain how Gea2 binds to the Golgi membrane surface. We find the GEF domain adopts two different conformations compatible with different stages of the Arf activation reaction. The structure of a Gea2-Arf1 activation intermediate suggests the movement of the GEF domain primes Arf1 for membrane insertion upon GTP binding. We propose that conformational switching of Gea2 during the nucleotide exchange reaction promotes membrane insertion of Arf1.

category: cell biology


title: Thermal acclimation to warmer temperatures can protect host populations from both further heat stress and the potential invasion of pathogens

abstract: Phenotypic plasticity in response to shifts in temperature, known as thermal acclimation, is an essential component of the ability of a species to cope with environmental change. Not only does this process potentially improve an individuals thermal tolerance, it will also act simultaneously on various fitness related traits that determine whether a population increases or decreases in size. In light of global change, thermal acclimation therefore has consequences for population persistence that extend beyond simply coping with heat stress. This particularly important when we consider the additional threat of parasitism associated with global change, as the ability of a pathogen to invade a host population depends on both its capacity to proliferate within a host and spread between hosts, and thus the supply of new susceptible hosts in a population. Here, we use the host Daphnia magna and its bacterial pathogen Pasteuria ramosa to investigate how thermal acclimation may impact various aspects of host and pathogen performance at the scale of both an individual and the population. We independently test the effect of maternal thermal acclimation and direct thermal acclimation on host thermal tolerance, measured as knockdown times, as well as host fecundity and lifespan, and pathogen infection success and spore production. We find that direct thermal acclimation enhances host thermal tolerance and intrinsic rates of population growth, despite a decline observed for host fecundity and lifespan. Pathogens, on the other hand, faired consistently worse at warmer temperatures at the within-host scale, and also in their potential to invade a host population. Our results suggest that hosts could benefit more from warming than their pathogens, but highlight that considering both within- and between-host thermal performance, including thermal tolerance and fitness traits, is needed to fully appreciate how increasing thermal variability will impact host and pathogen populations.

category: ecology


title: Impact of Salmonella genome rearrangement on gene expression

abstract: In addition to nucleotide variation, many bacteria also undergo changes at a much larger scale via rearrangement of their genome structure around long repeat sequences. These rearrangements result in genome fragments shifting position and/or orientation in the genome without necessarily affecting the underlying nucleotide sequence. To date, scalable techniques have not been applied to genome structure (GS) identification, so it remains unclear how extensive this variation is and the extent of its impact upon gene expression. However, the emergence of multiplexed, long-read sequencing overcomes the scale problem, as reads of several thousand bases are routinely produced that can span long repeat sequences to identify the flanking chromosomal DNA, allowing GS identification. Genome rearrangements were generated in Salmonella enterica serovar Typhi through long-term culture at ambient temperature. Colonies with rearrangements were identified via long-range PCR and subjected to long-read nanopore sequencing to confirm genome variation. Four rearrangements were investigated for differential gene expression using transcriptomics. All isolates with changes in genome arrangement relative to the parent strain were accompanied by changes in gene expression. Rearrangements with similar fragment movements demonstrated similar changes in gene expression. The most extreme rearrangement caused a large imbalance between the origin and terminus of replication and was associated with differential gene expression as a factor of distance moved towards or away from the origin of replication. Genome structure variation may provide a mechanism through which bacteria can quickly adapt to new environments and warrants routine assessment alongside traditional nucleotide level measures of variation.

category: genomics


title: Three-dimensional envelope and subunit interactions of the plastid-encoded RNA polymerase from Sinapis alba

abstract: RNA polymerases (RNAPs) involved in gene transcription are found in all living organisms with degrees of complexity ranging from single polypeptide chains to multimeric enzymes. In the chloroplasts, the nuclear-encoded RNA polymerase and the plastid-encoded RNA polymerase (PEP) are both involved in the selective transcription of the plastid genome. The PEP is a prokaryotic-type multimeric RNAP found in different states depending on light stimuli and cell identity. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa regarded as essential for chloroplast biogenesis. A purification procedure compatible with structural analysis was used to enrich the native PEP from Sinapis alba chloroplasts. Mass spectrometry (MS)-based proteomic analysis identified the core components, the PAPs and additional members, and coupled to cross-linking (XL-MS) provided initial structural information about the relative position of PEP subunits. Sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that the overall shape of the catalytic core and the residues essential for the catalytic activity are conserved. However, variations are observed at the surface of the core, some of them corresponding to PAP binding sites as suggested by XL-MS experiments. Using negative stain electron microscopy, the PEP 3D envelope was calculated. 3D classification shows that the protrusions which we attribute to the PAPs are firmly associated with the catalytic core. Overall, the shape of the S. alba PEP envelope is different from that of RNAPs.


title: Functional brain region-specific neural spheroids for modeling neurological diseases and therapeutics screening

abstract: 3D spheroids have emerged as powerful drug discovery tools given their high-throughput screening (HTS) compatibility. Here, we established a method for generating functional neural spheroids with differentiated human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes at cell type compositions mimicking specific regions of the human brain. Recordings of intracellular calcium oscillations were used as functional assays, and the utility of this spheroids system was shown through disease modelling, drug testing, and formation of assembloids to model neurocircuitry. We developed disease models for Alzheimers and Parkinsons Disease through incorporating genetically engineered cells into spheroids, and Opioid Use Disorder by chronically treating spheroids with DAMGO. We reversed baseline functional deficits in each disease model with clinically approved treatments and showed that assembloid activity can be chemogenetically manipulated. Here, we lay the groundwork for brain region-specific spheroids as a robust functional assay platform for HTS studies.

category: bioengineering


title: Force Field Dependent DNA Breathing Dynamics: A Case Study of Hoogsteen Base Pairing in A6-DNA

abstract: The Hoogsteen (HG) base pairing (bp) conformation, commonly observed in damaged and mutated DNA helix, facilitates DNA repair mechanism and DNA recognition by proteins and small molecules. The free energy difference of HG and Watson-Crick (WC) base pairs has been computed in previous computational studies. But, the mechanism of the conformational transition is not well understood. A detailed understanding of the process of WC to HG base pair transition can provide deeper understanding of DNA repair and recognition. In an earlier study, we explored the free energy land-scape for this process using extensive computer simulation with CHARMM36 force field for the nucleic acid. In this work, we study the impact of force field models in describing the WC to HG base pairing transition using the meta-eABF enhanced sampling, quasi-harmonic entropy calculation, and non-bonded energy analysis. The secondary structures of the both base pairing forms and the topology of the free energy landscapes were consistent over different force field models, although the relative free energy, entropy and the interaction energies tend to vary. The relative stability of WC and HG conformation is dictated by a delicate balance between the enthalpic stabilization and the reduced entropy of the structurally rigid HG structure. These findings provide a holistic view on the impact of force fields on the DNA base pair dynamics leading to the formation of HG conformation and will facilitate future computational investigations of DNA repair and recognition mechanism in physiologically relevant conditions.

category: biophysics


title: The MIX domain is required for T6SS-mediated effector secretion

abstract: Bacteria use diverse classes of secreted polymorphic toxins to outcompete bacterial rivals. The MIX domain defines a widespread class of polymorphic type VI secretion system (T6SS) effectors, many of which are horizontally shared. Although several MIX-effectors have been investigated, the role of the MIX domain and the activity of many fused toxin domains remain unknown. Here, we use the Vibrio parahaemolyticus MIX-effector VPA1263 to demonstrate that MIX is necessary for T6SS-mediated secretion; however, we find that it is dispensable for interaction with the secreted T6SS tail tube component, VgrG. We also identify a C-terminal HNH nuclease toxin domain in VPA1263, and we demonstrate that it functions as a DNase during interbacterial competition. Finally, we identify variants of the genomic island containing vpa1263 in various vibrios, each carrying a different cargo of antibacterial T6SS effectors or anti-phage defense systems. We propose that these genomic islands are hotspots for bacterial defensive weapons.


title: Polaramycin B, and not physical interaction, is the signal that rewires fungal metabolism in the Streptomyces - Aspergillus interaction.

abstract: Co-culturing the bacterium Streptomyces rapamycinicus and the ascomycete Aspergillus nidulans has previously been shown to trigger the production of orsellinic acid and its derivates in the fungal cells. Based on these studies it was assumed that direct physical contact between fungal and bacterial cells is a prerequisite for the metabolic reaction of the fungus that involves an amino acid starvation response and concomitant activating chromatin modifications at the affected biosynthetic gene clusters (BGCs). Here we show that not physical contact, but a guanidine containing macrolide, named Polaramycin B, triggers the response. This metabolite is produced constitutively by the bacterium and production does not need stimulation by a fungal co-culture. Polaramycin B freely diffuses in an aqueous environment and provokes the production of orsellinic acid above a certain concentration. In addition to orsellinic acid, the production of several other secondary metabolites was triggered by Polaramycin B. Our genome-wide transcriptome analysis showed that Polaramycin B treatment causes down-regulation of fungal genes necessary for membrane stability as well as genes involved in general metabolism and growth. A compensatory genetic response can be observed in the fungus that included up-regulation of BGCs and genes necessary for ribosome biogenesis, translation and membrane stability. Our work discovered a specific chemical communication in which Polaramycin B, an antifungal metabolite constitutively produced by different Streptomycete species, leads to the production of fungal defence chemicals and to the up-regulation of genes necessary to compensate for the cellular damage caused by Polaramycin B.


title: Physics of Self-Assembly and Morpho-Topological Changes of Klebsiella Pneumoniae in Desiccating Sessile Droplets

abstract: We have investigated the flow and desiccation-driven self-assembly of Klebsiella Pneumoniae in the naturally evaporating sessile droplets. Klebsiella Pneumoniae exhibits extensive changes in its morphology and forms unique patterns as the droplet dries, revealing hitherto unexplored rich physics governing its survival and infection strategies. Self-assembly of bacteria at the droplet contact line is characterized by order-to-disorder packing transitions with high packing densities and excessive deformations (bacteria deforms nearly twice its original length scales). In contrast, thin-film instability-led hole formation at the center of the droplet engenders spatial packing of bacteria analogous to honeycomb weathering. The varying physical forces acting on bacteria based on their respective spatial location inside the droplet cause an assorted magnitude of physical stress. Self-assembly favors the bacteria at the rim of the droplet, leading to enhanced viability and pathogenesis on the famously known "coffee ring" of the droplet compared to the bacteria present at the center of the droplet residue. Mechanistic insights gained via our study can have far-reaching implications for bacterial infection through droplets, e.g., through open wounds.


title: A systematic approach to study protein-substrate specificity enables the identification of Ssh1 substrate range

abstract: Many cellular functions are carried out by protein pairs, or families, providing robustness alongside functional diversity. For such processes, it remains a challenge to map the degree of specificity versus promiscuity. Protein-protein interactions (PPIs) can be used to inform on these matters as they highlight cellular locals, regulation and, in cases where proteins affect other proteins - substrate range. However, methods to study transient PPIs systematically are underutilized. In this study we create a novel approach to study stable as well as transient PPIs in yeast. Our approach, Cel-lctiv (CELlular biotin-Ligation for Capturing Transient Interactions in Vivo), uses high- throughput pairwise proximity biotin ligation for uncovering PPIs systematically and in vivo. As a proof of concept we study the homologous translocation pores Sec61 and Ssh1. We show how Cel-lctiv can uncover the unique substrate range for each translocon allowing us to pinpoint a specificity determinator driving interaction preference. More generally this demonstrates how CEl-lctiv can provide direct information on substrate specificity even for highly homologous proteins.


title: High-throughput single-cell sequencing for retroviral reservoir characterization

abstract: During the course of infection, human immunodeficiency virus (HIV) maintains a stably integrated reservoir of replication-competent proviruses within the host genome that are unaffected by antiretroviral therapy. Curative advancements rely heavily on targeting the reservoir, though determinants of its evolutionary origins remain ill-supported through current strategies and are often limited by sample variety. Here, we describe a single-cell deoxyribonucleic acid sequencing (scDNA-seq) method, optimized for sequencing of proviral and host DNA from a treatment-interrupted HIV animal model. We report its benefits for improving viral reservoir resolution to support critical evolutionary events otherwise considered unreliable using traditional viral envelope gene signal alone, as well as comparative advantages to existing near-full-length genome sequencing methods. Given the variety of proviral characteristics that may influence viral rebound, scDNA-seq holds immense value in its ability to streamline many of the present-day applications available in viral reservoir studies, such as integration status and putative replication competency.


title: Cirrhosis and Inflammation Regulates CYP3A4 Mediated Chemoresistance in Vascularized Hepatocellular Carcinoma-on-a-chip

abstract:
Understanding the effects of inflammation and cirrhosis on the regulation of drug metabolism during the progression of hepatocellular carcinoma (HCC) is critical for developing patient-specific treatment strategies. In this work, we created novel three-dimensional vascularized HCC-on-a-chips (HCCoC), composed of HCC, endothelial, stellate, and Kupffer cells tuned to mimic normal or cirrhotic liver stiffness. HCC inflammation was controlled by tuning Kupffer macrophage numbers, and the impact of cytochrome P450-3A4 (CYP3A4) was investigated by culturing HepG2 HCC cells transfected with CYP3A4 to upregulate expression from baseline. This model allowed for the simulation of chemotherapeutic delivery methods such as intravenous injection and transcatheter arterial chemoembolization (TACE). We showed that upregulation of metabolic activity, incorporation of cirrhosis and inflammation, increase vascular permeability due to upregulated inflammatory cytokines leading to significant variability in chemotherapeutic treatment efficacy. Specifically, we show that further modulation of CYP3A4 activity of HCC cells by TACE delivery of doxorubicin provides an additional improvement to treatment response and reduces chemotherapy-associated endothelial porosity increase. The HCCoCs were shown to have utility in uncovering the impact of the tumor microenvironment (TME) during cancer progression on vascular properties, tumor response to therapeutics, and drug delivery strategies.

Statement of SignificanceRegulation of drug metabolism during the cancer progression of hepatocellular carcinoma (HCC) can be influential to develop personalized treatment strategies. We created novel vascularized hepatocellular carcinoma-chip (HCCoC) composed of tunable collagen and four main liver-specific cell lines to be used as a preclinical tool. In this model, we found cancer evolution states such as inflammation and cirrhosis increases vascular permeability progressively as a result of increased inflammatory cytokines. Furthermore, delivery of doxorubicin only with embolization improved treatment efficacy by decreasing CYP3A4 activity, which can modulate treatment outcome. Overall, we found different disease states can be influential on CYP3A4, thus its targeting can improve HCC treatment outcome.


title: Microbial population dynamics decouple nutrient affinity from environmental concentration

abstract: How the growth rate of a microbial population depends on the availability of chemical nutrients and other resources in the environment is a fundamental question in microbiology. Models of this dependence, such as the well-known Monod model, are characterized by a threshold concentration (or affinity) of the resource, above which the population can grow near its maximum rate. Even though this concept is a core element of microbiological and ecological modeling, there is little empirical and theoretical evidence for the evolved diversity of these thresholds. In particular, does resource scarcity drive populations to evolve commensurately lower thresholds? To address this question, we perform the largest-to-date meta-analysis of resource threshold data across a wide range of organisms and resources, substantially expanding previous surveys. We find that the thresholds vary across orders of magnitude, even for the same organism and resource. To explain this variation, we develop an evolutionary model to show that demographic fluctuations (genetic drift) can constrain the adaptation of resource thresholds. We find that this effect fundamentally differs depending on the type of population dynamics: populations undergoing periodic bottlenecks of fixed size will adapt their thresholds in proportion to the environmental resource concentration, but populations undergoing periodic dilutions of fixed size will evolve thresholds that are largely decoupled from the environmental concentration. Our model not only provides testable predictions for laboratory evolution experiments, but it also reveals how an evolved resource threshold may not reflect the organisms environment. In particular, this explains how organisms in nutrient-rich environments can still evolve facultative growth at low resource concentrations. Altogether our results demonstrate the critical role of population dynamics in shaping fundamental ecological traits.


title: Spt6 directly interacts with Cdc73 and is required for Paf1C recruitment to active genes

abstract: Paf1C is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C recruitment to Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 remain to be elucidated. Using an in vivo site-specific protein cross-linking strategy, we identified direct interactions between Cdc73 and two components of the elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Through in vitro protein binding assays and crosslinking/mass spectrometry, we show that Cdc73 and Spt6 can interact in the absence of additional factors and propose a binding interface. Rapid depletion of Spt6 dissociated Paf1 from chromatin and altered patterns of Paf1C-dependent histone modifications genome-wide. These results reveal previously unrecognized interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to Paf1C recruitment to active genes in Saccharomyces cerevisiae.


title: Best practice recommendations for sample preservation in metabarcoding studies: a case study on diatom environmental samples

abstract:
The development of DNA metabarcoding and High-Throughput Sequencing for diatoms is nowadays offering an interesting approach to assess their communities in freshwater and marine ecosystems. In the context of the implementation of these genomic methods to environmental monitoring, protocol constraints are moving from scientific to operational applications, requiring operational guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies.

The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design included four freshwater and two marine samples from sites of diverse ecological characteristics. The impact of the preservation and storage was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, community composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflects the site origin. Only rare taxa (below 100 reads) differed among methods and durations. Thus, importance of preservation method choice is important for low-density species (rare, invasive, threatened or toxic species). However, for biomonitoring purposes, freshwater ecological index values were not affected whatever the preservation method and duration considered (including ethanol preservation), reflecting the site ecological status.

This study proved that diatom metabarcoding is robust enough to replace or complement the current approach based on morphotaxonomy, paving the way to new possibilities for biomonitoring. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring.


title: Elemental content of a host-parasite relationship in the threespine stickleback

abstract: Parasite infections are ubiquitous and their effects on hosts may play a role in ecosystem processes. Ecological stoichiometry provides a framework to study linkages between consumers and ecosystem process, but the stoichiometric traits of host-parasite associations are rarely quantified. Specifically, whether parasites elemental ratios closely resemble those of their host or if infection is related to host stoichiometry remains less known. To answer such questions, we measured the elemental content (%C, %N, and %P) and ratios (C:N, C:P, and N:P) of parasitized and unparasitized Gasterosteus aculeatus (three-spined stickleback) and their cestode parasite, Schistocephalus solidus. Host and parasite elemental content were distinct from each other, and parasites were generally higher in %C and lower in %N and %P. Parasite infections were related to some elemental ratios, specifically C:N, with more intense parasite infections corresponding to hosts with lower C:N ratio. Parasite stoichiometry was independent of their host and there was no relationship between host and parasite stoichiometry. Instead, parasite body mass and parasite density were important drivers of parasite stoichiometry where larger parasites had lower %C, %N, and %P. Overall, these potential effects of parasite infections on host stoichiometry along with parasites distinct elemental compositions suggest parasites may further contribute to how hosts store and cycle nutrients.


title: Global genomic epidemiology, resistome, virulome and plasmidome of the Extraintestinal Pathogenic Escherichia coli (ExPEC) ST38 lineage

abstract: Most of the extraintestinal human infections worldwide are determined by some specific Extraintestinal Pathogenic Escherichia coli (ExPEC) lineages, which also present a zoonotic character. One of these lineages belongs to ST38, which is considered a uropathogenic/enteroaggregative E. coli hybrid and a high-risk globally disseminated ExPEC. To get insights on the aspects of ST38 epidemiology and evolution as a multidrug resistant and pathogenic lineage, this study performed a global comparative genomic analysis on ST38 genomes. A total of 376 genomes recovered from environments, humans, livestock, wild and domestic animals in all continents throughout three decades were analyzed considering the ST38 phylogenomics, resistome, plasmidome, and virulome. In general, our analyses revealed that, independently of host sources and geographic origin, these genomes were distributed in two clusters comprising clonal and non-clonal genomes. Moreover, the ST38 accessory genome was not strictly associated with clusters and sub-clusters. Interestingly, the High Pathogenicity Island (HPI) prevailed in the major cluster (cluster 2), which comprised most of the genomes from human origin recovered worldwide (2000 to 2020). In addition, the ExPEC ST38 harbors a huge and diverse plasmidome that might contribute to the evolution of this lineage, reflecting in ST38 ability to live in a diversity of niches as a commensal or pathogenic organism.


title: Thermal adaptation and fatty acid profiles of bone marrow and muscles in mammals: implications of a study of caribou (Rangifer tarandus caribou)

abstract: Mammals have evolved several physiological mechanisms to cope with changes in ambient temperature. Particularly critical among them is the process of keeping cells in a fluid phase to prevent metabolic dysfunction. In this paper, we examine variation in the fatty acid composition of bone marrow and muscle tissues in the cold-adapted caribou (Rangifer tarandus caribou) to determine whether there are systematic differences in fatty acid profiles between anatomical regions that could potentially be explained by thermal adaptation. Our results indicate that the bone marrow and muscle tissues from the appendicular skeleton are more unsaturated than the same tissues in the axial skeleton, a finding that is consistent with physiological adaptation of the appendicular regions to thermal challenges. Because mechanisms of thermal adaptation appear to be widely shared among terrestrial mammals, we suggest that the same patterns may prevail in other species, possibly including humans.

category: zoology


title: A potent and selective inhibitor for the modulation of MAGL activity in the neurovasculature

abstract: Chronic inflammation and blood-brain barrier dysfunction are key pathological hallmarks of neurological disorders such as multiple sclerosis, Alzheimers disease and Parkinsons disease. Major drivers of these pathologies include pro-inflammatory stimuli such as prostaglandins, which are produced in the central nervous system by the oxidation of arachidonic acid in a reaction catalyzed by the cyclooxygenases COX1 and COX2. Monoacylglycerol lipase hydrolyzes the endocannabinoid signaling lipid 2-arachidonyl glycerol, enhancing local pools of arachidonic acid in the brain and leading to cyclooxygenase-mediated prostaglandin production and neuroinflammation. Monoacylglycerol lipase inhibitors were recently shown to act as effective anti-inflammatory modulators, increasing 2-arachidonyl glycerol levels while reducing levels of arachidonic acid and prostaglandins, including PGE2 and PGD2. In this study, we characterized a novel, highly selective, potent and reversible monoacylglycerol lipase inhibitor (MAGLi 432) in a mouse model of lipopolysaccharide-induced blood-brain barrier permeability and in both human and mouse cells of the neurovascular unit: brain microvascular endothelial cells, pericytes and astrocytes. We confirmed the expression of monoacylglycerol lipase in specific neurovascular unit cells in vitro, with pericytes showing the highest expression level and activity. However, MAGLi 432 did not ameliorate lipopolysaccharide-induced blood-brain barrier permeability in vivo or reduce the production of pro-inflammatory cytokines in the brain. Our data confirm monoacylglycerol lipase expression in mouse and human cells of the neurovascular unit and provide the basis for further cell-specific analysis of MAGLi 432 in the context of blood-brain barrier dysfunction caused by inflammatory insults.

category: neuroscience


title: The Tryptophan Metabolizing Enzyme Indoleamine 2,3-Dioxygenase 1 Regulates Polycystic Kidney Disease Progression

abstract: Autosomal dominant polycystic kidney disease (ADPKD), the most common monogenic nephropathy, is characterized by phenotypic variability exceeding genic effects. Dysregulated metabolism and immune cell function are key disease modulators. The tryptophan metabolites, kynurenines, produced through IDO1, are known immunomodulators. Here, we study the role of tryptophan metabolism in PKD using an orthologous disease model (C57Bl/6J Pkd1RC/RC). We found elevated kynurenine and IDO1 levels in Pkd1RC/RC kidneys versus wildtype. Further, IDO1 levels were increased in ADPKD cell lines and patient cyst cells. Genetic Ido1 loss in Pkd1RC/RC animals resulted in reduced PKD severity as measured by %kidney weight/body weight and cystic index. Consistent with a immunomodulatory role of kynurenines, Pkd1RC/RC;Ido1-/- mice presented with significant changes in the cystic immune microenvironment (CME) versus controls. Of note, kidney macrophage numbers decreased and CD8+ T cell numbers increased, both known PKD modulators. Also, pharmacological IDO1 inhibition using a tryptophan analog in Pkd1RC/RC animals resulted in less severe PKD versus controls with similar changes in the CME as in the genetic model. Together, our data suggest that tryptophan metabolism is dysregulated in ADPKD and that its inhibition results in changes to the CME and slows disease progression, making IDO1 a novel therapeutic target for ADPKD.


title: The H2A.Z.1/PWWP2A/NuRD-associated protein HMG20A controls early head and heart developmental transcription programs

abstract: Abstract/SummarySpecialized chromatin-binding proteins are required for DNA-based processes during development. We recently established PWWP2A as direct histone variant H2A.Z interactor involved in mitosis and cranial-facial development. Here, we identify the H2A.Z/PWWP2A-associated protein HMG20A as part of several chromatin-modifying complexes including NuRD, and show that it localizes to genomic regulatory regions. Hmg20a depletion causes severe head and heart developmental defects in Xenopus laevis. Our data indicate that craniofacial malformations are caused by defects in neural crest cell (NCC) migration and cartilage formation. These developmental defects are pheno-copied in HMG20A-depleted mESCs, which show inefficient differentiation into NCCs and cardiomyocytes (CMs). Accordingly, loss of HMG20A caused striking deregulation of transcription programs involved in epithelial- mesenchymal transition (EMT) and cardiac differentiation, thereby providing insights into the regulatory circuits controlled by HMG20A. Collectively, our findings implicate HMG20A as part of the H2A.Z/PWWP2A/NuRD-axis and reveal it as a key modulator of the intricate developmental transcription programs that guide NCC and cardiomyocyte differentiation.


title: Expression and functional evaluation of recombinant human ABO blood group antigen cleaving glycoside hydrolases α-N-acetylgalactosaminidase, α-galactosidase, and endo-β-galactosidase produced in Escherichia coli

abstract: Blood transfusions are an integral component of healthcare; however, availability of viable blood is limited by patient-donor blood type specificity, which contributes to seasonal shortages as well as shortages worldwide, especially in developing countries, and during pandemics or natural disasters. Attempts to increase blood supply with commercial incentives have raised ethical concerns, and current proposed artificial blood substitutes are unable to fully replicate the function of native red blood cells (RBCs). In this study, we explore the potential strategy of alleviating blood shortages through enzymatic conversion of A, B, and AB blood types to blood type O. In theory, this process eliminates ABO patient-donor incompatibility, which increases the supply of universal donor blood. Three glycoside hydrolases, -N-acetylgalactosaminidase, -galactosidase, and endo-{beta}-galactosidase, were selected to act as molecular scissors to cleave terminal residues on A and B RBC surface antigens and catalyze the conversion process. These enzymes were recombinantly expressed in BL21(DE3) Escherichia coli and purified through nickel ion affinity chromatography. A combination of colorimetric substrate assays, thin-layer chromatography, and mass spectroscopy were utilized to evaluate enzyme functionality. Enzyme efficiency was modeled using Michaelis-Menten kinetics. Partial enzymatic A-to-O blood type conversion on porcine red blood cells was observed with slide agglutination tests. Results confirm recombinant enzyme-mediated blood type conversion as a potential strategy for alleviating blood shortages.


title: AptaMat: a matrix-based algorithm to compare single-stranded oligonucleotides secondary structures

abstract:
Comparing single-stranded nucleic acids (ssNAs) secondary structures is fundamental when investigating their function and evolution and predicting the effect of mutations on the ssNAs structures. Many comparison metrics exist, although they are either too elaborate or not enough sensitive to distinguish close ssNAs structures.

In this context, we developed AptaMat, a simple and sensitive algorithm for ssNAs secondary structures comparison based on matrices representing the ssNAs secondary structures and a metric built upon the Manhattan distance in the plane. We applied AptaMat to several examples and compared the results to those obtained by the most frequently used metrics, namely the Hamming distance and the RNAdistance, and by a recently developed image-based approach. We showed that AptaMat is able to discriminate between similar sequences, outperforming all the other here considered metrics.

category: bioinformatics


title: Identification of neoantigens in esophageal adenocarcinoma

abstract: Esophageal adenocarcinoma (EAC) has a relatively poor long-term survival and limited treatment options. Promising targets for immunotherapy are short peptide neoantigens containing tumor mutations, presented to cytotoxic T-cells by human leukocyte antigen molecules (HLA). Despite an association between putative neoantigen abundance and therapeutic response across cancers, immunogenic neoantigens are challenging to identify. Here we characterized the mutational and immunopeptidomic landscapes of tumors from a cohort of seven patients with EAC. We directly identified one HLA-I presented neoantigen from one patient, and report functional T-cell responses from a predicted HLA-II neoantigen in a second patient. The predicted class II neoantigen contains both HLA I and II binding motifs. Our exploratory observations are consistent with previous neoantigen studies in finding that neoantigens are rarely directly observed, and an identification success rate following prediction in the order of 10%. However, our identified putative neoantigen is capable of eliciting strong T-cell responses, emphasizing the need for improved strategies for neoantigen identification.

category: cancer biology


title: High-resolution mass spectrometry reveals environmentally relevant uptake, elimination, and metabolic alterations following early embryonic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish

abstract:
Dioxin and dioxin-like compounds are ubiquitous environmental contaminants that induce toxicity by binding to the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. The zebrafish model has been used to define the developmental toxicity observed following exposure to exogenous AHR ligands such as the potent agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD). While the model has successfully identified cellular targets of TCDD and molecular mechanisms mediating TCDD-induced phenotypes, fundamental information such as the body burden produced by standard exposure paradigms is still unknown. We performed targeted gas chromatography (GC) high-resolution mass spectrometry (HRMS) in tandem with non-targeted liquid chromatography (LC) HRMS to quantify TCDD uptake, model the elimination dynamics of TCDD, and determine how TCDD exposure affects the zebrafish metabolome. We found that 10 ppb, 1 ppb, and 50 ppt waterborne exposures during early embryogenesis produced environmentally relevant body burden of TCDD: 38 {+/-} 4.34, 26.6 {+/-} 1.2, and 8.53 {+/-} 0.341 pg/embryo, respectively, at 24 hours post fertilization. In addition, we discovered that TCDD exposure was associated with the dysregulation of several metabolic pathways that are critical for brain development and function including glutamate metabolism, chondroitin sulfate biosynthesis, and tyrosine metabolism pathways. Together, these data demonstrate that existing exposure paradigms produce environmentally relevant body burdens of TCDD in zebrafish and provide insight into the biochemical pathways impacted by toxicant-induced AHR activation.

HIGHLIGHTSO_LIHistorical TCDD exposure paradigms produce environmentally relevant body burdens in zebrafish embryos.
C_LIO_LITCDD elimination for high doses can be modeled using an exponential regression.
C_LIO_LIExposure to TCDD alters metabolic pathways that are essential for brain development and function.
C_LI

GRAPHICAL ABSTRACT

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category: pharmacology and toxicology


title: Visualizing cell-cell interactions between immune effector and tumor cells induced by antibodies using a proximity-dependent biosensor system

abstract: Therapeutic antibodies can engage innate (NK) and adaptive (T) immune cells to eliminate pathogenic or malignant cells. Activated NK or T cells exert cytotoxic effects dependent upon direct cell-cell contact and subsequent formation of immunological synapses (IS) with target cells. Therefore, understanding antibody-mediated cell-cell interactions is crucial to optimizing antibody pharmacology and efficacy across all therapeutic areas. To date, most investigations around antibody-mediated cell-cell interactions have primarily focused on molecular-scale interactions. In this study, we developed a biosensor system to evaluate direct cell-cell contact and interactions and the formation of IS at the cell population level. In this biosensor system, two structural complementary luciferase units (SmBit and LgBit) were respectively expressed on immune effector and target cell membranes with optimized spacer lengths. Upon cell-cell contact, two subunits come together to form active NanoLuc, generating a luminescent signal for longitudinal monitoring. The selectivity of the system were optimized by adjusting the spacer lengths to assure the signal is from stable cell-cell interactions with minimal interference from nonspecific and transient intercellular contact. This system was then applied to longitudinally quantify cell-cell interactions between NK and target cells induced by an anti-CD20 antibody (rituximab) and between T and target cells induced by a bispecific (anti-CD3/CD19) T cell engaging antibody (blinatumomab) in a three-dimensional cell culture system. This biosensor system shows promise for monitoring cell-cell interactions in physiologically relevant environments and provides insights into the environmental factors that impede cell-cell interaction and antibody efficacy.


title: A computational toolbox to investigate the metabolic potential and resource allocation in fission yeast

abstract: The fission yeast Schizosaccharomyces pombe is a popular eukaryal model organism for cell division and cell cycle studies. With this extensive knowledge of its cell and molecular biology, S. pombe also holds promise for use in metabolism research and industrial applications. However, unlike the bakers yeast Saccharomyces cerevisiae, a major workhorse in these areas, cell physiology and metabolism of S. pombe remain less explored. One way to advance understanding of organism-specific metabolism is construction of computational models and their use for hypothesis testing. To this end, we leverage existing knowledge of S. cerevisiae to generate a manually-curated high-quality reconstruction of S. pombes metabolic network, including a proteome-constrained version of the model. Using these models, we gain insights into the energy demands for growth, as well as ribosome kinetics in S. pombe. Furthermore, we predict proteome composition and identify growth-limiting constraints that determine optimal metabolic strategies under different glucose availability regimes, and reproduce experimentally determined metabolic profiles. Notably, we find similarities in metabolic and proteome predictions of S. pombe with S. cerevisiae, which indicate that similar cellular resource constraints operate to dictate metabolic organization. With these use cases, we show, on the one hand, how these models provide an efficient means to transfer metabolic knowledge from a well-studied to a lesser-studied organism, and on the other, how they can successfully be used to explore the metabolic behaviour and the role of resource allocation in driving different strategies in fission yeast.

category: systems biology


title: Morphological evolution and diversity of pectoral fin skeletons in teleosts

abstract: The Teleostei class has the most species of the fishes. Members of this group have paired rostral appendages and pectoral fins, enabling refined movements in the water. Although teleosts live in a diverse set of environments, the skeletal pattern of pectoral fins in teleosts is considered to show little morphological variability. Here, in order to elucidate variations in pectoral fin skeletons and to identify their evolutionary processes, we compared the pectoral fin skeletons from 27 species of teleosts. We identified several variations and a diversity of pectoral fin skeletal patterns within some teleost groups. Taken together with previous reports on teleost skeletons, our findings reveal that in the course of teleost evolution, there are a mixture of conserved and non- conserved components in the pectoral fin skeletons of teleosts, and that teleosts may have experienced the variation and conservation of the number and shape of the proximal radials, the loss of the mesocoracoid, and the change in the distal radial-fin ray relationship.


title: Ribotoxic collisions on CAG expansions disrupt proteostasis and stress responses in Huntington's Disease

abstract: Huntingtons disease (HD) is a devastating neurodegenerative disorder caused by CAG trinucleotide repeat expansions encoding a polyglutamine (polyQ) tract in the Huntingtin (HTT) gene1. Although mutant HTT (mHTT) protein tends to aggregate, the exact causes of neurotoxicity in HD remain unclear2. Here we show that altered elongation kinetics on CAG expansions cause ribosome collisions that trigger ribotoxicity, proteotoxicity and maladaptive stress responses. CAG expansions cause an elongation rate conflict during HTT translation, when ribosomes rapidly decoding the optimal polyQ encounter a flanking slowly-decoded polyproline tract. The ensuing ribosome collisions lead to premature termination and release of aggregation-prone mHTT fragments. Due to the presence of a stress-responsive upstream open reading frame (uORF), HTT translation and aggregation are limited under normal conditions but enhanced under stress, seeding a vicious cycle of dysfunction. mHTT further exacerbates ribotoxicity by progressively sequestering eIF5A, a key regulator of translation elongation, polyamine metabolism and stress responses. eIF5A depletion in HD cells leads to widespread ribosome pausing on eIF5A-dependent sites, impaired cotranslational proteostasis, disrupted polyamine metabolism and maladaptive stress responses. Importantly, drugs that reduce translation initiation attenuate ribosome collisions and mitigate this escalating cascade of ribotoxic stress and dysfunction in HD.


title: Behavioral and brain responses to verbal stimuli reveal transient periods of cognitive integration of external world in all sleep stages

abstract: Sleep has long been considered as a state of disconnection from the environment, with absent reactivity to external stimuli. Here, we questioned this sleep disconnection dogma by directly investigating behavioral responsiveness in 49 napping subjects (27 with narcolepsy and 22 healthy volunteers) engaged in a lexical decision task. Participants were instructed to frown or smile depending on the stimulus type (words vs pseudo-words). We found accurate behavioral responses, visible via contractions of the corrugator or zygomatic muscles, in all sleep stages in both groups (except slow-wave sleep for healthy volunteers). Stimuli presented during states with high (vs. low) values of neural markers indexing rich cognitive states more often yielded responses. Our findings suggest that transient windows of reactivity to external stimuli exist in all sleep stages, even in healthy individuals. Such windows of reactivity could be used to probe sleep-related mental and cognitive processes in real-time across all sleep stages.


title: Perceptual cycles travel across retinotopic space

abstract: Visual perception waxes and wanes periodically as a function of the phase of low-frequency brain oscillations (theta, 4-7 Hz; alpha, 8-13 Hz) [1-9]. Perceptual cycles are defined as the corresponding periodic modulation of perceptual performance (review [10, 11]). Here, using psychophysics, we tested the hypothesis that brain oscillations travel across the visual cortex, leading to predictable perceptual consequences across the visual field, i.e., perceptual cycles travel across the retinotopic visual space. An oscillating disk (inducer) was presented in the periphery of the visual field to induce brain oscillations at low frequencies (4, 6, 8 or 10 Hz) at a specific retinotopic cortical location. Target stimuli at threshold (50% detection) were displayed at random times during the periodic disk stimulation, at one of three possible distances from the disk. Electroencephalography (EEG) was recorded while participants performed a detection task. EEG analyses showed that the periodic stimulation produced a complex brain oscillation composed of the induced frequency and its first harmonic likely due to the overlap of the periodic response and the neural response to individual stimuli. This complex oscillation, which originated from a precise retinotopic position, modulated detection performance periodically at each target position and at each frequency. Critically, the optimal behavioral phase, i.e., of highest performance, of the 8 Hz and 10 Hz oscillations (alpha range) consistently shifted across target distance to the inducer. Together, the results demonstrate that alpha-induced perceptual cycles traveled across the retinotopic space in human observers at a propagation speed between 0.2 and 0.4 m/s.


title: Task prioritization modulates low frequency EEG dynamics reflecting proactive cognitive control

abstract:
Most neuroscientific studies investigating mental effort apply unspecific effort allocation paradigms. In contrast, the present EEG study targets specific effort allocation during task prioritization.

Twenty-eight participants performed a cued number classification task during the retention interval of a working memory task including retrospective cues. One of two possible number classifications was done per trial. Each trial started with a cue indicating which of the two tasks would be more important in the upcoming trial. Subjects were told to engage in both tasks, but to concentrate on the important one. Feedback given at the end of each trial was calculated based on task performance, with scores obtained from the relevant task being tripled.

Participants performed significantly better in either task when it was important compared to when not.Task prioritization modulates theta, alpha and beta oscillations, predominantly during task preparation. Multivariate pattern analysis revealed that the exact type of the two possible number classifications was decodable, however, decoding accuracy did not depend on task importance. Hemispheric alpha power asymmetries indicating attentional orienting between working memory representations also did not depend on task importance. The findings suggest that task prioritization primarily affects proactive cognitive control on a superordinate level.


title: Expression of GDNF Receptors Within Nucleus Ambiguus During Rat Development

abstract:
ObjectiveUpregulation of GDNF and its receptors is observed during laryngeal reinnervation after nerve injury. In contrast, little is known regarding the expression of GDNF receptors in the formation of the nucleus ambiguus (NA) and its innervation of the larynx during embryogenesis. Differences may suggest therapeutic targets after nerve injury.

Study DesignLaboratory experiment.

MethodsRat brainstems at E14, E16, E18, E20, adult (4 animals/timepoint) were sectioned and stained for GDNF receptors: GFR-1, GFR-2, GFR-3, and Ret. Islet1 and ChAT were used as markers for motoneuron cell bodies. Sections were observed using Zeiss Axio Imager M2 Microscope and quantified using Image J.

ResultsExpression of all four GDNF receptors was identified within the nucleus ambiguus, as well as hypoglossal and facial nuclei of the adult rat brainstem. During rat development, GFR-3 and Ret exhibited upregulation within the nucleus ambiguus at E14 whereas GFR-1 began showing upregulation at E20. GFR-2 exhibited no upregulation at embryonic timepoints. Conclusion: Upregulation of the GDNF receptors within the nucleus ambiguus occur after laryngeal muscles innervation during development and may associated with maturation and maintenance of the neuromuscular synapses of the larynx before and after birth. No differences among ambiguus, hypoglossal, and facial nuclei was observed.


title: Plasma miRNA-214 is a predictive candidate biomarker of progression speed in patients with ALS

abstract: This study was designed to develop and validate a reliable biomarker to predict the progression speed reflecting immune function of amyotrophic lateral sclerosis (ALS). After establishing the induced microglia model (iMGs) derived from peripheral blood monocytes, comparative studies to find factors related to phagocytic differences between iMGs of patients with rapidly progressive ALS [ALS(R)-iMGs, n = 15] and those of patients with slowly progressive ALS [ALS(S)-iMGs, n = 14] were conducted in the discovery cohort. To validate discovered candidate and whether it could be used as a reliable biomarker predicting the progression speed of ALS, we recruited 132 patients with ALS and 30 age-matched healthy controls as the validation cohort. ALS(R)-iMGs showed impaired phagocytic function. Transcriptomic analysis revealed that the perturbed phagocytosis in ALS(R)-iMGs was related to the decreased expression of NCKAP1 (NCK-associated protein 1) and NCKAP1 overexpression rescued the impaired phagocytic function. miRNA-214-3p targeting NCKAP1 in ALS-iMGs was correlated with progression speed in the discovery cohort. The validation cohort revealed that plasma miRNA-214-3p levels were significantly increased in ALS patients (p < 0.0001, AUC = 0.839), correlated with disease progression speed (p = 0.0005), and distinguished the rapidly progressive subgroup (Q1) from the slowly progressive (Q4, p = 0.029), respectively. Plasma miRNA-214-3p can predict the progression speed in ALS. Plasma miRNA-214-3p could be used as a simple and easily accessible biomarker for predicting the future progression speed related to phagocytic dysfunction in ALS patients.


title: A pro-oxidant combination of resveratrol and copper down-regulates multiple biological hallmarks of ageing and neurodegeneration

abstract: Several hundred billion to a trillion cells die in the body every day, and cell-free chromatin particles (cfChPs) that are released from them enter into the extracellular compartments of the body, including into the circulation. We have earlier reported that cfChPs can readily enter into healthy cells to damage their DNA, activate apoptotic pathways and induce inflammatory cytokines. We hypothesized that repeated lifelong assault on healthy cells by cfChPs is the underlying cause of ageing, and that the ageing process could be retarded by deactivating cfChPs. The latter can be effected by oxygen radicals that are generated upon admixing the nutraceuticals resveratrol (R) and copper (Cu). Using confocal microscopy and antibodies against DNA and histone we detected copious presence of extra-cellular cfChPs in brain of ageing mice, and observed that these were deactivated / eradicated following prolong oral administration of small quantities of R-Cu. Deactivation / eradication of cfChPs was associated with down-regulation of several biological hallmarks of ageing in brain cells which included reduction in: 1) telomere attrition, 2) amyloid deposition, 3) DNA damage, 4) apoptosis, 5) inflammation, 6) senescence, 7) aneuploidy and 8) mitochondrial dysfunction. At a systemic level, R- Cu treatment led to significant reduction in blood levels of glucose, cholesterol and C-reactive protein. These results suggest that cfChPs may be global instigators of ageing and neurodegeneration, and that therapeutic use of R-Cu may help to retard the process of ageing.


title: Retinoic acid-gated BDNF synthesis in neuronal dendrites drives presynaptic homeostatic plasticity

abstract: Homeostatic synaptic plasticity is a non-Hebbian synaptic mechanism that adjusts synaptic strength to maintain network stability while achieving optimal information processing. Among the molecular mediators shown to regulate this form of plasticity, synaptic signaling through retinoic acid (RA) and its receptor, RAR, has been shown to be critically involved in the homeostatic adjustment of synaptic transmission in both hippocampus and sensory cortices. In this study, we explore the molecular mechanism through which postsynaptic RA and RAR regulates presynaptic neurotransmitter release during prolonged synaptic inactivity at excitatory synapses. We show that RAR binds to a subset of dendritically sorted brain-derived neurotrophic factor (BDNF) mRNA splice isoforms and represses their translation. The RA-mediated translational de-repression of postsynaptic BDNF results in the retrograde activation of presynaptic Tropomyosin receptor kinase B (TrkB) receptors, facilitating presynaptic homeostatic compensation through enhanced presynaptic release. Together, our study illustrates a RA-mediated retrograde synaptic signaling pathway through which postsynaptic protein synthesis during synaptic inactivity drives compensatory changes at presynaptic site.


title: Multivariate Analysis of PET Pharmacokinetic Parameters

abstract:
PurposeIn positron emission tomography (PET) quantification, multiple pharmacokinetic parameters are typically estimated from each time activity curve. Conventionally, all but the parameter of interest are discarded before performing subsequent statistical analysis. However, we assert that these discarded parameters also contain relevant information which can be exploited to improve the precision and power of statistical analyses on the parameter of interest. Properly taking this into account can thereby draw more informative conclusions without collecting more data.

MethodsBy applying a hierarchical multifactor multivariate Bayesian approach, all estimated parameters from all regions can be analysed at once. We refer to this method as PuMBA (Parameters undergoing Multivariate Bayesian Analysis). We simulated patientcontrol studies with different radioligands, varying sample sizes and measurement error to explore its performance, comparing the precision, statistical power, false positive rate and bias of estimated group differences relative to univariate analysis methods.

ResultsWe show that PuMBA improves the statistical power for all examined applications relative to univariate methods without increasing the false positive rate. PuMBA improves the precision of effect size estimation, and reduces the variation of these estimates between simulated samples. Furthermore, we show that PuMBA yields performance improvements even in the presence of substantial measurement error. Remarkably, owing to its ability to leverage information shared between pharmacokinetic parameters, PuMBA even shows greater power than conventional univariate analysis of the true binding values from which the parameters were simulated. Across all applications, PuMBA exhibited a small degree of bias in the estimated outcomes, however this was small relative to the variation in estimated outcomes between simulated datasets.

ConclusionPuMBA improves the precision and power of statistical analysis of PET data without requiring the collection of additional measurements. This makes it possible to study new research questions in both new and previously collected data. PuMBA therefore holds great promise for the field of PET imaging.


title: KIF21B binds Myosin Va for Spine Entry and regulates Actin Dynamics to control Homeostatic Synaptic Downscaling

abstract: Homeostatic synaptic plasticity adjusts the strength of synapses to restrain neuronal activity within a physiological range. Postsynaptic GKAP controls the bidirectional synaptic scaling of AMPA receptors (AMPARs) however how chronic activity triggers postsynaptic protein remodeling to downscale synaptic transmission is barely understood. Here we report that the microtubule-dependent kinesin motor KIF21B interacts with GKAP and likewise enters dendritic spines in a myosin Va- and activity-dependent manner. We observed that under conditions of chronic activity KIF21B regulates actin dynamics in spines, triggers spine removal of GluA2-containing AMPA receptors, and mediates homeostatic synaptic downscaling of AMPA receptor-mediated mEPSC amplitudes. Our data highlight a myosin-kinesin interaction that enables the entry of the microtubule-dependent motor KIF21B into actin-rich spine compartments. A slow actin turnover rate might be beneficial for efficient protein removal from excitatory synapses, suggesting a functional role of KIF21B in a GKAP- and AMPA receptor-dependent mechanism, underlying homeostatic downscaling of neuronal firing.


title: Chronic lymphocytic leukemia includes a tumor subset resembling memory B cells that develop early and persist

abstract: Single-cell RNAseq/VDJseq of tumor cells and normal residual B (NRB) cells from peripheral blood of chronic lymphocytic leukemia (CLL) patients identified three distinct tumor subsets according to phenotype, transcriptome, and immunoglobulin-V-gene (IgV)-mutations. Two major subsets share a typical CLL phenotype but differ in signaling, metabolism and cell cycle control, indicating that the circulating CLL pool is shaped by two states of activity. The third CLL subset shows the phenotype, proliferation capacity and extensive IgV-mutation diversity of normal CD5+ memory B cells. This previously unrecognized CLL tumor subset, which intermingles with NRB cells, was confirmed in 33 IgV-mutated (M-CLL) and IgV-unmutated (U-CLL) cases. Longitudinal IgV-mutation phylogenetics suggest that these NRB-associated CLL cells are generated pathogenetically early, mostly in germinal center reactions, and archive the individual IgV-diversification program, which is conserved throughout CLL course. Our study suggests that diversity is established early in CLL, that each tumor is composed of multiple subclonal expansions, and subclonal evolution can be depicted by IgV-mutation phylogenetics.


title: The radio-protective effects of (-)-Epigallocatechin-3-gallate (EGCG): regulating macrophage function in radiation-induced intestinal injury.

abstract: Accidental and medical radiation exposure may cause radiation-induced intestinal injury (RIII), a catastrophic disease requiring efficient therapies. (-)-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea, has been shown to have potent biological activity as well as strong anti-inflammation effects. Here, we demonstrate that EGCG treatment not only protects mice against total body irradiation (TBI)-induced toxicity and weight loss but also alleviates whole abdominal irradiation (WAI)-induced intestinal injury. EGCG promotes proliferation and survival of intestinal stem cells, inhibits radiation-induced apoptosis and inflammation. At the same time, EGCG preserves the composition of the gut microbiota in WAI mice. In vitro, we demonstrate that EGCG regulates the release of radiation-induced inflammatory factors by inhibiting inflammatory pathways such as toll-like receptor signaling pathway in peritoneal macrophages. Mechanistically, the radioprotective effect of EGCG was likely attributable to its preservation on macrophages and the colonized gut microbiota composition, thus relieving the intestinal inflammation. EGCG provides a novel strategy to mitigate RIII and improve the prognosis of patients after radiotherapy.


title: Inhibiting 5-lipoxygenase prevents skeletal muscle atrophy by targeting organogenesis signaling and insulin-like growth factor-1

abstract:
BackgroundSkeletal muscle atrophy can occur in response to numerous factors, such as aging and certain medications, and produces a major socioeconomic burden. At present, there are no approved drugs for treating skeletal muscle atrophy. Arachidonate 5-lipoxygenase (Aox5) is a drug target for a number of diseases. However, pharmacological targeting of Alox5, and its role in skeletal muscle atrophy, is unclear.

MethodsThe potential effects of gene knockdown and pharmacological targeting of Alox5 on skeletal muscle atrophy was investigated using cell-based models, animal models, and human skeletal muscle tissue cultures. Malotilate, a clinically safe drug developed for enhancing liver regeneration and Alox5 inhibitor, was investigated as a repurposing candidate. Mechanism(s) of action in skeletal muscle atrophy were assessed by measuring the expression level or activation status of key regulatory pathways, and validated using gene knockdown and RNA sequencing.

ResultsMyotubes treated with the atrophy-inducing glucocorticoid, dexamethasone, were protected from catabolic responses by treatment with malotilate (+41.29%, P < 0.01). Similar anti-atrophy effects were achieved by gene knockdown of Alox5 (+30.4%, P < 0.05). Malotilate produced anti-atrophy effects without affecting the myogenic differentiation program. In an in vivo model of skeletal muscle atrophy, malotilate treatment enhanced muscle performance (Grip strength: +35.72%, Latency to fall: +553.1%, P < 0.05), increased mass and fiber cross sectional area (Quadriceps: +23.72%, Soleus: +33.3%, P < 0.01), and down-regulated atrogene expression (Atrogin-1: -61.58%, Murf-1: -66.06%, P < 0.01). Similar, beneficial effects of malotilate treatment were observed in an aging muscle, which also showed the preservation of fast twitch fibers (Type 2a: +56.48%, Type 2b: +37.32%, P < 0.01). Leukotrine B4, a product of Alox5 activity with inflammatory and catabolic functions, was found to be elevated in skeletal muscle undergoing atrophy (Quadriceps: +224.4%, P < 0.001). Cellular transcriptome analysis showed that targeting Alox5 upregulated biological processes regulating organogenesis and increased the expression of insulin-like growth factor-1, a key anti-atrophy hormone (+226.5%, P < 0.05). Interestingly, these effects were restricted to the atrophy condition and not observed in normal skeletal muscle cultures with Alox5 inhibition. Human skeletal muscle tissue was also protected from atrophy by pharmacological targeting of Alox5 (+23.68%, P < 0.05).

ConclusionThese results shed new light on novel drug targets and mechanisms underpinning skeletal muscle atrophy. Alox5 is a regulator and drug target for muscle atrophy, and malotilate is an attractive compound for repurposing studies to treat this disease.


title: An autonomous mathematical model for the mammalian cell cycle

abstract:
A mathematical model for the mammalian cell cycle is developed as a system of 13 coupled nonlinear ordinary differential equations. The variables and interactions included in the model are based on detailed consideration of available experimental data. Key features are that the model is autonomous, except for dependence on external growth factors; variables are continuous in time, without instantaneous resets at phase boundaries; cell cycle controllers and completion of tasks associated with cell cycle progression are represented; mechanisms to prevent rereplication are included; and cycle progression is independent of cell size. Eight variables represent cell cycle controllers: Cyclin D1 in complex with Cdk4/6, APCCdh1, SCF{beta}Trcp, Cdc25A, MPF, NUMA, securin-separase complex, and separase. Five variables represent task completion, with four for the status of origins and one for kinetochore attachment. The model predicts distinct behaviors consistent with each main phase of the cell cycle. The response to growth factors shows restriction-point behavior. These results imply that the main features of the mammalian cell cycle can be accounted for in a quantitative mechanistic way based on known interactions among cycle control factors and their coupling to tasks involved in replication of DNA. The model is robust to parameter changes, in that cycling is maintained over at least a five-fold range of each parameter when varied individually. The most sensitive parameters are those associated with the initiation and completion of mitosis. The model is suitable for exploring how extracellular factors affect cell cycle progression, including responses to metabolic conditions and to anti-cancer therapies.

Author SummaryWe created a model, that is, a set of mathematical equations, to represent the entire cell cycle in mammals. All terms in our equations correspond to actual biological mechanisms. We solved the equations and verified that they show similar behavior to real-life cell cycles. We designed our model to cycle only when enough external growth factors stimulate it, as real cells do. Our model helps us better understand how the cell cycle is controlled. One very important aspect of control is how the cell ensures that its DNA is copied only once per cell cycle. If "rereplication" (copying a section of DNA twice in the same cycle) occurs, it can cause harmful DNA damage. We considered several mechanisms that some biologists believe play a role, and found that most could not explain how rare rereplication is. Two mechanisms did work well, and we used them to make two variants of the model, which show similar behavior. The model has many potential applications. The cell cycle is altered in most cancer cells, so understanding how these changes affect control is useful. Many widely-used drugs affect the cell cycle, and our model can be used to study these effects.


title: Modelling the spread and mitigation of an emerging vector-borne pathogen:citrus greening in the U.S.

abstract: Predictive models, based upon epidemiological principles and fitted to surveillance data, play an increasingly important role in shaping regulatory and operational policies for emerging outbreaks. Data for parameterising these strategically important models are often scarce when rapid actions are required to change the course of an epidemic invading a new region. We provide a flexible toolkit for landscape-scale disease management, which is applicable to a range of emerging pathogens including vector-borne pathogens for both endemic and invading epidemic vectors. We use the toolkit to analyse and predict the spread of Huanglongbing disease or citrus greening in the U.S. We estimate epidemiological parameters using survey data from one region (Texas) and show how to transfer and test parameters to construct predictive spatio-temporal models for another region (California). The models are used to screen effective coordinated and reactive management strategies for different regions.


title: Effects of microplastics mixed with natural particles on Daphnia magna populations

abstract:
The toxicity of microplastics on Daphnia magna as key model for freshwater zooplankton is well described. While several studies predict population-level effects based on short-term, individual-level responses, only very few have validated these predictions experimentally. Thus, we exposed D. magna populations to irregular polystyrene microplastics and diatomite as natural particle (both [≤]63 {micro}m) over 50 days. We used mixtures of both particle types at fixed particle concentrations (50,000 mL-1) and recorded the overall population density, the size of the individual animals, and resting egg production. Particle exposure adversely affected the population density and structure and induced resting egg production. The terminal population size was 31-42% lower in exposed compared to control populations. Interestingly, mixtures containing diatomite induced stronger effects than microplastics alone highlighting that natural particles are not per se less toxic than microplastics. Our results demonstrate that an exposure to synthetic and natural particles has negative population-level effects on zooplankton. Understanding the mixture toxicity of microplastics and natural particles is important given that aquatic organisms will experience exposure to both. Just as for chemical pollutants, better knowledge of such joint effects is essential to fully understand the environmental risks of complex particle mixtures.

Environmental ImplicationsWhile microplastics are commonly considered hazardous based on individual-level effects, there is a dearth of information on how they affect populations. Since the latter is key for understanding the environmental impacts of microplastics, we investigated how particle exposures affect the population size and structure of Daphnia magna. In addition, we used mixtures of microplastics and natural particles because neither occurs alone in nature and joint effects can expected in an environmentally realistic scenario. We show that such mixtures adversely affect daphnid populations and highlight that population-level and mixture-toxicity designs are one important step towards more environmental realism in microplastics research.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/490562v1_ufig1.gif" ALT="Figure 1">
View larger version (26K):
org.highwire.dtl.DTLVardef@13cf281org.highwire.dtl.DTLVardef@552921org.highwire.dtl.DTLVardef@ff461corg.highwire.dtl.DTLVardef@153f479_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIDaphnia populations exposed to mixtures of microplastics and diatomite
C_LIO_LIEffects on population density, structure, and resting egg production
C_LIO_LIDiatomite as natural particle was more toxic than microplastics
C_LIO_LIParticle mixtures induce negative population-level effects
C_LIO_LIParticle mixtures represent more realistic exposure scenario
C_LI


title: Genome mining as a biotechnological tool for the discovery of novel biosynthetic genes in lichens

abstract: The ever-increasing demand for novel drugs highlights the need for bioprospecting unexplored taxa for their biosynthetic potential. Lichen-forming fungi (LFF) are a rich source of natural products but their implementation in pharmaceutical industry is limited, mostly because the genes corresponding to a majority of their natural products is unknown. Furthermore, it is not known to what extent these genes encode structurally novel molecules. Advance in next-generation sequencing technologies has expanded the range of organisms that could be exploited for their biosynthetic potential. In this study, we mine the genomes of nine lichen-forming fungal species of the genus Umbilicaria for biosynthetic genes, and categorize the BGCs as "associated product structurally known", and "associated product putatively novel". We found that about 25-30% of the biosynthetic genes are divergent when compared to the global database of BGCs comprising of 1,200,000 characterized biosynthetic genes from planta, bacteria and fungi. Out of 217 total BGCs, 43 were only distantly related to known BGCs, suggesting they encode structurally and functionally unknown natural products. Clusters encoding the putatively novel metabolic diversity comprise PKSs (30), NRPSs (12) and terpenes (1). Our study emphasizes the utility of genomic data in bioprospecting microorganisms for their biosynthetic potential and in advancing the industrial application of unexplored taxa. We highlight the untapped structural metabolic diversity encoded in the lichenized fungal genomes. To the best of our knowledge, this is the first investigation identifying genes coding for NPs with potentially novel therapeutic properties in LFF.


title: Evaluation of Cognitive Function in the Dog Aging Project: Associations with Baseline Canine Characteristics

abstract:
Canine Cognitive Dysfunction (CCD) is a neurodegenerative disease in aging dogs. It has been described previously in relatively small cohorts of dogs using multiple different rating scales. This study aimed to use a minimally modified CCD rating scale developed by previous researchers to describe the prevalence of CCD more thoroughly in a large, nationwide cohort of companion dogs participating in the Dog Aging Project (DAP). Associations between various canine characteristics, predicted lifespan quartiles, and CCD were examined using univariable and multivariable logistic regression models and Receiver Operating Curve (ROC) analysis.

When controlling for all other characteristics, the odds of CCD increased 52% with each additional year of age. Among dogs of the same age, health status, breed type, and sterilization status, odds of CCD were 6.47 times higher in dogs who were not active compared to those who were very active. When controlling for age, breed type, activity level, and other comorbidities, dogs with a history of neurological, eye, or ear disorders had higher odds of CCD. Lifespan quartile analysis showed excellent discriminating ability between CCD positive and negative dogs. Weight-based lifespan quartile estimation could therefore serve as a tool to inform CCD screening by veterinarians.

category: animal behavior and cognition


title: Head-direction tracks internally directed selective attention in visual working memory

abstract: We shift our gaze even when we orient attention internally to visual representations in working memory. Here, we show the bodily orienting response associated with internal selective attention is widespread as it also includes the head. In three virtual reality (VR) experiments, participants remembered two visual items. After a working memory delay, a central colour cue indicated which item needed to be reproduced from memory. After the cue, head movements became biased in the direction of the memorised location of the cued memory item - despite there being no items to orient towards in the external environment. The head-direction bias had a distinct temporal profile from the gaze bias. Our findings reveal that directing attention within the spatial layout of visual working memory bears a strong relation to the overt head orienting response we engage when directing attention to sensory information in the external environment. The head-direction bias further demonstrates common neural circuitry is engaged during external and internal orienting of attention.