title: Monash DaCRA fPET-fMRI: A DAtaset for Comparison of Radiotracer Administration for high temporal resolution functional FDG-PET

abstract:
Background Functional [18F]-fluorodeoxyglucose positron emission tomography (FDG-fPET) is a new approach for measuring glucose uptake in the human brain. The goal of FDG-fPET is to maintain a constant plasma supply of radioactive FDG in order to track, with high temporal resolution, the dynamic uptake of glucose during neuronal activity that occurs in response to a task or at rest. FDG-fPET has most often been applied in simultaneous BOLD-fMRI/FDG-fPET (blood oxygenation level dependent functional MRI fluorodeoxyglucose functional positron emission tomography) imaging. BOLD-fMRI/FDG-fPET provides the capability to image the two primary sources of energetic dynamics in the brain, the cerebrovascular haemodynamic response and cerebral glucose uptake.

FindingsIn this Data Note, we describe an open access dataset, Monash DaCRA fPET-fMRI, which contrasts three radiotracer administration protocols for FDG-fPET: bolus, constant infusion, and hybrid bolus/infusion. Participants (n=5 in each group) were randomly assigned to each radiotracer administration protocol and underwent simultaneous BOLD-fMRI/FDG-fPET scanning while viewing a flickering checkerboard. The Bolus group received the full FDG dose in a standard bolus administration; the Infusion group received the full FDG dose as a slow infusion over the duration of the scan, and the Bolus-Infusion group received 50% of the FDG dose as bolus and 50% as constant infusion. We validate the dataset by contrasting plasma radioactivity, grey matter mean uptake, and task-related activity in the visual cortex.

ConclusionsThe Monash DaCRA fPET-fMRI dataset provides significant re-use value for researchers interested in the comparison of signal dynamics in fPET, and its relationship with fMRI task-evoked activity.


title: High temperature perception in leaves promotes vascular regeneration in distant tissues

abstract: Cellular regeneration in response to wounding is fundamental to maintain tissue integrity. Various internal factors including hormones and developmental pathways affect wound healing but little is known about how external factors influence regeneration. To better understand how the environment affects regeneration, we investigated the effects of temperature using the horticulturally relevant process of plant grafting. We found that elevated temperatures accelerated vascular regeneration of Arabidopsis thaliana and tomato (Solanum lycopersicum) grafts. Leaves were critical for this effect since blocking auxin transport or mutating PHYTOCHROME INTERACTING FACTOR4 (PIF4) or YUCCA2/5/8/9 in the cotyledons abolished the temperature enhancement. However, these perturbations had no effect upon graft healing at ambient temperatures and mutations in PIF4 did not affect the temperature enhancement of callus formation or tissue adhesion, suggesting that leaf-derived auxin was specific for enhancing vascular regeneration in response to elevated temperatures. Tissue-specific perturbations of auxin response using a BODENLOS (BDL) mutant revealed an asymmetric effect of temperature upon regeneration: the presence of bdl above the cut prevented temperature enhancement whereas the presence of bdl below the cut prevented graft healing regardless of temperature. Promotion of tissue regeneration by elevated temperatures was not specific for graft healing and we found that elevated temperatures accelerated xylem formation between the parasite Phtheirospermum japonicum and host Arabidopsis thaliana, and this effect required shoot-derived auxin from the parasite. Taken together, our results identify a pathway by which elevated temperatures accelerate vascular development which could be of relevance for improving regeneration and better understanding inter-plant vascular connections.

category: plant biology


title: Myth-busting the provider-user relationship for digital sequence information

abstract: The United Nations Convention on Biological Diversity (CBD) formally recognized the sovereign rights of nations over their biological diversity. Implicit within the treaty is the idea that mega-biodiverse countries will provide genetic resources and grant access to them and scientists in high-income countries will use these resources and share back benefits. However, little research has been conducted on how this framework is reflected in real-life scientific practice. Currently, parties to the CBD) are debating whether or not digital sequence information (DSI) should be regulated under a new benefit-sharing framework. At this critical time point in the upcoming international negotiations, we test the fundamental hypothesis of provision and use by looking at the global patterns of access and use in scientific publications. Our data reject the provider-user relationship and suggest far more complex information flow for digital sequence information. Therefore, any new policy decisions on digital sequence information should be aware of the high level of use of DSI across low- and middle-income countries and seek to preserve open access to this crucial common good.

category: scientific communication and education


title: Mitotic Spindle Positioning (MISP) is an actin bundler that selectively stabilizes the rootlets of epithelial microvilli

abstract: Microvilli are conserved actin-based surface protrusions that have been repurposed throughout evolution to fulfill diverse cell functions. In the case of transporting epithelia, microvilli are supported by a core of actin filaments bundled in parallel by villin, fimbrin, and espin. Remarkably, microvilli biogenesis persists in mice lacking all three of these factors, suggesting the existence of unknown bundlers. We identified Mitotic Spindle Positioning (MISP) as an actin binding factor that localizes specifically to the rootlet end of the microvillus. MISP promotes rootlet elongation in cells, and purified MISP exhibits potent filament bundling activity in vitro. MISP-bundled filaments also recruit fimbrin, which further elongates and stabilizes bundles. MISP confinement to the rootlet is enforced by ezrin, which prevents decoration of the membrane-wrapped distal end of the core bundle. These discoveries reveal how epithelial cells optimize apical membrane surface area and offer insight on the remarkable robustness of microvilli biogenesis.

category: cell biology


title: Gene-dependent yeast cell death pathway requires AP-3 vesicle trafficking leading to vacuole membrane permeabilization

abstract: Unicellular eukaryotes are suggested to undergo self-inflicted destruction. However, molecular details are sparse by comparison to the mechanisms of cell death known for human cells and animal models. Here we report a molecular pathway in Saccharomyces cerevisiae leading to vacuole/lysosome membrane permeabilization and cell death. Following exposure to heat-ramp conditions, a model of environmental stress, we observed that yeast cell death occurs over several hours, suggesting an ongoing molecular dying process. A genome-wide screen for death-promoting factors identified all subunits of the AP-3 adaptor complex. AP-3 promotes stress-induced cell death through its Arf1-GTPase-dependent vesicle trafficking function, which is required to transport and install proteins on the vacuole/lysosome membrane, including a death-promoting protein kinase Yck3. Time-lapse microscopy revealed a sequence of events where AP-3-dependent vacuole permeability occurs hours before the loss of plasma membrane integrity. An AP-3-dependent cell death pathway appears to be conserved in the human pathogen Cryptococcus neoformans.


title: Heme stimulates platelet mitochondrial oxidant production via the activation of toll-like receptor 4 signaling to mediate targeted granule secretion

abstract:
Hemolysis is a pathological component of many diseases and is associated with thrombosis and vascular dysfunction. Hemolytic products, including cell-free hemoglobin and free heme directly activate platelets. However, the effect of hemolysis on platelet degranulation, a central process in not only thrombosis, but also inflammatory and mitogenic signaling, remains less clear. Our group showed that hemoglobin-induced platelet activation involved the production of mitochondrial reactive oxygen species (mtROS). However, the molecular mechanism by which extracellular hemolysis induces platelet mtROS production, and whether the mtROS regulate platelet degranulation remains unknown. Here, we demonstrate using isolated human platelets that cell free heme is a more potent agonist for platelet activation than hemoglobin, and stimulates the release of a specific set of molecules from the -granule of platelets, including the glycoprotein thrombospondin-1 (TSP-1). We uncover the mechanism of heme-mediated platelet mtROS production which is dependent on the activation of platelet TLR4 signaling and leads to the downstream phosphorylation of complex-V by the serine kinase Akt. Notably, inhibition of platelet TLR4 or Akt, or scavenging mtROS prevents heme-induced granule release in vitro. Further, heme-dependent granule release is significantly attenuated in vivo in mice lacking TLR4 or those treated with the mtROS scavenger MitoTEMPO. These data elucidate a novel mechanism of TLR4-mediated mitochondrial regulation, establish the mechanistic link between hemolysis and platelet degranulation, and begin to define the heme and mtROS-dependent platelet secretome. These data have implications for hemolysis-induced thrombo-inflammatory signaling and for the consideration of platelet mitochondria as a therapeutic target in hemolytic disorders.

Key pointsO_LIHeme induces platelet mtROS production by inhibiting complex-V activity via TLR4 signaling.
C_LIO_LIHeme stimulated platelet granule secretion is regulated by mtROS.
C_LI


title: Mis-regulation of the Nucleoporins 98 and 96 lead to defects in protein synthesis that promote hallmarks of tumorigenesis.

abstract:
The Nucleoporin 98KD (Nup98) is one of the most promiscuous translocation partners in hematological malignancies, contributing to at least 31 different truncation-fusion proteins. To date, nearly all disease models of Nup98 translocations involve ectopic expression of transgenes recapitulating the fusion protein under study, leaving the endogenous Nup98 loci unperturbed. Overlooked in these approaches is that translocation leads to the loss of one copy of normal Nup98 in addition to the loss of Nup96 - a second Nucleoporin encoded within the same mRNA and reading frame as Nup98. Nup98 and 96 are also mutated in a number of other cancer types and are located near a tumor suppressor region known to be epigenetically silenced, suggesting that their disruption is not limited to blood cancers. We found that reducing Nup98-96 function via an RNAi approach in Drosophila melanogaster (where the Nup98-96 shared mRNA and reading frame gene structure is conserved) de-regulates the cell cycle. We find evidence of over-proliferation in Nup98-96 deficient tissues, counteracted by elevated apoptosis and aberrant Wingless and JNK signaling associated with chronic wound healing. When the knockdown of Nup98-96 is combined with inhibition of apoptosis, we see synergism leading to dramatic tissue overgrowth, consistent with a tumor-suppressor function for endogenous Nup98 and 96. To understand how growth and proliferation become mis-regulated when Nup98-96 levels are reduced, we performed RNAseq and uncovered a gene expression signature consistent with defects in ribosome biogenesis. We found that reducing Nup 98 and 96 function limits nuclear export of the ribosome component RpL10A, leading to defects in protein synthesis. Defects in protein synthesis are sufficient to trigger JNK signaling that contributes to compensatory proliferation and hallmarks of tumorigenesis when apoptosis is inhibited. Based upon our data, we suggest that the partial loss of Nup98 and Nup96 function in translocations could de-regulate protein synthesis leading to stress signaling that cooperates with other mutations in cancer to promote tumorigenesis.

HighlightsO_LICompromising Nups 98 and 96 triggers cell death and compensatory proliferation via JNK signaling that becomes tumorigenic when apoptosis is blocked
C_LIO_LIReducing Nup 98 and 96 function limits nuclear export of the ribosome stalk component RpL10A, leading to defects in protein synthesis which cause stress signaling via JNK.
C_LIO_LIReduced protein synthesis coupled with increased JNK signaling, paradoxically leads to more rapid proliferation with a gene expression signature that resembles a chronic wounding response.
C_LIO_LIOverexpression of Nup98, which occurs in oncogenic fusions, leads to similar defects in protein synthesis and JNK activation.
C_LI

category: developmental biology


title: The DNA methylation landscape of five pediatric-tumor types

abstract: Fewer DNA mutations have been identified in pediatric tumors than adult tumors, suggesting that alternative tumorigenic mechanisms, including aberrant DNA methylation, may play a prominent role in pediatric tumors. Methylation is an epigenetic process of regulating gene expression in which methyl groups are attached to DNA molecules, often in promoter regions. In Wilms tumors and acute myeloid leukemias, increased levels of epigenetic silencing have been associated with worse patient outcomes. However, to date, researchers have studied methylation primarily in adult tumors and for specific genes but not on a pan-pediatric cancer scale. We addressed these gaps first by aggregating methylation data from 309 noncancerous samples and establishing baseline expectations for each gene. Even though these samples represent diverse tissue types and population ancestral groups, methylation levels were highly consistent for most genes. Second, we compared tumor methylation levels against these baseline values for five pediatric cancer types--Wilms tumors, clear cell sarcomas of the kidney, rhabdoid tumors, neuroblastomas, and osteosarcomas. Hypermethylation was more common than hypomethylation--as many as 11.8% of genes were hypermethylated in a given tumor, compared to a maximum of 4.8% for hypomethylated genes. For each cancer type, genes with the highest variance exhibited consistently divergent methylation patterns for distinct patient subsets. We evaluated whether genomic and epigenomic abnormalities contribute to pediatric tumorigenesis in a mutually exclusive manner but did not find evidence of this phenomenon. Furthermore, even though oncogenes are commonly upregulated in tumors, and tumor-suppressor genes are commonly downregulated in tumors, we did not find statistical evidence that methylation drives such patterns on a broad scale in pediatric tumors.

category: genomics


title: The timing of natural killer cell response in coronavirus infection: a concise model perspective

abstract: Coronaviruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2 cause respiratory diseases with remarkably heterogeneous progression. This in part reflects the viral ability to influence the cytokine secretion and thereby the innate immune system. Especially the viral interference of IFN-I signaling and the subsequent deficiency of innate immune response in the early phase have been associated with rapid virus replication and later excessive immune responses. We propose a mathematical framework to analyze IFN-I signaling and its impact on the interaction motif between virus, NK cells and macrophages. The model recapture divergent dynamics of coronavirus infections including the possibility for elevated secretion of IL-6 and IFN-{gamma} as a consequence of exacerbated macrophage activation. Dysfunction of NK cells recruitment increase disease severity by leading to a higher viral load peak, the possibility for excessive macrophage activation, and an elevated risk of the cytokine storm. Thus the model predicts that delayed IFN-I signaling could lead to pathogenicity in the latter stage of an infection. Reversely, in case of strong NK recruitment from infected cells we predict a possible chronic disease state with moderate and potentially oscillating virus/cytokine levels.

category: biophysics


title: Protective mucosal immunity against SARS-CoV-2 after heterologous systemic RNA-mucosal adenoviral vector immunization

abstract: Several effective SARS-CoV-2 vaccines are currently in use, but in the light of waning immunity and the emergence of novel variants, effective boost modalities are needed in order to maintain or even increase immunity. Here we report that intranasal vaccinations with adenovirus 5 and 19a vectored vaccines following a systemic DNA or mRNA priming result in strong systemic and mucosal immunity in mice. In contrast to two intramuscular injections with an mRNA vaccine, the mucosal boost with adenoviral vectors induced high levels of IgA and tissue-resident memory T cells in the respiratory tract. Mucosal neutralization of virus variants of concern was also enhanced by the intranasal boosts. Importantly, priming with mRNA provoked a more comprehensive T cell response consisting of circulating and tissue-resident memory T cells after the boost, while a DNA priming induced mostly mucosal T cells. Concomitantly, the intranasal boost strategies provided protection against symptomatic disease. Therefore, a mucosal booster immunization after mRNA priming is a promising approach to establish mucosal immunity in addition to systemic responses.

category: immunology


title: Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit.

abstract: Three highly pathogenic betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks are likely to occur. Evidence suggests that neutralizing antibodies are important for protection against infection with CoVs. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-coronavirus protection. To this end, we previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 fusion machinery, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and displays cross reactive binding to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is also maintained against the Alpha, Delta and Gamma variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region on sarbecovirus spikes. A 1.74[A] crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Thus, CV3-25 defines a novel site of sarbecovirus vulnerability that will inform pan-CoV vaccine development.


title: Vaccinia virus-based vaccines confer protective immunity against SARS-CoV-2 virus in Syrian hamsters

abstract: COVID-19 in humans is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that belongs to the beta family of coronaviruses. SARS-CoV-2 causes severe respiratory illness in 10-15% of infected individuals and mortality in 2-3%. Vaccines are urgently needed to prevent infection and to contain viral spread. Although several mRNA- and adenovirus-based vaccines are highly effective, their dependence on the "cold chain" transportation makes global vaccination a difficult task. In this context, a stable lyophilized vaccine may present certain advantages. Accordingly, establishing additional vaccine platforms remains vital to tackle SARS- CoV-2 and any future variants that may arise. Vaccinia virus (VACV) has been used to eradicate smallpox disease, and several attenuated viral strains with enhanced safety for human applications have been developed. We have generated two candidate SARS-CoV-2 vaccines based on two vaccinia viral strains, MVA and v-NY, that express full-length SARS-CoV-2 spike protein. Whereas MVA is growth-restricted in mammalian cells, the v-NY strain is replication-competent. We demonstrate that both candidate recombinant vaccines induce high titers of neutralizing antibodies in C57BL/6 mice vaccinated according to prime-boost regimens. Furthermore, our vaccination regimens generated TH1-biased immune responses in mice. Most importantly, prime-boost vaccination of a Syrian hamster infection model with MVA-S and v-NY-S protected the hamsters against SARS-CoV-2 infection, supporting that these two vaccines are promising candidates for future development. Finally, our vaccination regimens generated neutralizing antibodies that partially cross-neutralized SARS-CoV-2 variants of concern.


title: Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern

abstract: Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remained effective against emerging variants of concern in vitro and in vivo. CV3-1 bound to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the "RBD-up" position and triggered potent shedding of the S1 subunit. In contrast, CV3-25 inhibited membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among {beta}-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

category: microbiology


title: SARS-CoV-2 B.1.351 (beta) variant shows enhanced infectivity in K18-hACE2 transgenic mice and expanded tropism to wildtype mice compared to B.1 variant

abstract: SARS-CoV-2 variants display enhanced transmissibility and/or immune evasion and can be generated in humans or animals, like minks, thus generating new reservoirs. The continuous surveillance of animal susceptibility to new variants is necessary to predict pandemic evolution. In this study we demonstrate that, compared to the B.1 SARS-CoV-2 variant, K18-hACE2 transgenic mice challenged with the B.1.351 variant displayed a faster progression of infection. Furthermore, we also report that B.1.351 can establish infection in wildtype mice, while B.1 cannot. B.1.351-challenged wildtype mice showed a milder infection than transgenic mice, confirmed by detectable viral loads in oropharyngeal swabs and tissues, lung pathology, immunohistochemistry and serology. In silico models supported these findings by demonstrating that the Spike mutations in B.1.351 resulted in increased affinity for both human and murine ACE2 receptors. Overall, this study highlights the plasticity of SARS-CoV-2 animal susceptibility landscape, which may contribute to viral persistence and expansion.


title: BWA-mem is not the best aligner for ancient DNA short reads.

abstract:
Xu and colleagues (Xu et al., 2021) recently suggested a new parameterisation of BWA-mem (Li, 2013) as an alternative to the current standard BWA-aln (Li and Durbin, 2009) to process ancient DNA sequencing data. The authors tested several combinations of the -k and -r parameters to optimise BWA-mems performance with degraded and contaminated ancient DNA samples. They report that using BWA-mem with -k 19 -r 2.5 parameters results in a mapping efficiency comparable to BWA-aln with -I 1024 -n 0.03 (i.e. a derivation of the standard parameters used in ancient DNA studies; (Schubert et al., 2012)), while achieving significantly faster run times.

We recently performed a systematic benchmark of four mapping software (i.e. BWA-aln, BWA-mem, NovoAlign (http://www.novocraft.com/products/novoalign), and Bowtie2 (Langmead and Salzberg, 2012) for ancient DNA sequencing data and quantified their precision, accuracy, specificity, and impact on reference bias (Oliva et al., 2021). Notably, while multiple parameterisations were tested for BWA-aln, NovoAlign, and Bowtie2, we only tested BWA-mem with default parameters.

Here, we use the alignment performance metrics from Oliva et al. to directly compare the recommended BWA-mem parameterisation reported in Xu et al. with the best performing alignment methods determined in the Oliva et al. benchmarks, and we make recommendations based on the results.

category: bioinformatics


title: The Ca2+ concentration in vitro impacts the cytokine production of mouse and human lymphoid cells and the polarization of human macrophages

abstract: Various aspects of the in vitro culture conditions can impact the functional response of immune cells. For example, it was shown that a Ca2+ concentration of at least 1.5 mM during in vitro stimulation is needed for optimal cytokine production by conventional {beta} T cells. Here we extend these findings by showing that also unconventional T cells (invariant Natural Killer T cells, mucosal-associated invariant T cells, {gamma}{delta} T cells), as well as B cells, show an increased cytokine response following in vitro stimulation in the presence of elevated Ca2+ concentrations (approx. 1.8 mM). This effect appeared more pronounced with mouse than with human lymphoid cells and did not influence their survival. A similarly increased cytokine response due to elevated Ca2+ levels was observed with primary human monocytes. In contrast, primary human monocyte-derived macrophages, either unpolarized (M0) or polarized into M1 or M2 macrophages, displayed increased cell death in the presence of elevated Ca2+ concentrations. Furthermore, elevated Ca2+ concentrations promoted phenotypic M1 differentiation by increasing M1 markers on M1 and M2 macrophages and decreasing M2 markers on M2 macrophages. However, the cytokine production of macrophages, again in contrast to the lymphoid cells, was unaltered by the Ca2+ concentration. In summary, our data demonstrate that the Ca2+ concentration during in vitro cultures is an important variable to be considered for functional experiments and that elevated Ca2+ levels can boost cytokine production by both mouse and human lymphoid cells.


title: Biomorphogenic feedbacks and the spatial organisation of a dominant grass steer dune development

abstract: Nature-based solutions to mitigate the impact of future climate change depend on restoring biological diversity and natural processes. Coastal foredunes represent the most important natural flood barriers along coastlines worldwide, but their area has been squeezed dramatically because of a continuing urbanisation of coastlines, especially in Europe. Dune development is steered by the development of vegetation in interaction with sand fluxes from the beach. Marram grass (Calamagrostis arenaria, formerly Ammophila arenaria) is the main dune building species along most European coasts, but also in other continents where the species was introduced. Engineering of coastal dunes, for instance by building dunes in front of dikes, needs to be based on a solid understanding of the species interactions with the environment. Only quantitative approaches enable the further development of mechanistic models and coastal management strategies that encapsulate these biomorphogenic interactions. We here provide a quantitative review of the main biotic and physical interactions that affect marram grass performance, their interactions with sand fluxes and how they eventually shape dune development. Our review highlights that the species spatial organisation is central to dune development. We further demonstrate this importance by means of remote sensing and a mechanistic model and provide an outlook for further research on the use of coastal dunes as a nature-based solution for coastal protection.

category: ecology


title: A Chromosome-level Genome Assembly of the Reed Warbler (Acrocephalus scirpaceus)

abstract:
The reed warbler (Acrocephalus scirpaceus) is a long-distance migrant passerine with a wide distribution across Eurasia. This species has fascinated researchers for decades, especially its role as host of a brood parasite, and its capacity for rapid phenotypic change in the face of climate change. Currently, it is expanding its range northwards in Europe, and is altering its migratory behaviour in certain areas. Thus, there is great potential to discover signs of recent evolution and its impact on the genomic composition of the reed warbler. Here we present a high-quality reference genome for the reed warbler, based on PacBio, 10X and Hi-C sequencing. The genome has an assembly size of 1,075,083,815 bp with a scaffold N50 of 74,438,198 bp and a contig N50 of 12,742,779 bp. BUSCO analysis using aves_odb10 as a model showed that 95.7% of genes in the assembly were complete. We found unequivocal evidence of two separate macrochromosomal fusions in the reed warbler genome, in addition to the previously identified fusion between chromosome Z and a part of chromosome 4A in the Sylvioidea superfamily. We annotated 14,645 protein-coding genes, of which 97.5% were complete BUSCO orthologs. This reference genome will serve as an important resource, and will provide new insights into the genomic effects of evolutionary drivers such as coevolution, range expansion, and adaptations to climate change, as well as chromosomal rearrangements in birds.

Significance statementThe reed warbler (Acrocephalus scirpaceus) has been lacking a genomic resource, despite having been broadly researched in studies of coevolution, ecology and adaptations to climate change. Here, we generated a chromosome-length genome assembly of the reed warbler, and found evidence of macrochromosomal fusions in its genome, which are likely of recent origin. This genome will provide the opportunity for a deeper understanding of the evolution of genomes in birds, as well as the evolutionary path and possible future of the reed warbler.


abstract:
O_LILight-level geolocators have revolutionised the study of animal behaviour. However, lacking precision, they cannot be used to infer behaviour beyond large-scale movements. Recent technological developments have allowed the integration of barometers, magnetometers, accelerometers and thermometers into geolocator tags, offering new insights into the behaviour of species which were previously impossible to tag.
C_LIO_LIHere, we introduce an R toolbox for identifying behavioural patterns from multisensor geolocator tags, with functions specifically designed for data visualisation, calibration, classification and error estimation. Some functions are also tailored for identifying specific behavioural patterns in birds (most common geolocators-tagged species), but are flexible for other applications.
C_LIO_LIFinally, we highlight opportunities for applying this toolbox to other species beyond birds, the behaviours they might identify and their potential applications beyond behavioural analyses.
C_LI

Data archivingCurrently, the package is on github and will be submitted to CRAN after review. The supporting code (package manual) for this paper is also on https://kiranlda.github.io/PAMLrManual/index.html, but will later be hosted by the Swiss Ornithological Institute.

Summarypamlr: a toolbox for analysing animal behaviour using pressure, acceleration, temperature, magnetic and light data in R

category: animal behavior and cognition


title: The Effect of Blood Viscosity on Shear-Induced Hemolysis using a Magnetically Levitated Shearing Device

abstract:
IntroductionBlood contacting medical devices, including rotary blood pumps, can cause shear-induced blood damage that may lead to adverse effects in patients. Due in part to an inadequate understanding of how cell-scale fluid mechanics impact red blood cell membrane deformation and damage, there is currently not a uniformly accepted engineering model for predicting blood damage caused by complex flow fields within ventricular assist devices (VADs).

MethodsWe empirically investigated hemolysis in an axial Couette flow device typical of a rotary VAD to expand our current understanding of shear-induced blood damage in two ways. First, we used a magnetically levitated device to accurately control the shear rate and exposure time experienced by blood and to minimize the effects of other uncharacterized stresses. Second, we explored the effects of both hematocrit and plasma viscosity on shear-induced hemolysis to characterize blood damage based on the viscosity-independent shear rate, rather than on shear stress.

ResultsOver a shear rate range of 20,000-80,000 1/s, the Index of Hemolysis was found to be largely independent of hematocrit, bulk viscosity, or the suspension media viscosity.

ConclusionIt is recommended that future investigations of shear-induced blood damage report findings with respect to the viscosity-neutral term of shear rate, in addition to the bulk whole blood viscosity measured at an appropriate shear rate relevant to the flow conditions of the device.

category: bioengineering


title: Black Dots: Microcontact-Printed, Reference-Free Traction Force Microscopy

abstract:
Measuring the traction forces produced by cells provides insight into their behavior and physiological function. Here, we developed a technique (dubbed  black dots) that microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. To demonstrate our technique, we assessed human platelets, which can generate a large range of forces within a population. We find platelets that exert more force have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by this technique, we were able to evaluate multivariate mixed effects models with interaction terms and conduct a clustering analysis to identify clusters within our data. These statistical techniques demonstrated a complex relationship between spread area, circularity, F-actin dispersion, and platelet force, including cooperative effects that significantly associate with platelet traction forces.

GRAPHICAL ABSTRACT

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title: Multitask group Lasso for Genome Wide association Studies in admixed populations

abstract:
Genome-Wide Association Studies, or GWAS, aim at finding Single Nucleotide Polymorphisms (SNPs) that are associated with a phenotype of interest. GWAS are known to suffer from the large dimensionality of the data with respect to the number of available samples. Other limiting factors include the dependency between SNPs, due to linkage disequilibrium (LD), and the need to account for population structure, that is to say, confounding due to genetic ancestry.

We propose an efficient approach for the multivariate analysis of multi-population GWAS data based on a multitask group Lasso formulation. Each task corresponds to a subpopulation of the data, and each group to an LD-block. This formulation alleviates the curse of dimensionality, and makes it possible to identify disease LD-blocks shared across populations/tasks, as well as some that are specific to one population/task. In addition, we use stability selection to increase the robustness of our approach. Finally, gap safe screening rules speed up computations enough that our method can run at a genome-wide scale.

To our knowledge, this is the first framework for GWAS on diverse populations combining feature selection at the LD-groups level, a multitask approach to address population structure, stability selection, and safe screening rules. We show that our approach outperforms state-of-the-art methods on both a simulated and a real-world cancer datasets.


title: Multitask group Lasso for Genome Wide association Studies in diverse populations


title: Regulus, a transcriptional regulatory networks inference tool based on Semantic Web technologies

abstract:
MotivationTranscriptional regulation -a major field of investigation in life science-is performed by binding of specialized proteins called transcription factors (TF) to DNA in specific, context-dependent regulatory regions, leading to either activation or inhibition of gene expression. Relations between TF, regions and genes can be described as regulatory networks, which are basically knowledge graphs containing the relationships between the different entities. Current methods of transcriptional regulatory networks inference rarely use information about TF binding or regulatory regions, often require a large number of samples and most of time do not indicate if the TF-gene relation is an activation or an inhibition. The resulting networks may then contain inconsistent relations and the methods are not applicable for common experimental or clinical settings, where the number of samples is limited. Therefore, based on our previous experience of formalizing the Regulatory Circuits data-sets with Semantic Web Technologies, we decided to create a new tool for transcriptional networks inference, that could solve these issues. Results: Our tool, Regulus, provides candidate signed TF-gene relations computed from gene expressions, regulatory region activities and TF binding sites data, together with the genomic location of all entities. After creating expressions and activities patterns, data are integrated into a RDF endpoint. A dedicated SPARQL query retrieves all potential TF-region relations for a given gene expression pattern. These ternary TF-region-gene pattern relations are then filtered and signed using a logical consistency check translated from biological knowledge. Regulus compares favorably to its closest network inference method, provides signs which are consistent with public databases and, when applied to real biological data, identifies both known and potential new regulators. We also provide several means to more stringently filter the output regulators. Altogether, we propose a new tool devoted to transcriptional network inference in settings where samples are scarce and cell populations may be closely related.

AvailabilityThe Regulus package is available at https://gitlab.com/gcollet/regulus


title: SigTools: Exploratory Visualization for Genomic Signals

abstract:
MotivationWith the advancement of sequencing technologies, genomic data sets are constantly being expanded by high volumes of different data types. One recently introduced data type in genomic science is genomic signals, which are usually short-read coverage measurements over the genome. An example of genomic signals is Epigenomic marks which are utilized to locate functional and nonfunctional elements in genome annotation studies. To understand and evaluate the results of such studies, one needs to understand and analyze the characteristics of the input data.

ResultsSigTools is an R-based genomic signals visualization package developed with two objectives: 1) to facilitate genomic signals exploration in order to uncover insights for later model training, refinement, and development by including distribution and autocorrelation plots. 2) to enable genomic signals interpretation by including correlation, and aggregation plots. Moreover, Sigtools also provides text-based descriptive statistics of the given signals which can be practical when developing and evaluating learning models. We also include results from 2 case studies. The first examines several previously studied genomic signals called histone modifications. This use case demonstrates how SigTools can be beneficial for satisfying scientists curiosity in exploring and establishing recognized datasets. The second use case examines a dataset of novel chromatin state features which are novel genomic signals generated by a learning model. This use case demonstrates how SigTools can assist in exploring the characteristics and behavior of novel signals towards their interpretation. In addition, our corresponding web application, SigTools-Shiny, extends the accessibility scope of these modules to people who are more comfortable working with graphical user interfaces instead of command-line tools.

AvailabilitySigTools source code, installation guide, and manual is available on http://github.com/shohre73.

Contactshohre_masoumi@sfu.ca


title: New insights into the earlier evolutionary history of epiphytic macrolichens

abstract: Lichens are well known as pioneer organisms colonizing bare surfaces such as rocks and therefore have been hypothesized to play a role in the early formation of terrestrial ecosystems. Given the rarity of fossil evidence, our understanding of the evolutionary history of lichen-forming fungi is primarily based on molecular dating approaches. These studies suggest extant clades of macrolichens diversified after the K-Pg boundary. Here we corroborate the mid-Mesozoic fossil Daohugouthallus ciliiferus as an epiphytic macrolichen that predates the K-Pg boundary by 100 Mys. Based on new material and geometric morphometric analysis, we demonstrate that the Jurassic fossil is morphologically most similar to Parmeliaceae, but cannot be placed in Parmeliaceae or other similar family-level clades forming macrolichens as these evolved much later. Consequently, a new family, Daohugouthallaceae, is proposed here to accommodate this fossil, which reveals macrolichens may have been diverse long before the Cenozoic diversification of extant lineages.

category: paleontology


title: Controlled Administration of Dehydrochloromethyltestosterone in Humans: Urinary Excretion and Long-Term Detection of Metabolites for Anti-Doping Purpose

abstract:
Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol(R). It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail.

Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4-chloro-17{beta}-hydroxymethyl-17-methyl-18-nor-5-androstan-13-en-3-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite.

The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17-hydroxymethyl-17{beta}-methyl-18-nor-androsta-4,13-dien-3{beta}-ol. It may serve as additional confirmatory metabolite.

It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17-methyl steroids in general.

category: pharmacology and toxicology


title: A programmable probiotic encapsulation system enhances therapeutic delivery in vivo

abstract: Recent advances in therapeutic modulation of human microbiota have driven new efforts to engineer living microbial medicines using synthetic biology. However, a long-standing challenge for live bacterial therapies is balancing the high dose required to achieve robust efficacy with the potential for sepsis. Here, we developed a genetically encoded microbial encapsulation system with tunable and dynamic expression of surface capsular polysaccharides to enhance therapeutic delivery. Following a synthetic small RNA knockdown screen of the capsular biosynthesis pathway, we constructed synthetic gene circuits that regulate bacterial encapsulation based on sensing the levels of environmental inducer, bacterial density, and blood pH. The induced encapsulation system enabled tunable immunogenicity and survivability of the probiotic Escherichia coli, resulting in increased maximum tolerated dose and enhanced efficacy in murine cancer models. Furthermore, triggering in situ encapsulation was found to increase microbial translocation between mouse tumors, leading to efficacy in distal tumors. The programmable encapsulation system demonstrates a new approach to control microbial therapeutic profiles in vivo using synthetic biology.

category: synthetic biology


title: Resurrecting essential amino acid biosynthesis in a mammalian cell

abstract: Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids1. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly2,3, raising the possibility that these pathways were lost from organisms with access to abundant EAAs in the environment4,5. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in a mammalian cell. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese Hamster Ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation, and thus represents a successful example of metazoan EAA biosynthesis restoration. This prototrophic CHO line grows in valine-free medium, and metabolomics using labeled precursors verified de novo biosynthesis of valine. RNA-seq profiling of the valine prototrophic CHO line showed that the synthetic pathway minimally disrupted the cellular transcriptome. Furthermore, valine prototrophic cells exhibited transcriptional signatures associated with rescue from nutritional starvation. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, thus paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.


title: The two-component response regulator OrrA confers dehydration tolerance by regulating anaKa expression in the cyanobacterium Anabaena sp. strain PCC 7120

abstract: The aquatic cyanobacterium Anabaena sp. strain PCC 7120 exhibits dehydration tolerance. The regulation of gene expression in response to dehydration is crucial for the acquisition of dehydration tolerance, but the molecular mechanisms underlying dehydration responses remain unknown. In this study, the functions of the response regulator OrrA in the regulation of salt and dehydration responses were investigated. Disruption of orrA abolished or diminished the induction of hundreds of genes in response to salt stress and dehydration. Thus, OrrA is a principal regulator of both stress responses. In particular, OrrA plays a crucial role in dehydration tolerance because an orrA disruptant completely lost the ability to regrow after dehydration. Moreover, in the OrrA regulon, anaKa encoding a protein of unknown function was revealed to be indispensable for dehydration tolerance. OrrA and AnaK are conserved among the terrestrial cyanobacteria, suggesting their conserved functions in dehydration tolerance in cyanobacteria.


title: Clostridioides difficile binary toxin binding component (CDTb) increases virulence in a hamster model

abstract: Clostridioides difficile is the leading cause of hospital-acquired gastrointestinal infection, in part due to the existence of binary toxin (CDT)-expressing hypervirulent strains. We have previously shown that CDT interacts with the TLR2/6 heterodimer to induce inflammation, and in this study we further explore this interaction as well as the contribution of the separate components of CDT, CDTa and CDTb. We found that the binding component, CDTb, is capable of inducing inflammation. Additionally, complementation of a CDT-deficient C. difficile strain with CDTb alone restored virulence in a hamster model of C. difficile infection. Overall, this study demonstrates that the binding component of C. difficile binary toxin contributes to virulence during infection.


title: ZeBox: A novel non-intrusivecontinuous-use technology to trap and killairborne microbes

abstract: Preventing nosocomial infection is a major unmet need of our times. Existing air decontamination technologies suffer from demerits such as toxicity of exposure, species specificity, noxious gas emission, environment-dependent performance and high power consumption. Here, we present a novel technology called "ZeBox" that transcends the conventional limitations and achieves high microbicidal efficiency. In ZeBox, a non-ionizing electric field extracts naturally charged microbes from flowing air and deposits them on engineered microbicidal surfaces. The surfaces three dimensional topography traps the microbes long enough for them to be inactivated. The electric field and chemical surfaces synergistically achieve rapid inactivation of a broad spectrum of microbes. ZeBox achieved near complete kill of airborne microbes in challenge tests (5-9 log reduction) and >90% efficiency in a fully functional stem cell research facility in the presence of humans. Thus, ZeBox fulfills the dire need for a real-time, continuous, safe, trap-and-kill air decontamination technology.


title: Genome wide association studies define minimal set of loci for prediction of penicillin and tetracycline susceptibility in Neisseria gonorrhoeae

abstract:
BackgroundWhile Neisseria gonorrhoeae poses an urgent public health threat because of increasing antimicrobial resistance, much of the circulating population remains susceptible to historical treatment regimens. Point-of-care diagnostics that report susceptibility could allow for reintroduction of these regimens, but development of such diagnostics has been limited to ciprofloxacin, for which susceptibility can be predicted from a single locus.

MethodsWe assembled a dataset of 12,045 N. gonorrhoeae genomes with phenotypic resistance data for tetracycline (n = 3,611) and penicillin (n = 6,935). Using conditional genome wide association studies (GWAS), we sought to define genetic variants associated with susceptibility to penicillin and tetracycline. We evaluated the sensitivity and specificity of these variants for predicting susceptibility and non-resistance in our collection of gonococcal genomes.

FindingsIn our conditional penicillin GWAS, the presence of a genetic variant defined by a non-mosaic penA allele without an insertion at codon 345 was significantly associated with penicillin susceptibility and had the highest negative effect size of significant variants (p = 5.0 x 10-14, {beta} = -2.5). In combination with the absence of blaTEM, this variant predicted penicillin susceptibility with high specificity (99.8%) and modest sensitivity (36.7%). For tetracycline, the wild type allele at rpsJ codon 57, encoding valine, was significantly associated with tetracycline susceptibility (p = 5.6 x 10-16, {beta} = -1.6) after conditioning on the presence of tetM. The combination of rpsJ codon 57 allele and tetM absence predicted tetracycline susceptibility with high specificity (97.2%) and sensitivity (88.7%).

InterpretationAs few as two genetic loci can predict susceptibility to penicillin and tetracycline in N. gonorrhoeae with high specificity. Molecular point-of-care diagnostics targeting these loci have the potential to increase available treatments for gonorrhea.

FundingNational Institute of Allergy and Infectious Diseases, the National Science Foundation, and the Smith Family Foundation

Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed with the terms "Neisseria gonorrhoeae" and "diagnostic" or "assay" plus "penicillin" or "tetracycline" for reports in any language published up to July 1, 2021. We additionally searched for "Neisseria gonorrhoeae" and "genome wide association study". We found that previously proposed molecular diagnostics for penicillin and tetracycline susceptibility either exclusively focused on plasmid-mediated resistance (i.e., targeting blaTEM or tetM) or did not include variants in genes encoding antibiotic targets (e.g., did not include penA or rpsJ). Targets for molecular surveillance have focused on resistance-associated alleles rather than susceptibility-associated alleles. We did not find any previous penicillin or tetracycline conditional genome wide association studies (GWAS) in N. gonorrhoeae.

Added value of this studyTo identify targets for molecular diagnostics that predict penicillin and tetracycline susceptibility, we conducted GWAS conditioning on the presence of plasmid-mediated resistance determinants to detect chromosomal loci with the highest association with susceptibility. We discovered a sequence (penA_01) that differentiates susceptible isolates from those with a resistance-associated insertion at codon 345 and from those with mosaic penA alleles, which is associated with penicillin susceptibility. We also found that rpsJ codon 57 was the chromosomal locus contributing the most to tetracycline susceptibility. The combination of these chromosomal loci and the absence of plasmid encoded determinants predicts penicillin and tetracycline susceptibility with high specificity in both a large global collection of N. gonorrhoeae and a validation dataset consisting of recently published genomes from CDCs Gonococcal Isolate Surveillance Program (GISP) surveillance collected in 2018.

Implications of all the available evidenceThe chromosomal loci penA_01 and rpsJ codon 57 in combination with plasmid loci blaTEM and tetM are candidates for the development of point-of-care molecular diagnostics for penicillin and tetracycline susceptibility. The loci may be combined with the currently available ciprofloxacin susceptibility diagnostics to predict susceptibility to multiple antibiotics. Additionally, our study suggests that conditional GWAS focused on variants associated with susceptibility may be a promising approach to identify minimal sets of loci for molecular diagnostics and surveillance.


title: Loci for prediction of penicillin and tetracycline susceptibility in Neisseria gonorrhoeae: a genome wide association study


title: Identification of new Dickeya dadantii virulence factors secreted by the type 2 secretion system

abstract:
Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system (T2SS) named Out is necessary for bacterial virulence. Its study in D. dadantii showed that it secretes a wide range of plant cell wall degrading enzymes, including pectinases and a cellulase. However, the full repertoire of exoproteins it can secrete has probably not yet been identified. Secreted proteins are first addressed to the periplasm before their secretion by Out. No secretion signal present on the protein allows the identification of substrates of a T2SS. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced encoding a protein with a signal sequence. We identified four new Out-secreted proteins: the expansin YoaJ, the putative virulence factor VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that SvfA and SvfB are required for full virulence of D. dadantii and showed that svf genes are present in a variable number of copies in other Pectobacteriaceae, up to three in D. fanghzongdai. This work opens the way to the study of the role of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae.

IMPORTANCEThe plant pathogen Dickeya rely on a type 2 secretion system named Out for their pathogenicity. Importance of plant cell wall degrading enzymes secreted by this system has been well studied. However, existence and role of other Out-secreted proteins has barely been investigated. By mining D. dadantii transcriptome data, we identified four new Out-secreted proteins. We showed that two of them, SvfA and SvfB, are necessary for the full virulence of the bacteria. These findings show that identification of all the proteins secreted by the Dickeya Out system is necessary for a better knowledge of the virulence of these bacteria.


title: Global dissemination of tet(X3) and tet(X6) among livestock-associated Acinetobacter is sporadically mediated by highly diverse plasmidomes

abstract:
The emergence of plasmid-borne tet(X) genes mediated high-level resistance of tigecycline largely threatening its clinical effectiveness. Currently, the dissemination pattern of plasmid-borne tet(X) genes remains unclear. In this study, 684 fecal and environmental samples were collected at six livestock farms, and 15 tet(X)-positive Acinetobacter isolates were recovered, mainly including 9 tet(X3)- and 5 tet(X6)-positive A. towneri strains. A clonal dissemination of tet(X3)-positive A. towneri was detected in a swine farm, while the tet(X6)-positive A. towneri strains mainly sporadically disseminated in the same farm. A tet(X3)-carrying plasmid (pAT181) was self-transmissible from a tigecycline-susceptible A. towneri strain to A. baumannii ATCC17978, causing a 128-fold and 64-512-fold increase in the MIC values of tigecycline and the other tetracyclines, respectively. Worrisomely, pAT181 was stably maintained and increased the growth rate of ATCC17978. Further identification of tet(X)s in 10,680 Acinetobacter genomes retrieved from GenBank revealed that, tet(X3) (n=249) followed by tet(X5)-like (n=61) and tet(X6) (n=53) are the prevalent alleles mainly carried by four species, and most of them are livestock associated. Phylogenetic analysis showed that most of tet(X3)- and tet(X6)-positive isolates disseminate sporadically. The structures of tet(X3) and tet(X6) plasmidomes are highly diverse and no epidemic plasmids have emerged yet. However, cross-species and cross-region transmissions of tet(X3) might have been mediated by several plasmids in a small proportion of strains. Our study evidence that tet(X3) and tet(X6) currently disseminate sporadically in Acinetobacter. Continuous surveillance for tet(X)s in the context of One Health is necessary to prevent them from transmitting to humans.

ImportanceRecently identified plasmid-borne tet(X) genes highly challenged the efficiency of tigecycline, a last resort antibiotic for severe infection. Currently, the dissemination pattern of plasmid-borne tet(X) genes remains unclear. In this study, we first identified plasmid-borne tet(X)-positive Acinetobacter spp. strains from fecal and environmental samples collected at six livestock farms. A clonal dissemination of tet(X3)-positive A. towneri was detected in a swine farm, while the tet(X6)-positive A. towneri strains mainly disseminated sporadically in the same farm. A tet(X3)-carrying plasmid was found self-transmissible resulting in enhanced tigecycline resistance and growth rate. Further exploring a global dataset of tet(X)-positive Acinetobacter genomes retried from GenBank revealed that most of tet(X3) and tet(X6)-positive isolates share highly distant relationship, and the structures of tet(X3) and tet(X6) plasmidomes are highly diverse. Our study evidence that tet(X3) and tet(X6) disseminate sporadically in Acinetobacter and continuous surveillance for tet(X)s in the context of One Health is necessary.


title: Biofilm Interaction Mapping and Analysis (BIMA): A tool for deconstructing interspecific interactions in co-culture biofilms

abstract:
Pseudomonas species are ubiquitous in nature and include numerous medically, agriculturally and technologically beneficial strains of which the interspecific interactions are of great interest for biotechnologies. Specifically, co-cultures containing Pseudomonas stutzeri have been used for bioremediation, biocontrol, aquaculture management and wastewater denitrification. Furthermore, the use of P. stutzeri biofilms, in combination with consortia based approaches, may offer advantages for these processes. Understanding the interspecific interaction within biofilm co-cultures or consortia provides a means for improvement of current technologies. However, the investigation of biofilm based consortia has been limited. We present an adaptable and scalable method for the analysis of macroscopic interactions (colony morphology, inhibition and invasion) between colony forming bacterial strains using an automated printing method followed by analysis of the genes and metabolites involved in the interactions. Using Biofilm Interaction Mapping and Analysis (BIMA), these interactions were investigated between P. stutzeri strain RCH2, a denitrifier isolated from chromium (VI) contaminated soil, and thirteen other species of pseudomonas isolated from non-contaminated soil. The metabolites and genes associated with both active co-culture growth and inhibitory growth were investigated using mass spectrometry based metabolomics and mutant fitness profiling of a DNA-barcoded mutant library. One interaction partner, Pseudomonas fluorescens N1B4 was selected for mutant fitness profiling; with this approach four genes of importance were identified and the effects on interactions were evaluated with deletion mutants and metabolomics.

IMPORTANCEThe Biofilm Interaction Mapping and Analysis (BIMA) methodology provides a way to rapidly screen for positive and negative interspecific interactions, followed by an analysis of the genes and metabolites that may be involved. Knowledge of these may offer opportunities for engineered strains with improved function in biotechnology systems. P. stutzeri, an organism with wide-spread utilization in consortia based biotechnologies, was used to demonstrate the utility of this approach. Where little is known about the factors influencing biofilm based interactions, elucidation of the genes and metabolites involved allows for better control of the system for improved function or yield.


title: Dissemination and pathogenesis of toxigenic Clostridium perfringens strains linked to neonatal intensive care units and Necrotising Enterocolitis

abstract:
BackgroundClostridium perfringens is an anaerobic toxin-producing bacterium that has long been associated with intestinal diseases, particularly in neonatal humans and animals. More recently, infant gut microbiome studies have suggested an important link between C. perfringens and the devastating preterm-associated disease Necrotising Enterocolitis (NEC), but in-depth studies on this pathogen (genomics and mechanistic) are lacking.

Methods/MaterialsWe isolated and whole-genome sequenced 274 infant-associated C. perfringens isolates from 5 hospitals across the UK between 2011-2016 (including longitudinal samples from 31 individuals). We performed in-depth genomic analyses, phenotypically characterised pathogenic traits of 10 strains (including 4 C. perfringens from NEC patients) and established a novel oral-challenge C57BL/6 mouse infection model for microbe-host studies.

ResultsPore-forming toxin encoding genes pfoA and cpb2 were enriched within hypervirulent lineages that exclusively consisted of C. perfringens-associated NEC (CPA-NEC) strains, in addition to overabundance of colonisation factors. Importantly, we identified a circulating C. perfringens variant, eventually linked to a fatal CPA-NEC case. The variant was detected consistently within 6 individuals in two sister hospitals across a 40-day window, demonstrating for the first time the intra- and inter-hospital dissemination of C. perfringens. CPA-NEC isolates were determined phenotypically to be more virulent (linked with overabundance of gene pfoA) than isolates obtained from non-NEC preterm babies. In addition, two pfoA-positive CPA-NEC C. perfringens strains were confirmed to induce clinical inflammatory tissue lesions in vivo.

ConclusionsHypervirulent lineages are linked to CPA-NEC, potentially due to the production of pore-forming toxins, coupled with higher metabolic, transmission, and pathogenic capacities. These studies indicate C. perfringens is an important bacterial pathogen in preterm infants and highlights the requirement for further investigation into development of intervention and therapeutic strategies.


title: High Level of Interaction between Phages and Bacteria in an Artisanal Raw Milk Cheese Microbial Community

abstract: Endogenous starter cultures are used in the production of several cheeses around the world, such as Parmigiano-Reggiano, in Italy, Epoisses, in France, and Canastra, in Brazil. These microbial communities are responsible for many of the intrinsic characteristics of each of these cheeses. Bacteriophages are ubiquitous around the world, well known to be involved in the modulation of complex microbiological processes. However, little is known about phage-bacteria growth dynamics in cheese production systems, where phages are normally treated as problems, as the viral infections can negatively affect or even eliminate the starter culture during production. Furthermore, a recent metagenomic based meta-analysis has reported that cheeses contain a high abundance of phage-associated sequences. Here, we analyse the viral and bacterial metagenomes of Canastra cheese, a tradition artisanal cheese produced using an endogenous starter culture. We observe a very high phage diversity level, mostly composed of novel sequences. We detect several metagenomic assembled bacterial genomes at strain level resolution, and several putative phage-bacteria interactions, evidenced by the recovered viral and bacterial genomic signatures. We postulate that at least one bacterial strain detected could be endogenous to the Canastra region, in Brazil, and that its growth seems to be modulated by native phages present in this artisanal production system. This relationship is likely to influence the fermentation dynamics and ultimately the sensorial profile of these cheeses, with implication for all cheeses that employ similar production processes around the world.


title: Microbial byproducts determine reproductive fitness of free-living and parasitic nematodes

abstract: A distinguishing feature of Trichuris nematodes is that these parasitic worms reproduce within the digestive tracts of humans and other mammalian hosts shedding thousands of eggs daily, facilitating their sustained presence in the environment and hampering eradication efforts. Although this aspect of the lifecycle places Trichuris in a microbiota-rich environment, metabolic byproducts of bacteria that facilitate the reproductive development of parasites are unknown. Here, we employ a pipeline using the well-characterized free-living nematode C. elegans to identify microbial factors with conserved roles in the reproduction of nematodes. A screen for E. coli mutants that impair C. elegans fertility identified genes in fatty acid biosynthesis and ethanolamine utilization pathways, including fabH and eutN. Trichuris muris eggs displayed defective hatching in the presence of E. coli deficient in fabH or eutN due to reduction in arginine or elevated levels of aldehydes, respectively. Remarkably, T. muris reared in gnotobiotic mice colonized with these E. coli mutants displayed profound abnormalities including morphological defects and a failure to lay viable eggs. These findings indicate that microbial byproducts mediate evolutionarily conserved transkingdom interactions that impact the reproductive fitness of distantly-related nematodes.


title: Isolation and characterization of human monoclonal antibodies to pneumococcal capsular polysaccharide 3

abstract:
The current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective against Streptococcus pneumoniae serotype 3 (ST3), which remains a major cause of pneumococcal disease and mortality. Therefore, dissecting structure-function relationships of human PPS3 antibodies may reveal characteristics of protective antibodies. Using flow cytometry, we isolated PPS3-binding memory B cells from pneumococcal vaccine recipients and generated seven human PPS3-specific monoclonal antibodies (humAbs). Five humAbs displayed ST3 opsonophagocytic activity, four induced ST3 agglutination in vitro, and four mediated both activities. For two humAbs, C10 and C27, that used the same variable heavy (VH) and light (VL) chain domains (VH3-9*01/VL2-14*03), C10 had fewer VL somatic mutations, higher PPS3 affinity, more ST3 opsonophagocytic and agglutinating activity, whilst both humAbs altered ST3 gene expression in vitro. After VL swaps, C10VH/C27VL exhibited reduced ST3 binding and agglutination, but C27VH/C10VL binding was unchanged. In C57Bl/6 mice, C10 and C27 reduced nasopharyngeal colonization with ST3 A66 and a clinical strain, B2, and prolonged survival following lethal A66 intraperitoneal infection, but only C10 protected against lethal intranasal infection with the clinical strain. Our findings, associate efficacy of PPS3-specific humAbs with ST3 agglutination and opsonophagocytic activity and reveal an unexpected role for the VL in functional activity in vitro and in vivo. These findings also provide insights that may inform antibody-based therapy and identification of surrogates of vaccine efficacy against ST3.

IMPORTANCEDespite the global success of pneumococcal conjugate vaccination, serotype 3 (ST3) pneumococcus remains a leading cause of morbidity and mortality. In comparison to other vaccine-included serotypes, the ST3 pneumococcal capsular polysaccharide (PPS3) induces a weaker opsonophagocytic response, which is considered a correlate of vaccine efficacy. Previous studies of mouse PPS3 monoclonal antibodies identified ST3 agglutination as a correlate of reduced ST3 nasopharyngeal colonization in mice, however neither the agglutinating ability of human vaccine-elicited PPS3 antibodies nor their ability to prevent experimental murine nasopharyngeal colonization has been studied. We generated and analysed the functional and in vivo efficacy of human vaccine-elicited PPS3 monoclonal antibodies and found that ST3 agglutination associated with antibody affinity, protection in vivo, and limited somatic mutations in the light chain variable region. These findings provide new insights that may inform the development of antibody-based therapies and next generation vaccines for ST3.


title: A survey of Chinese pig farms and human healthcare isolates reveals separate human and animal MRSA populations

abstract:
There has been increasing concern that the overuse of antibiotics in livestock farming is contributing to the burden of antimicrobial resistance in people. Farmed animals in Europe and North America, particularly pigs, provide a reservoir for livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA, ST398) found in people. This study was designed to investigate the contribution of MRSA from Chinese pig farms to human infection and carriage.A collection of 603 S. aureus were isolated from 55 pig farms and 4 hospitals (MRSA= 285, 198; MSSA= 50, 70) in central China, a high pig farming density area, during 2017-2018. CC9 MRSA accounting for 93% of all farm MRSA isolates, while no was found in hospitals. ST398 isolates were found on three farms (n = 23) and three hospitals (n = 12). None of the ST398 from this study belong to the livestock clade of the LA-MRSA commonly found in Europe and North America. The hospital ST398 MRSA isolates formed a clade that was clearly separate from the farm ST398 MRSA and MSSA isolates, and all possessed human immune evasion cluster genes which were absent from all the pig farm ST398 isolates. Despite the presence of high levels of MRSA found on Chinese pig farms we found no evidence of them spilling over to the human population. Nevertheless, the ST398 MRSA obtained from human samples appear to be part of a widely distributed lineage in China. And the new animal adapted ST398 lineage that emerged in China should also be alarmed.

ImportanceWe disclosed the fact that although the high MRSA positive rate in Chinese hospitals and pig farms should be alarmed, they might be two separate issues. The new CC398 clades we identified highlight that the host adaption of the MRSA lineage is kept changing. These results suggest that continued surveillance of MRSA in livestock is necessary. We found that the pig farm MRSA isolates had unique antimicrobial resistance genes while most of the hospital MRSA isolates had human immune evasion cluster genes. These features could be used to distinguish the pig farm associated S. aureus in clinical laboratories. The policies of reducing antimicrobials use in livestock were implemented in China since 2020. Our study described the situation of MRSA populations in pig farms and hospitals in Central China before 2020, which provides a potential opportunity for future studies to evaluate the effects of the policies.


title: Dynamic evolution of bacterial ligand recognition by formyl peptide receptors

abstract: The detection of invasive pathogens is critical for host immune defense. Cell surface receptors play a key role in the recognition of diverse microbe-associated molecules, triggering leukocyte recruitment, phagocytosis, release of antimicrobial factors, and cytokine production. The intense selective forces acting on innate immune receptor genes has led to their rapid diversification across plant and animal species. However, the impacts of this genetic variation on immune functions are often unclear. Formyl peptide receptors (FPRs) are a family of animal G-protein coupled receptors which are activated in response to a variety of ligands including formylated bacterial peptides, microbial virulence factors, and host-derived peptides. Here we investigate patterns of gene loss, sequence diversity, and ligand recognition among primate and carnivore FPRs. We observe that FPR1, which plays a critical role in innate immune defense in humans, has been lost in New World primates. Patterns of amino acid variation in FPR1 and FPR2 suggest a history of repeated positive selection acting on extracellular domains involved in ligand binding. To assess the consequences of FPR variation on bacterial ligand recognition, we measured interactions between primate FPRs and the FPR agonist Staphylococcus aureus enterotoxin B, as well as S. aureus FLIPr-like which functions as an FPR inhibitor. We find that comparatively few sequence differences between great ape FPRs are sufficient to modulate recognition of S. aureus ligands, further demonstrating how genetic variation can act to tune FPR activation in response to diverse microbial binding partners. Together this study reveals how rapid evolution of host immune receptors shapes the detection of diverse microbial molecules.

category: evolutionary biology


title: Crystal structure of the potato leafroll virus coat protein and implications for viral assembly

abstract: Luteoviruses, poleroviruses, and enamoviruses are insect-transmitted, agricultural pathogens that infect a wide array of staple food crops. Previous cryo-electron microscopy studies of virus-like particles indicate that luteovirid viral capsids are built from a structural coat protein that organizes with T=3 icosahedral symmetry. Here we present the crystal structure of a truncated version of the coat protein monomer from potato leafroll virus at 1.57-[A] resolution. In the crystal lattice, monomers pack into flat sheets that preserve the two-fold and three-fold axes of icosahedral symmetry and show minimal structural deviations when compared to the full-length subunits of the assembled virus-like particle. These observations have important implications in viral assembly and maturation, suggesting that the CP N-terminus and its interactions with RNA serve as a key driver for generating capsid curvature.

category: biochemistry


title: The structural coverage of the human proteome before and after AlphaFold

abstract:
The protein structure field is experiencing a revolution. From the increased throughput of techniques to determine experimental structures, to developments such as cryo-EM that allow us to find the structures of large protein complexes or, more recently, the development of artificial intelligence tools, such as AlphaFold, that can predict with high accuracy the folding of proteins for which the availability of homology templates is limited.

Here we quantify the effect of the recently released AlphaFold database of protein structural models in our knowledge on human proteins. Our results indicate that our current baseline for structural coverage of 48%, considering experimentally-derived or template-based homology models, elevates up to 76% when including AlphaFold predictions. At the same time the fraction of dark proteome is reduced from 26% to just 10% when AlphaFold models are considered. Furthermore, although the coverage of disease-associated genes and mutations was near complete before AlphaFold release (69% of Clinvar pathogenic mutations and 88% of oncogenic mutations), AlphaFold models still provide an additional coverage of 3% to 13% of these critically important sets of biomedical genes and mutations. Finally, we show how the contribution of AlphaFold models to the structural coverage of non-human organisms, including important pathogenic bacteria, is significantly larger than that of the human proteome.

Overall, our results show that the sequence-structure gap of human proteins has almost disappeared, an outstanding success of direct consequences for the knowledge on the human genome and the derived medical applications.


title: Controlling reversible cell differentiation for labor division in microbial consortia

abstract: We address the problem of regulating and keeping at a desired balance the relative numbers between cells exhibiting a different phenotype within a monostrain microbial consortium. We propose a strategy based on the use of external control inputs, assuming each cell in the community is endowed with a reversible, bistable memory mechanism. Specifically, we provide a general analytical framework to guide the design of external feedback control strategies aimed at balancing the ratio between cells whose memory is stabilized at either one of two equilibria associated to different cell phenotypes. We demonstrate the stability and robustness properties of the control laws proposed and validate them in silico by means of a realistic agent-based implementation enabling cooperative bioproduction in microbial consortia. The proposed control framework may be used to allow long term coexistence of different populations, with both industrial and biotechnological applications.


title: Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

abstract: Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multipleturnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been characterized - but only from archaeal methanogens that are anaerobic, O2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli {Delta}thiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, the use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.


title: Reconstruction of phospholipid synthesis by combing in vitro fatty acid synthesis and cell-free gene expression

abstract: Phospholipid synthesis is a fundamental process that promotes cell propagation and, presently, is the most challenging issue in artificial cell research aimed at reconstituting living cells from biomolecules. Here, we constructed a cell-free phospholipid synthesis system that combines in vitro fatty acid synthesis and a cell-free gene expression system that synthesizes acyltransferases for phospholipid synthesis. Fatty acids were synthesized from acetyl-CoA and malonyl-CoA, then continuously converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of synthetic intermediates that suppress the reaction, the yield of phospholipid has significantly improved from previous schemes (up to 400 {micro}M). Additionally, by adding enzymes for recycling CoA, we synthesized phosphatidic acids from acetic acid and bicarbonate as carbon sources. The constructed system is available to express the genes from pathogenic bacteria and to analyze the synthesized phospholipids. By encapsulating our system inside giant vesicles, it would be possible to construct the artificial cells in which the membrane grows and divides sustainably.


title: Broadening the toolkit for quantitatively evaluating noncanonical amino acid incorporation in yeast

abstract:
Genetic code expansion is a powerful approach for advancing critical fields such as biological therapeutic discovery. However, the machinery for genetically encoding noncanonical amino acids (ncAAs) is only available in limited plasmid formats, constraining potential applications. In extreme cases, the introduction of two separate plasmids, one containing an orthogonal translation system (OTS) to facilitate ncAA incorporation and a second for expressing a ncAA-containing protein of interest, is not possible due to lack of convenient selection markers. One strategy to circumvent this challenge is to express the OTS and protein of interest from a single vector. For what we believe is the first time in yeast, we describe here several sets of single plasmid systems (SPSs) for performing genetic code manipulation and compare the ncAA incorporation capabilities of these plasmids against the capabilities of previously described dual plasmid systems (DPSs). For both dual fluorescent protein reporters and yeast display reporters tested with multiple OTSs and ncAAs, measured ncAA incorporation efficiencies with SPSs were determined to be equal to or improved relative to efficiencies determined with DPSs. Click chemistry on yeast cells displaying ncAA-containing proteins was also shown to be feasible in both formats, although differences in reactivity between formats suggest the need for caution when using such approaches. Additionally, we investigated whether these reporters would support separation of yeast strains known to exhibit distinct ncAA incorporation efficiencies. Model sorts conducted with mixtures of two strains transformed with the same SPS or DPS led to enrichment of a strain known to support higher efficiency ncAA incorporation, suggesting that these reporters will be suitable for conducting screens for strains exhibiting enhanced ncAA incorporation efficiencies. Overall, our results confirm that SPSs are well-behaved in yeast and provide a convenient alternative to DPSs. In particular, SPSs are expected to be invaluable for conducting high-throughput investigations of the effects of genetic or genomic changes on ncAA incorporation efficiency and, more fundamentally, the eukaryotic translation apparatus.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/454837v1_ufig1.gif" ALT="Figure 1">
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title: Microbiota-dependent and independent production of L-Dopa in the gut of Daphnia magna

abstract: The host-microbiome interactions are essential for the physiological and ecological performance of the host, yet these interactions are challenging to identify. Neurotransmitters are commonly implicated in these interactions, but we know very little about the mechanisms of their involvement, especially in invertebrates. Here, we report a peripheral Catecholamine (CA) pathway involving the gut microbiome of the model species Daphnia magna. We demonstrate that: (1) tyrosine hydroxylase and dopa decarboxylase enzymes are present in the gut wall; (2) DOPA decarboxylase gene is expressed in the gut by the host, and its expression follows the molt cycle peaking after ecdysis; (3) biologically active L-Dopa, but not Dopamine, is present in the gut lumen; and (4) gut bacteria produce L-Dopa in a concentration-dependent manner when provided L-Tyrosine as a substrate. Impinging on gut bacteria involvement in host physiology and ecologically relevant traits, we suggest L-Dopa as a communication agent in the host-microbiome interactions in daphnids and, possibly, other crustaceans.


title: The sRNA MicC downregulates hilD translation to control the SPI1 T3SS in Salmonella enterica serovar Typhimurium

abstract:
Salmonella enterica serovar Typhimurium invades the intestinal epithelium and induces inflammatory diarrhea using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). Expression of the SPI1 T3SS is controlled by three AraC-like regulators, HilD, HilC and RtsA, which form a feed-forward regulatory loop that leads to activation of hilA, encoding the main transcriptional regulator of the T3SS structural genes. This complex system is affected by numerous regulatory proteins and environmental signals, many of which act at the level of hilD mRNA translation or HilD protein function. Here, we show that the sRNA MicC blocks translation of the hilD mRNA by base pairing near the ribosome binding site. This binding blocks translation but does not induce degradation of the hilD message. Our data indicate that micC is transcriptionally activated by SlyA, and SlyA feeds into the SPI1 regulatory network solely through MicC. Transcription of micC is negatively regulated by the OmpR/EnvZ two-component system, but this regulation is dependent on SlyA. OmpR/EnvZ control SPI1 expression partially through MicC, but also affect expression through other mechanisms. MicC-mediated regulation plays a role during infection, as evidenced by an increase in Salmonella fitness in the intestine in the micC deletion mutant that is dependent on the SPI1 T3SS. These results further elucidate the complex regulatory network controlling SPI1 expression and add to the list of sRNAs that control this primary virulence factor.

IMPORTANCEThe Salmonella SPI1 T3SS is the primary virulence factor required for causing intestinal disease and initiating systemic infection. The system is regulated in response to a large variety of environmental and physiological factors such that the T3SS is expressed at only the appropriate time and place in the host during infection. Here we show how the sRNA MicC affects expression of the system. This work adds to our detailed mechanistic studies aimed at a complete understanding of the regulatory circuit.


title: Taxonomy of Rhizobiaceae revisited: proposal of a new framework for genus delimitation

abstract: The alphaproteobacterial family Rhizobiaceae is highly diverse, with 168 species with validly published names classified into 17 genera with validly published names. Most named genera in this family are delineated based on genomic relatedness and phylogenetic relationships, but some historically named genera show inconsistent distribution and phylogenetic breadth. Most problematic is Rhizobium, which is notorious for being highly paraphyletic, as most newly described species in the family being assigned to this genus without consideration for their proximity to existing genera, or the need to create novel genera. In addition, many Rhizobiaceae genera lack synapomorphic traits that would give them biological and ecological significance. We propose a common framework for genus delimitation within the family Rhizobiaceae. We propose that genera in this family should be defined as monophyletic groups in a core-genome gene phylogeny, that are separated from related species using a pairwise core-proteome average amino acid identity (cpAAI) threshold of approximately 86%. We further propose that the presence of additional genomic or phenotypic evidence can justify the division of species into separate genera even if they all share greater than 86% cpAAI. Applying this framework, we propose to reclassify Rhizobium rhizosphaerae and Rhizobium oryzae into the new genus Xaviernesmea gen. nov. Data is also provided to support the recently proposed genus "Peteryoungia", and the reclassifications of Rhizobium yantingense as Endobacterium yantingense comb. nov., Rhizobium petrolearium as Neorhizobium petrolearium comb. nov., Rhizobium arenae as Pararhizobium arenae comb. nov., Rhizobium tarimense as Pseudorhizobium tarimense comb. nov., and Rhizobium azooxidefex as Mycoplana azooxidifex comb. nov. Lastly, we present arguments that the unification of the genera Ensifer and Sinorhizobium in Opinion 84 of the Judicial Commission is no longer justified by current genomic and phenotypic data. We thus argue that the genus Sinorhizobium is not illegitimate and now encompasses 17 species.


title: QT-AMP: Quanti-Tray-based amplicon sequencing for simultaneous quantification and identification of enterococci for microbial source tracking

abstract:
Enterococcus is ubiquitous in human feces and has been adopted as a useful indicator of human fecal pollution in water. Although regular enterococci monitoring only examines their numbers, identification of human-specific Enterococcus species or genotypes could help in the discrimination of human fecal contamination from other environmental sources. We documented a new approach to characterize enterococci using an amplicon sequencing platform from Quanti Trays after following the counting of most probable numbers (MPN) of enterococci. We named this method as QT-AMP (Quanti-Tray-based amplicon sequencing). We tested surface water samples collected from three rivers in southwest Florida. We detected 11 Enterococcus species from 45 samples in 1.1 million sequence reads. The method detected three rare species and eight cosmopolitan species (Enterococcus faecalis, E.faecium, E. casseliflavus, E. hirae, E. mundtii, E. gallinarum, E. avium, and E. durans) which have been commonly documented in various enterococci isolation studies. It is likely that the approximate detection level of QT-AMP is four orders of magnitude higher than regular 16S rRNA gene amplicon sequencing. QT-AMP revealed that a majority of Enterolert positive signals are actually the mixture of both enterococci and other facultative aerobes and anaerobes. QT-AMP may have the potential to monitor not only enterococci but also other pathogenic bacteria commonly found in natural environments. This QT-AMP could be a powerful tool to streamline the quantification and identification of enterococci and allows us to do more accurate and efficient microbial source tracking in various water management projects and human health risk assessment.

HighlightsO_LIA selected primer set (27f-519r) can differentiate over 50 Enterococcus species and is suitable for Illumina amplicon sequencing.
C_LIO_LIThe median of the relative contribution of enterococci reads among total sequencing reads was 82.7% and ranged between 0% and 100%.
C_LIO_LIEnterolert signals are most likely the mixture of enterococci and other facultative aerobes and anaerobes.
C_LIO_LIWe identified eight cosmopolitan enterococci species and three rare species.
C_LIO_LIThe median of the relative contribution of non-enterococci reads among total sequencing reads was 17.3%, respectively, and ranged between 0% and 100%.
C_LI


title: A clinically validated human saliva metatranscriptomic test for global systems biology studies

abstract:
We report here the development of a high throughput, automated, inexpensive, and clinically validated saliva metatranscriptome test that requires less than 100 microliters of saliva. The RNA in the samples is preserved at the time of collection. Samples can then be stored and transported at ambient temperatures for up to 28 days without significantly affecting the test results. Another important feature of the sample preservative is that it inactivates all classes of pathogens, enabling safe transportation globally. Given the unique set of convenience, low cost, safety, and technical performance, this saliva metatranscriptomic test can be integrated into longitudinal, global scale, systems biology studies that will lead to an accelerated development of precision medicine diagnostic and therapeutic tools.

Method SummaryThis report describes an important improvement to saliva transcriptome analysis. While current methods are complicated and expensive, the method reported here includes at-home sample collection, global shipping at ambient temperatures and pathogen inactivation at the point of collection; it uses fully automated, clinically validated and licensed laboratory and bioinformatic analyses.


title: Personalized mathematical oncology: Challenges and opportunities

abstract:
An outstanding challenge in the clinical care of cancer is moving from a one-size-fits-all approach that relies on population-level statistics towards personalized therapeutic design. Mathematical modeling is a powerful tool in treatment personalization, as it allows for the incorporation of patient-specific data so that treatment can be tailor-designed to the individual. Herein, we work with a mathematical model of murine cancer immunotherapy that has been previously-validated against the average of an experimental dataset. We ask the question: what happens if we try to use this same model to perform personalized fits, and therefore make individualized treatment recommendations? Typically, this would be done by choosing a single fitting methodology, and a single cost function, identifying the individualized best-fit parameters, and extrapolating from there to make personalized treatment recommendations. Our analyses show the potentially problematic nature of this approach, as predicted personalized treatment response proved to be sensitive to the fitting methodology utilized. We also demonstrate how a small amount of the right additional experimental measurements could go a long way to improve consistency in personalized fits. Finally, we show how quantifying the robustness of the average response could also help improve confidence in personalized treatment recommendations.

Author summaryAs we enter the era of healthcare where personalized medicine becomes a more common approach to treating cancer patients, harnessing the power of mathematical models will only become more essential. Using a preclinical dataset on cancer immunotherapy, we explore the challenges and limitations that arise when trying to move from fitting and making predictions for the population-level average, to fitting and making predictions for an individual. We find that the standard of approach of picking a single fitting methodology and a single cost function may end up having limited predictive value when applied to individual data. We also show how having a small amount of the right additional experimental data, and establishing the robustness of average treatment response, can help improve confidence in personalized model predictions.

category: cancer biology


title: From fitting the average to fitting the individual: A cautionary tale for mathematicalmodelers