title: Hairy root transformation system as a tool for CRISPR/Cas9-directed genome editing in oilseed rape (Brassica napus)

abstract:
Our study examined the mutation efficiency of the CRISPR/Cas9 method for tryptophan aminotransferase BnaTAA1 genes involved in the auxin biosynthesis pathway. We made nine CRISPR/Cas9 constructs with various promoters driving the expression of a Cas9 from Staphylococcus aureus (SaCas9) or a plant codon-optimized Streptococcus pyogenes Cas9 (pcoCas9). We developed a fast and efficient system for evaluating the variety and frequency of mutations caused by each construct using Brassica napus hairy roots. We showed that pcoCas9 is more efficient in mutating the targeted loci than SaCas9 and the presence of the NLS signal enhanced the chance of mutagenesis by 25%. The mutations were studied further in regenerated lines, and we determined the BnaTAA1 gene expression and heritability of the gene modifications in transgenic plants. Hairy root transformation combined with CRISPR/Cas9-mediated gene editing represents a fast and straightforward system for studying target gene function in the important oilseed crop Brassica napus.

One-sentence summaryThe hairy root transformation system of Brassica napus generates stable transformants and is a tool for efficiently identifying CRISPR/Cas9-induced genome editing.


title: Bacterial produce membrane-binding small molecules to regulate horizontal gene transfer in vesicles

abstract:
The exchange of bacterial extracellular vesicles facilitates molecular exchange between cells, including the horizontal transfer of genetic material. Given the implications of such transfer events on cell physiology and adaptation, some bacterial cells have likely evolved mechanisms to regulate vesicle exchange. Past work has identified mechanisms that regulate the formation of extracellular vesicles, including the production of small molecules that modulate membrane structure, however whether these mechanisms also regulate vesicle uptake and have an overall impact on the rate of vesicle exchange is unknown. Here we show that membrane-binding molecules produced by microbes regulate both the formation and uptake of extracellular vesicles and have the overall impact of increasing the vesicle exchange rate within a bacterial coculture. In effect, production of compounds that influence vesicle exchange rates enable cells to steal genes from neighboring cells. The ability of several membrane-binding compounds to regulate vesicle exchange was demonstrated. Three of these compounds, nisin, colistin, and polymyxin B, are antimicrobial peptides added at sub-inhibitory concentrations. These results suggest that a key function of exogenous compounds that bind to membranes may be the regulation of vesicle exchange between cells.

ImportanceThe exchange of bacterial extracellular vesicles is one route of gene transfer between bacteria, although it was unclear if bacteria developed strategies to regulate the rate of gene transfer within vesicles. In eukaryotes, there are many examples of specialized molecules that have evolved to facilitate the production, loading, and uptake of vesicles. Recent work with bacteria has shown that some small molecules influence membrane curvature and induce vesicle formation. Here we show that similar compounds facilitate vesicle uptake, thereby regulating the overall rate of vesicle exchange within bacterial populations. The addition of membrane-binding compounds, several of them antibiotics at sub-inhibitory concentrations, to a bacterial co-culture increased the rate of horizontal gene transfer via vesicle exchange.

category: microbiology


title: Resetting H3K4me3, H3K27ac, H3K9me3 and H3K27me3 during the maternal-to-zygotic transition and blastocyst lineage specification in bovine embryos

abstract: It remains poorly understood how histone modifications regulate changes in gene expression during preimplantation development. Using a bovine model, we profiled changes in two activating (H3K4me3 and H3K27ac) and two repressive (H3K9me3 and H3K27me3) marks in oocytes, 2-, 4- and 8-cell embryos (that developed in the presence or absence of the transcription inhibitor a-amanitin), morula, blastocysts, inner cell mass cells and trophectoderm. In oocytes, we find that broad bivalent domains of H3K4me3 and H3K27me3 mark developmental genes, and that prior to genome activation, H3K9me3 and H3K27me3 co-occupy gene bodies. During genome activation, chromatin accessibility is established before canonical H3K4me3 and H3K27ac, and although embryonic transcription is required for this active remodeling, it is dispensable for maintenance of pre-established histone marks. Finally, blastocyst lineages are defined by differential Polycomb repression and transcription factor activity. Overall, these results further support the use of bovine as a more appropriate model system than the mouse to study genome activation and cell lineage specification during human preimplantation development.

category: developmental biology


title: The intra-mitochondrial contact site formed by Cqd1 and the Por1-Om14 complex modulates architecture and morphology of mitochondria

abstract: Mitochondria are essential organelles of eukaryotic cells that are characterized by their unique and complex membrane system. They are confined from the cytosol by an envelope consisting of two membranes. Signals, metabolites, proteins and lipids have to be transferred across these membranes via proteinaceous contact sites to keep mitochondria functional. In the present study we identified a novel mitochondrial contact site that is formed by Cqd1 in the inner membrane and the outer membrane complex Por1-Om14. Cqd1 is a member of the highly conserved UbiB protein kinase-like family (also called aarF domain containing kinases). It was recently shown that Cqd1 in cooperation with Cqd2 controls the cellular distribution of coenzyme Q by a yet unknown mechanism. Our data suggest that Cqd1 besides its known function in coenzyme Q homeostasis is involved in the homeostasis of phospholipids, the organization of the mitochondrial inner membrane and the interaction of mitochondria with the endoplasmic reticulum.

category: biochemistry


title: Probing Notch1-Dll4 Signaling in Regulating Osteogenic Differentiation of Human Mesenchymal Stem Cells using Single Cell Nanobiosensor

abstract: Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroids differentiation were also investigated. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Specifically, Notch1-Dll4 signaling is active during osteogenic differentiation. Our results also showed that Dll4 is a molecular signature of differentiated hMSCs during osteogenic induction. Notch inhibition mediated osteogenic differentiation with reduced Alkaline Phosphatase (ALP) activity. Lastly, we elucidated the role of Notch1-Dll4 signaling during osteogenic differentiation in a 3D spheroid model. Our results showed that Notch1-Dll4 signaling is required and activated during osteogenic differentiation in hMSCs spheroids. Inhibition of Notch1-Dll4 signaling mediated osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid sizes. Taken together, the capability of LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis in 2D and 3D microenvironment. These findings will provide new insights to improve cell-based therapies and organ repair techniques.

category: bioengineering


title: Lipidoid nanoparticles increase ATP uptake into hypoxic brain endothelial cells

abstract:
Lipidoid nanoparticles (LNPs) are clinically successful carriers for nucleic acid delivery to liver and muscle targets. Their ability to load and deliver small molecule drugs has not been reported yet. We propose that the delivery of adenosine triphosphate (ATP) to brain endothelial cells (BECs) lining the blood-brain barrier may increase cellular energetics of the injured BECs. We formulated and studied the physicochemical characteristics of ATP-loaded LNPs using the C12-200 ionizable cationic lipid and other helper lipids. Polyethylene glycol-dimyristoyl glycerol (PEG-DMG), one of the helper lipids, played a crucial role in maintaining colloidal stability of LNPs over time whereas the inclusion of both ATP and PEG-DMG maintained the colloidal stability of LNPs in the presence of serum proteins. ATP-LNPs formulated with PEG-DMG resulted in a 7.7- and 6.6-fold increased uptake of ATP into normoxic and hypoxic BECs, respectively. Altogether, our results demonstrate the potential of LNPs as a novel carrier for the delivery of small molecular mass actives to BECs--a CNS target.

HighlightsO_LILNPs were formulated with ATP, a small molecule drug
C_LIO_LIPEG-DMG plays a critical role in maintaining particle stability over tim
C_LIO_LIATP and PEG-DMG play a critical role in maintaining particle stability in 10% serum
C_LIO_LIATP-LNPs were internalized by normoxic and hypoxic brain endothelial cells (BECs)
C_LIO_LILNP delivery to BECs broadens its applicability to CNS targets
C_LI

Graphical Abstract

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title: Mapping CAR T-cell design space using agent-based models

abstract: Chimeric antigen receptor (CAR) T-cell therapy shows promise for treating liquid cancers and increasingly for solid tumors as well. While potential design strategies exist to address translational challenges, including the lack of unique tumor antigens and the presence of an immunosuppressive tumor microenvironment, testing all possible design choices in vitro and in vivo is prohibitively expensive, time consuming, and laborious. To address this gap, we extended the modeling framework ARCADE (Agent-based Representation of Cells And Dynamic Environments) to include CAR T-cell agents (CAR T-cell ARCADE, or CARCADE). We conducted in silico experiments to investigate how clinically relevant design choices and inherent tumor features--CAR T-cell dose, CD4+:CD8+ CAR T-cell ratio, CAR-antigen affinity, cancer and healthy cell antigen expression--individually and collectively impact treatment outcomes. Our analysis revealed that tuning CAR affinity modulates IL-2 production by balancing CAR T-cell proliferation and effector function. It also identified a novel multi-feature tuned treatment strategy for balancing selectivity and efficacy and provided insights into how spatial effects can impact relative treatment performance in different contexts. CARCADE facilitates deeper biological understanding of treatment design and could ultimately enable identification of promising treatment strategies to accelerate solid tumor CAR T-cell design-build-test cycles.


title: Threshold Values for the Gini Variable Importance: A Empirical Bayes Approach

abstract:
Random Forests (RF) are a very widely used modelling tool. Lundberg et al. (2019) concludes that no nonlinear model had a more widespread popularity, from health care to academia to industry, than random forests and decision trees. The bounds of the methodology are still being extended. Bayat et al. (2020) give an example with 80 million variables.

It is highly desirable that RF models be made more interpretable and a large part of that is a better understanding of the characteristics of the variable importance measures generated by the RF. Due to its speed and ease of calculation, we consider the mean decrease in node "impurity" (MDI) variable importance (VI) and address the question of setting a significance level.

The report is organized as follows:

O_LIwe first consider the question of multiple testing in the case of multiple measurements made on two groups (the standard microarray set up Efron (2008)). We show that some standard approaches for multiple testing can fail severely due to the correlation structure of the measurement and other modelling failures. We show that deriving the null distribution by permutation does not fix the problem. This point applies to determining the null distribution of variable importances as well as many other statistical tests;
C_LIO_LIwe show that variable correlation can either increase or decrease the (MDI) of variables in different settings. We also show that there is an additional problem with the permutation null due to the functional relationships between the statistics;
C_LIO_LIWe consider the empirical Bayes argument of Efron (2005) and model the VI as a mixture of two distribution, a null and a non-null distribution. We find that unlike the relatively well behaved case considered in Efrons papers, there are a number of issues here:
C_LI

- the distribution may be multi-modal, which creates modelling difficulties;
- the null distribution is not of an obvious form, as it is not symmetric.

O_LIwe resolve these issues to derive a fast, plausible, empirical Bayes method for selecting significant variables while controlling the false discovery rate.
C_LI

category: bioinformatics


title: Genotype error biases trio-based estimates of haplotype phase accuracy

abstract: Haplotypes can be estimated from unphased genotype data using statistical methods. When parent-offspring data are available for inferring true phase from Mendelian inheritance rules, the accuracy of statistical phasing is usually measured by the switch error rate, which is the proportion of pairs of consecutive heterozygotes that are incorrectly phased. We present a method for estimating the genotype error rate from parent-offspring trios and a method for estimating the bias in the observed switch error rate that is caused by genotype error. We apply these methods to 485,301 genotyped UK Biobank samples that include 899 White British trios and to 38,387 sequenced TOPMed samples that include 217 African Caribbean trios and 669 European American trios. We show that genotype error inflates the observed switch error rate and that the relative bias increases with sample size. For the UK Biobank White British trios, we estimate that the observed switch error rate in the trio offspring is 2.4 times larger than the true switch error rate (1.41 x 10-3 vs 5.79 x 10-4) and that the average distance between phase errors is 64 megabases.


title: Drug combination prioritization for cancer treatment using single-cell RNA-seq based transfer learning

abstract: Precision oncology seeks to match patients to the optimal pharmacological regimen; yet, due to tumor heterogeneity, this is challenging. Numerous studies have been conducted to produce clinically relevant pharmacological response forecasts by integrating modern machine learning algorithms and several data types. Insufficient patient numbers and lack of knowledge of the molecular targets for each drug under study limit their use. As a proof of concept, we use single-cell RNA-seq based transfer learning to contextualize patients tumor cells in terms of their more similar cell lines with known susceptibility to drug combinations. Our objective is to maximize the translational potential of in-vitro assays for identifying synergistic drug combinations and prioritizing them for clinical use. Consistent findings in a cohort of breast cancer patients corroborated our understanding of the diseases molecular subtypes. To aid in creating personalized treatments and data-driven clinical trials, we identified the most prevalent cell lines and prioritized synergistic combinations based on tumor compositions at various resolution levels.


title: Functional recovery from tactile stimulation after perinatal cortical injury is mediated by FGF-2

abstract: The consequences of perinatal brain injury can be devastating but currently there are few effective treatments. We sought to determine if tactile stimulation (TS) or exogenous application of fibroblast growth factor-2 (FGF-2) following injury could reverse the behavioral loss. Infant rats received frontal cortex removals on postnatal day 4 (P4) or a sham surgery. The TS animals received thrice-daily 15-minute bouts of stimulation (Experiment 1) on the day following surgery until weaning. In Experiment 2, treated animals received subcutaneous injections of FGF-2 once daily for one week, postsurgery. Behavioral testing began on postnatal day 60. Brains were later processed for Golgi analysis. We show in Experiment 1, that tactilely stimulating infant rats with perinatal cortical injury stimulates functional recovery and reverses injury-related changes in neuronal morphology in the cerebral cortex. The TS induction of recovery is associated with changes in expression of FGF-2 in both the skin and brain. Direct administration of FGF-2 (Experiment 2) is also effective in facilitating recovery, although not as completely as TS. These results suggest that early behavioral intervention after perinatal cortical injury can stimulate plastic neuronal changes that can underlie functional recovery and that these changes are mediated, in part, through an upregulation of FGF-2.

category: neuroscience


title: A descending interneuron with depolarized resting membrane potential controls C. elegans motor states

abstract: Mutually exclusive behaviors in animals are often driven by independent motor subcircuits that directly or indirectly inhibit each other. For example, in the nematode C. elegans, motor circuits for forward and backward locomotion are gated by premotor interneurons AVB and AVA respectively, which are thought to be connected via reciprocal inhibition. In this study, we dissect the interactions between forward and motor subcircuits in C. elegans by genetic manipulation of AVAs activity via cell-specific characterization of a two-pore potassium channel (TWK-40). AVA has an unusually depolarized resting membrane potential (RMP). Perturbations to its RMP through AVA-specific TWK-40 loss-of-function and AVA-specific TWK-40 gain-of-function, led to dramatic changes in both forward and back-ward motor activity. AVA, thus regulated both backward and forward locomotion in C. elegans, functioning as a master neuron for overall locomotion. These effects required chemical transmission from AVA and were dependent on AVB neurons. We were able to reconcile results of genetic perturbation with optogenetic manipulation by carefully designed stimulation protocols. Finding that phasic optogenetic activation of AVA leads to backward locomotion, while tonic optogenetic activation potentiates forward locomotion. These results propose that a single neuron can regulate the activity of motor circuits generating two mutually exclusive behaviors.


title: The presynaptic active zone protein Bassoon as a marker for synapses between Type lll cells and afferent nerve fibers in taste buds

abstract: Taste buds are receptor organs for gustation. Two types of taste receptor cells have been identified in taste buds: Type II and Type III cells. Type III cells connect with afferent fibers through conventional chemical synapses. In the present study on taste buds, we used immunocytochemistry to examine the distribution pattern of Bassoon, a scaffolding protein of the cytomatrix at the active zones of conventional synapses. Bassoon was predominantly detected as small puncta in Type III cells. Bassoon-immunoreactive puncta were observed in proximity to or partially overlapping with intragemmal nerve fibers. Immunoelectron microscopy showed Bassoon at the active zones of the conventional synapses of Type III cells. The present results demonstrated that Bassoon is a marker for synapses between Type III cells and afferent fibers in taste buds.


title: Neural representation of intraoral olfactory and gustatory signals by the mediodorsal thalamus in alert rats

abstract:
The mediodorsal thalamus is a higher-order thalamic nucleus involved in a variety of cognitive behaviors, including olfactory attention, odor discrimination, and the hedonic perception of flavors. Although it forms connections with principal regions of the olfactory and gustatory networks, its role in processing olfactory and gustatory signals originating from the mouth remains unclear. Here, we recorded single-unit activity in the mediodorsal thalamus of behaving rats during the intraoral delivery of individual odors, individual tastes, and odor-taste mixtures. Our results are the first to demonstrate that neurons in the mediodorsal thalamus dynamically encode chemosensory signals originating from the mouth. This chemoselective population is broadly tuned, responds with excitation and suppression, and represents odor-taste mixtures differently than their odor and taste components. Furthermore, a subset of chemoselective neurons encoded taste palatability. Our results further demonstrate the multidimensionality of the mediodorsal thalamus and provides additional evidence of its involvement in processing chemosensory information important for consummatory behaviors.

SIGNIFICANCE STATEMENTThe perception of food relies upon the concurrent processing of olfactory and gustatory signals originating from the mouth. The mediodorsal thalamus is a higher-order thalamic nucleus involved in a variety of chemosensory-dependent behaviors and connects the olfactory and gustatory cortices with prefrontal cortex. However, it is unknown how neurons in the mediodorsal thalamus process intraoral chemosensory signals. Using tetrode recordings in alert rats, our results are the first to show that neurons in the mediodorsal thalamus dynamically represent olfactory and gustatory signals from the mouth. Our findings suggest that the mediodorsal thalamus is a key node between sensory and higher-order cortical areas for processing chemosensory information underlying consummatory behavior.


title: Single nuclei RNAseq stratifies multiple sclerosis patients into three distinct white matter glia responses

abstract: The lack of understanding as to the cellular and molecular basis of clinical and genetic heterogeneity in progressive multiple sclerosis (MS) has hindered the search for new effective therapies and biomarkers. Here, to address this gap, we analysed 740,000 single nuclei RNAseq profiles of 165 samples of white matter (WM) lesions, normal appearing WM, grey matter (GM) lesions and normal appearing GM from 55 MS patients and 28 controls. We find that gene expression changes in response to MS are highly cell-type specific in WM and GM lesions but are largely shared within an individual cell-type across lesions, following a continuum rather than discrete lesion-specific molecular programs. The major biological determinants of variability in gene expression in MS samples relate to individual patient effects, rather than to lesion types or other metadata. Using multi-omics factor analysis (MOFA+), we identify three subgroups of MS patients with distinct oligodendrocyte composition and WM glial gene expression signatures, suggestive of engagement of different pathological/regenerative processes. The discovery of these three patterns significantly advances our mechanistic understanding of progressive MS, provides a framework to use molecular biomarkers to stratify patients for best therapeutic approaches for progressive MS, and highlights the need for precision-medicine approaches to address heterogeneity among MS patients.


title: Cbln1 directs axon targeting by corticospinal neurons specifically toward thoraco-lumbar spinal cord.

abstract:
Corticospinal neurons (CSN) are centrally required for skilled voluntary movement, which necessitates that they establish precise subcerebral connectivity with the brainstem and spinal cord. However, molecular controls regulating specificity of this projection targeting remain largely unknown. We previously identified that developing CSN subpopulations exhibit striking axon targeting specificity in the spinal white matter. These CSN subpopulations with segmentally distinct spinal projections are also molecularly distinct; a subset of differentially expressed genes between these distinct CSN subpopulations function as molecular controls regulating differential axon projection targeting. Rostrolateral CSN extend axons exclusively to bulbar-cervical segments (CSNBC-lat), while caudomedial CSN (CSNmedial) are more heterogeneous, with distinct, intermingled subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. Here, we report that Cerebellin 1 (Cbln1) is expressed specifically by CSN in medial, but not lateral, sensorimotor cortex. Cbln1 shows highly dynamic temporal expression, with Cbln1 levels in CSN highest during the period of peak axon extension toward thoraco-lumbar segments. Using gain-of-function experiments, we identify that Cbln1 is sufficient to direct thoraco-lumbar axon extension by CSN. Mis-expression of Cbln1 in CSNBC-lat either by in utero electroporation, or in postmitotic CSNBC-lat by AAV-mediated gene delivery, re-directs these axons past their normal bulbar-cervical targets toward thoracic segments. Further, Cbln1 overexpression in postmitotic CSNmedial increases the number of CSNmedial axons that extend past cervical segments into the thoracic cord. Collectively, these results identify that Cbln1 functions as a potent molecular control over thoraco-lumbar CSN axon extension, part of an integrated network of controls over segmentally-specific CSN axon projection targeting.

Significance StatementCorticospinal neurons (CSN) exhibit remarkable diversity and precision of axonal projections to targets in the brainstem and distinct spinal segments; the molecular basis for this targeting diversity is largely unknown. CSN subpopulations projecting to distinct targets are also molecularly distinguishable. Distinct subpopulations degenerate in specific motor neuron diseases, further suggesting that intrinsic molecular differences might underlie differential vulnerability to disease. Here, we identify a novel molecular control, Cbln1, expressed by CSN extending axons to thoraco-lumbar spinal segments. Cbln1 is sufficient, but not required, for CSN axon extension toward distal spinal segments, and Cbln1 expression is controlled by recently identified, CSN-intrinsic regulators of axon extension. Our results identify that Cbln1, together with other regulators, coordinates segmentally precise CSN axon targeting.


title: Single and complex spikes relay distinct frequency-dependent circuit information in the hippocampus

abstract: Hippocampal neurons generate either single spikes or stereotyped bursts of spikes known as complex spikes. Although single and complex spikes co-occur in the same neuron, their contribution to information processing remains unclear. We analyzed hippocampal CA1 neurons in awake mice and in behaving rats, combining cellular membrane voltage imaging with optogenetics and extracellular recordings. We found that network-driven subthreshold membrane rhythms in the theta versus gamma frequencies preferably entrained complex versus single spikes in individual neurons. Optogenetic membrane perturbation revealed a causal link between subthreshold theta and gamma power and the initiation of complex versus single spikes. Further, single and complex spikes exhibited different place field properties and frequency-dependent coding during spatial navigation. Thus, individual hippocampal neurons do not integrate theta and gamma rhythms into a combined spike timing code, but instead, transmit frequency-specific information as distinct output modes of single versus complex spikes during spatial cognition.


title: Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina.

abstract: Direct reprogramming of retinal Muller glia is a promising avenue for replacing photoreceptors and retinal ganglion cells lost to retinal dystrophies. However, questions have recently been raised about the accuracy of studies claiming efficient glia-to-neuron reprogramming in retina that were conducted using GFAP mini promoter-driven adeno-associated virus (AAV) vectors. In this study, we have addressed these questions using GFAP mini promoter-driven AAV constructs to simultaneously overexpress the mCherry reporter and candidate transcription factors predicted to induce glia-to-neuron conversion, in combination with prospective genetic labeling of retinal Muller glia using inducible Cre-dependent GFP reporters. We find that, while control GFAP-mCherry constructs express faithfully in Muller glia, 5 out of 7 transcription factor overexpression constructs tested are predominantly expressed in amacrine and retinal ganglion cells. However, genetic cell lineage analysis shows no evidence for glia-to-neuron conversion. These findings demonstrate strong insert-dependent effects on AAV-based GFAP mini promoter specificity that preclude its use in inferring cell lineage relationships when studying glia-to-neuron conversion in retina.


title: Chitin oligomers directly promote lymphoid innate and adaptive immune cell activation

abstract:
BackgroundChitin is a highly abundant N-acetyl-glucosamine (GlcNAc) polysaccharide which has been linked to immune responses in the context of fungal infections and allergic asthma, especially T helper 2 (Th2) immune responses. Unfortunately, due to the frequent use of crude chitin preparations of unknown purity and degree of polymerization there is still great uncertainty how chitin activates different parts of the human immune system.

ObjectiveWe recently identified chitin oligomers of six GlcNAc units as the smallest immunologically active chitin motif and the innate immune receptor TLR2 as primary chitin sensor on human and murine myeloid cells, but the response of lymphoid cells to oligomeric chitin has not been investigated and was addressed here.

MethodsFlow cytometric analysis of primary human immune cells upon stimulation with oligomeric chitin. For detailed Methods, please see the Methods section in this articles Online Repository at www.jacionline.org.

ResultHere we report that chitin oligomers also activate immune responses by both primary lymphoid innate and adaptive immune cells: Notably, chitin oligomers activated Natural Killer (NK) cells and B lymphocytes. Moreover, the maturation of dendritic cells enabled CD8 T cell recall responses.

ConclusionsOur results suggest that chitin oligomers not only trigger immediate innate responses in limited range of myeloid cells but exert critical activities across the entire human immune system.

Key messagesChitin oligomers are microbe-associated molecular patterns involved in TLR2-dependent fungal recognition by myeloid immune cells.

Here we show that lymphoid cells, most notably Natural Killer (NK) cells and B cells, are activated by oligomeric chitin.

Oligomeric chitin also promotes antigen-presenting cell (APC) maturation and CD8 T cell recall responses.

Capsule summaryOligomeric chitin is a novel microbe-associated molecular patterns involved in TLR2-dependent fungal recognition by myeloid innate immune cells. Here we show that lymphoid innate and adaptive immune responses are also promoted by oligomeric chitin.

category: immunology


title: Platelet response to influenza vaccination reflects effects of aging

abstract: Platelets are uniquely positioned as mediators of not only hemostasis but also innate immunity, but how age and alterations in functional status such as frailty influence platelet function during an immune response remains unclear. We assessed the platelet transcriptome in younger (age 21-35) and older (age [≥] 65) adults (including frail and non-frail individuals) following influenza vaccination. Prior to vaccination, we identified an age- and frailty-associated increase in expression of platelet activation and mitochondrial RNAs and decrease in RNAs encoding proteins mediating translation. Using tensor decomposition analysis, we also elucidated dynamic post-vaccination platelet activation and translation signatures associated with age and frailty. At the protein level, enhanced platelet activation was found in non-frail older adults, compared to young individuals both prior to and post-vaccine; but frail adults showed decreased platelet activation compared to non-frail that could reflect the influence of decreased translation RNA expression. Our results reveal an age-dependent alteration in platelet function prior to and post-vaccination that may contribute to age-associated chronic inflammation.


title: A macroecological perspective on genetic diversity in the human gut microbiome

abstract: While the human gut microbiome has been intensely studied, we have yet to obtain a sufficient understanding of the genetic diversity harbored by this complex system. This difficulty is compounded by the confluence of forces operating within the gut, where the dynamics of a given allele can be driven by evolutionary forces (drift, selection, etc.) or by the ecology of the strain on which said allele is a constituent. To make progress towards this goal, I identified and characterized statistically invariant patterns of genetic diversity in a large cohort of unrelated hosts across 22 prevalent microbial species. The existence of said patterns allows for the set of feasible models to be pared down to those that can reproduce observed patterns without the use of free parameters. Using a feasible distribution of allele frequencies, I evaluated the extent that a model of ecological dynamics was capable of predicting the fraction of hosts harboring a given allele (i.e., prevalence), a statistic that allows for the effect of sampling to be taken into account. I found that the Stochastic Logistic Model (SLM), an ecological model that has been shown to successfully predict the dynamics of strains, succeeded across all species. The accuracy of the predictions was correlated with independent estimates of strain structure, validating the ecological implications of the SLM. This work indicates that a large number of detectable genetic variants in the human gut are subject to ecological, as opposed to evolutionary, dynamics.

category: ecology


title: Hazel dormouse in managed woodland select for young, dense, and species-rich tree stands

abstract:
In fragmented forest landscapes, population persistence of arboreal species with limited dispersal ability may strongly depend on the quality of the remaining forest habitat. Using the hazel dormouse (Muscardinus avellanarius) as a model species, we studied habitat selection at two spatial scales (home range and within home range) in intensely managed woodlands at its northern distributional range in Denmark. We modelled selection at home range level as the conditional probability of occupancy of 588 nest boxes and nest tubes in 15 managed forests relative to habitat variables measured within 25 m radius. Habitat selection within home ranges was modelled by comparing habitat variables within 3 m radius of triangulated locations by 19 radio-tracked individuals (12 M, 7 F) when active at night with regularly distributed available locations within their home ranges.

At both spatial scales, hazel dormice strongly selected sites with high abundance-weighted species richness and high vegetation density of woody plants. On home range level, they furthermore selected for young tree vegetation, while they within home ranges selected for intermediate aged tree stands (maximum trunk circumference: 1.50 m). The predicted probability of presence in nest boxes or nest tubes varied from less than 1% to more than 99% as a combined function of three habitat variables. From May to October, selection for abundance-weighted species richness of woody plants of radio-tagged individuals decreased with date and body weight, suggesting that a diverse food base is particularly important early in their season of activity and for lean and small (growing) individuals. Selection for dense vegetation increased with body mass and mean available vegetation density within home ranges, indicating behavioural variability related to changes in energy expenditure and need for safety among individuals.

The study demonstrates that the hazel dormouse has specific habitat requirements related to food and safety that can be accommodated with relatively simple means in managed forests.

Graphical abstract

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title: Assessing the importance of resistance, persistence and hyper-mutation for antibiotic treatment success with stochastic modelling

abstract: Antimicrobial resistance poses a rising threat to global health, making it crucial to understand the routes of bacterial survival during antimicrobial treatments. Treatment failure can result from genetic or phenotypic mechanisms, which diminish the effect of antibiotics. By assembling empirical data, we find that, for example, Pseudomonas aeruginosa infections in cystic fibrosis patients frequently contain persisters, transiently non-growing and antibiotic-refractory subpopulations, and hyper-mutators, mutants with elevated mutation rates and thus higher probability of genetic resistance emergence. Resistance, persistence and hyper-mutation dynamics are difficult to disentangle experimentally. Hence, we use stochastic population modelling and deterministic fitness calculations of bacterial evolution under antibiotic treatment to investigate how genetic resistance and phenotypic mechanisms affect treatment success. We find that treatment failure is caused by resistant mutants at lower antibiotic concentrations (with high final bacterial numbers), but by persistence phenotypes at higher antibiotic concentrations (with low final bacterial numbers). Facilitation of resistance occurs through hyper-mutators during treatment, but through persistence only after treatment is discontinued, which allows for persisters to resume growth and evolve resistance in the absence of antibiotics. Our findings highlight the time- and concentration-dependence of different bacterial mechanisms to escape antibiotic killing, which should be considered when designing  resistance-proof antimicrobial treatments.

category: evolutionary biology


title: Analysis of flavonol regulator evolution in the Brassicaceae reveals MYB12, MYB111 and MYB21 duplications associated with MYB11 and MYB24 gene loss

abstract:
BackgroundFlavonols are the largest subgroup of flavonoids, possessing multiple functions in plants including protection against ultraviolet radiation, antimicrobial activities, and flower pigmentation together with anthocyanins. They are of agronomical and economical importance because the major off-taste component in rapeseed protein isolates is a flavonol derivative, which limits rapeseed protein use for human consumption. Flavonol production in Arabidopsis thaliana is mainly regulated by the subgroup 7 (SG7) R2R3-MYB transcription factors MYB11, MYB12, and MYB111. Recently, the SG19 MYBs MYB21, MYB24, and MYB57 were shown to regulate flavonol accumulation in pollen and stamens. The members of each subgroup are closely related, showing gene redundancy and tissue-specific expression in A. thaliana. However, the evolution of these flavonol regulators inside the Brassicaceae, especially inside the Brassiceae, which include the rapeseed crop species, is not fully understood.

ResultsWe studied the SG7 and SG19 MYBs in 44 species, including 31 species of the Brassicaceae, by phylogenetic analyses followed by synteny and gene expression analyses. Thereby we identified a deep MYB12 and MYB111 duplication inside the Brassicaceae, which likely occurred before the divergence of Brassiceae and Thelypodieae. These duplications of SG7 members were followed by the loss of MYB11 after the divergence of Eruca vesicaria from the remaining Brassiceae species. Similarly, MYB21 experienced duplication before the emergence of the Brassiceae family, where the gene loss of MYB24 is also proposed to have happened. Due to the overlapping spatio-temporal expression patterns of the SG7 and SG19 MYB members in B. napus, the loss of MYB11 and MYB24 is likely to be compensated by the remaining homologs.

ConclusionsWe identified a duplication of MYB12, MYB111, and MYB21 inside the Brassicaceae which is associated with MYB11 and MYB24 gene loss inside the tribe Brassiceae. We propose that gene redundancy and meso-polyploidization events have shaped the evolution of the flavonol regulators in the Brassicaceae, especially in the Brassiceae.


title: The regulatory landscape of multiple brain regions in outbred heterogeneous stock rats

abstract: Heterogeneous Stock (HS) rats are a genetically diverse outbred rat population that is widely used for studying genetics of behavioral and physiological traits. Mapping Quantitative Trait Loci (QTL) associated with transcriptional changes would help to identify mechanisms underlying these traits. We generated genotype and transcriptome data for five brain regions from 88 HS rats. We identified 21,392 cis-QTLs associated with expression and splicing changes across all five brain regions. Surprisingly, use of a linear mixed model had little impact on the eQTLs identified compared to linear regression. We also characterized empirical properties of these QTLs and validated their effects using allele specific expression data. We identified 80 cases where eQTLs were colocalized with GWAS results from nine physiological traits, demonstrating the utility of these eQTL data. Comparing our dataset to human data from the Genotype-Tissue Expression (GTEx) project, we found that the HS rat data yields twice as many significant eQTLs as a similarly sized human dataset. We also identified a modest but highly significant correlation between genetic regulatory variation among orthologous genes. Surprisingly, we found less genetic variation in gene regulation in HS rats relative to humans, though we still found eQTLs for the orthologs of many human genes for which eQTLs had not been found. These data are available from the RatGTEx data portal (RatGTEx.org) and will enable new discoveries of the genetic influences of complex traits.

category: genetics


title: A Novel Caenorhabditis elegans Gene Network Uncovers Mechanisms of Mitochondrial Maintenance

abstract:
Mitochondria play key roles in cellular health and metabolism and are a critical determinant of the activation of multiple cell death processes. Although several pathways for regulating and re-establishing mitochondrial homeostasis have been identified within the past twenty years, large gaps remain in our understanding of how cells keep mitochondria healthy.

To address this limitation, have developed a network of genes that underlie mitochondrial health. We began by compiling a list of frequently mutated genes using publicly available data from multiple human cancer cell lines. RNAi was used to disrupt orthologous genes in the model organism Caenorhabditis elegans in a series of assays to evaluate these genes ability to support mitochondrial health, as evidenced by precocious activation of mitochondrial autophagy and sensitivity to acute mitochondrial damage. Iterative screening of ~1000 genes yielded a network of 139 genes showing significant connectivity.

Functional validation of a panel of genes from the network indicated that disruption of each gene triggered at least one phenotype consistent with mitochondrial dysfunction, including increased fragmentation of the mitochondrial network, abnormal steady-state levels of ATP, NADH, or ROS, and altered oxygen consumption. Importantly, RNAi-mediated knockdown of these genes often exacerbated -synuclein aggregation in a C. elegans model of Parkinsons disease, indicating significant changes to cellular health. Additionally, human orthologs of the final mitochondrial health gene network showed enrichment for roles in a number of human disorders identified in the OMIM database. This gene network provides a foundation for identifying new mechanisms that support mitochondrial and cellular homeostasis.


title: Studying meiosis in mitosis: Activating the meiosis-specific Red1-Hop1-Mek1 complex in mitotic budding yeast cells

abstract: In mitosis, sequences on sister chromatids are preferred as DNA repair templates, whereas in meiosis interhomolog-based repair is promoted. The switch of template preference during homologous recombinational (HR) repair of DNA breaks is a defining event in sexual reproduction. This preference is needed to establish linkages between homologous chromosomes that support meiotic chromosome segregation. In budding yeast, a central activity that enforces meiotic interhomolog bias is encoded in a meiosis-specific protein kinase complex, consisting of Red1, Hop1 and Mek1 (i.e., the RHM complex). Activation of Mek1 kinase in meiosis - dictated by complex formation and upstream DNA break-dependent signaling - leads to modification of HR factors and the establishment of interhomolog HR repair bias. How meiotic repair bias is established is a central question with implications for sexual reproduction, genetic diversity and genome stability. Studying the role of the RHM complex in DNA repair is complicated by the fact that Red1 and Hop1 are required for efficient meiotic DNA break formation. Here, we conditionally express RHM components in mitotically-dividing cells to show that these factors can autonomously establish the RHM complex outside of its physiological environment. In vivo analysis is complemented with in vitro biochemical reconstitution to analyze the composition of a Red1-Hop1 subcomplex. The RHM complex can be activated under DNA damaging conditions in mitotically-dividing cells, and activation depends on upstream Mec1 kinase function. We use this system to perform a structure-function analysis of RHM complex formation and Mek1 activation. Finally, we demonstrate that expressing active Mek1 in mitosis leads to rad51{Delta}-like DNA break sensitivity, suggesting that activation of the RHM complex is sufficient to reconstitute (parts of) its physiological function in mediating HR-based repair. This system should enable querying downstream effects of RHM complex action on DNA repair dynamics and template bias. Human homologs of Red1 and Hop1 are often aberrantly re-expressed in cancer cells. Our system has the potential to inform on (dys)functional effects of these genes on genome stability during human tumorigenesis.


title: Proteome-wide identification of amino acid substitutions deleterious for protein function

abstract: DNA sequencing has led to the discovery of millions of mutations that change the encoded protein sequences, but the impact of nearly all of these mutations on protein function is unknown. We addressed this scarcity of functional data by developing Miro, a proteomic technology that uses mistranslation to introduce amino acid substitutions and biochemical assays to quantify functional differences of thousands of protein variants by mass spectrometry. We apply this technology to the proteome of yeast to reveal amino acid substitutions that impact protein structure, ligand binding, protein-protein interactions, protein post-translational modifications, and protein thermal stability. Adapting Miro to human cells will provide a means to efficiently accelerate our mechanistic interpretation of genomic mutations to predict disease risk.

category: genomics


title: Multi-network analysis reveals context-dependent kin relationships in wild sulphur-crested cockatoos, Cacatua galerita

abstract:
O_LIA preference to associate with kin facilitates inclusive fitness benefits, and increased tolerance or cooperation between kin may be an added benefit of group living. Many species exhibit preferred associations with kin, however it is often hard to disentangle active preferences from passive overlap, for example caused by limited dispersal or inheritance of social position.
C_LIO_LIParrots are often thought to exhibit social complexity, and many species appear to show a multilevel society consisting of pair-bonded individuals moving between fission-fusion foraging groups, within larger communal roosts. Previous work has shown that, despite these fission-fusion dynamics, individuals can exhibit long-term preferred foraging associations outside their pair-bonds. Yet the underlying drivers of these social preferences remain largely unknown.
C_LIO_LIIn this study, we use a multi-layered social network approach to examine the influence of kinship on social associations and interactions in wild sulphur-crested cockatoos, Cacatua galerita. We recorded roost co-membership, social associations, and affiliative and aggressive interactions in 561 individually marked birds across three neighbouring roosts. We then collected genetic samples from 205 cockatoos, and conducted a relationship analysis to construct a kinship network. Finally, we tested correlations between kinship and four social networks: spatial proximity, affiliative, low-intensity aggression, and high-intensity aggressions. We examined these potential correlations at two levels - within roost groups and between roosts.
C_LIO_LIOur result showed that while roosting groups were clearly defined, they showed little genetic differentiation. Between roost movement was high, with juveniles, especially females, repeatedly moving between roosting sites. Both within and across these roosting communities, individuals preferentially associated with kin. Supporting this, individuals were also more likely to allopreen kin. However, against our expectation, individuals preferred to direct aggression towards kin within roosts, but avoided aggressing kin of different roosts.
C_LIO_LIOur study demonstrates that sulphur-crested cockatoos prefer to associate with kin, both within and between roosting groups, and exhibit behavioural and context-dependent social interaction rules towards kin. Our results help reveal the drivers of social association in this species, while adding to the evidence for social complexity in parrots.
C_LI

category: animal behavior and cognition


title: Computational Analysis of Effects of Clot Length on Acute Ischemic Stroke Recanalization under Different Cyclic Aspiration Loading Conditions

abstract: Acute ischemic stroke, the second leading cause of death worldwide, results from occlusion of a cerebral artery by a blood clot. Application of cyclic aspiration using an aspiration catheter is a current therapy for the removal of lodged clots. In this study, we perform finite element simulations to analyze deformation of long clots, having length to radius ratio of 2 to 10, which corresponds to clot-length of 2.85-14.25 mm, under peak-to-peak cyclic aspiration pressures of 10 to 50mmmHg, and frequencies of 0.5, 1 and 2 Hz. Our computational system comprises of a nonlinear viscoelastic solid clot, a hyperelastic artery, and a nonlinear viscoelastic cohesive zone, the latter modeling the clot-artery interface. We observe that clots having length-to-radius ratio approximately greater than two separate from the inner arterial surface somewhere between the axial and distal ends, irrespective of the cyclic aspiration loading conditions. The stress distribution within the clot shows large tensile stresses in the clot interior, indicating the possibility of simultaneous fragmentation of the clot. Thus, this study shows us the various failure mechanisms simultaneously present in the clot during cyclic aspiration. Similarly, the stress distribution within the artery implies a possibility of endothelial damage to the arterial wall near the end where the aspiration pressure is applied. This framework provides a foundation for further investigation to clot fracture and adhesion characterization.


title: PhenoComb: A discovery tool to assess complex phenotypes in high-dimension, single-cell datasets

abstract:
MotivationHigh-dimension cytometry assays can simultaneously measure dozens of markers, enabling the investigation of complex phenotypes. However, as manual gating relies on previous biological knowledge, few marker combinations are often assessed. This results in complex phenotypes with potential for biological relevance being overlooked. Here we present PhenoComb, an R package that allows agnostic exploration of phenotypes by assessing all combinations of markers.

DesignPhenoComb uses signal intensity thresholds to assign markers to discrete states (e.g. negative, low, high) and then counts the number of cells per sample from all possible marker combinations in a memory-safe manner. Time and disk space are the only constraints on the number of markers evaluated. PhenoComb also provides several approaches to perform statistical comparisons, evaluate the relevance of phenotypes, and assess the independence of identified phenotypes. PhenoComb allows users to guide analysis by adjusting several function arguments such as identifying parent populations of interest, filtering of low-frequency populations, and defining a maximum complexity of phenotypes to evaluate. We have designed PhenoComb to be compatible with local computer or server-based use.

ResultsIn testing of PhenoCombs performance on synthetic datasets, computation on 16 markers was completed in the scale of minutes and up to 26 markers in hours. We applied PhenoComb to two publicly available datasets: an HIV flow cytometry dataset (12 markers and 421 samples) and the COVIDome CyTOF dataset (40 markers and 99 samples). In the HIV dataset, PhenoComb identified immune phenotypes associated with HIV seroconversion, including those highlighted in the original publication. In the COVID dataset, we identified several immune phenotypes with altered frequencies in infected individuals relative to healthy individuals. Collectively, PhenoComb represents a powerful discovery tool for agnostically assessing high-dimension, single-cell data.

AvailabilityThe PhenoComb R package can be downloaded from https://github.com/SciOmicsLab/PhenoComb


title: Evaluation of in silico predictors on short nucleotide variants in HBA1, HBA2 and HBB associated with haemoglobinopathies

abstract:
IntroductionHaemoglobinopathies are the commonest monogenic diseases worldwide and are caused by variants in the globin gene clusters. With over 2400 variants detected to date, their interpretation using the ACMG/AMP guidelines is challenging, with computational evidence able to provide valuable input about their functional annotation. While many in silico predictors have already been developed, their performance varies for different genes and diseases.

Materials and MethodsWe evaluate 31 in silico predictors using a dataset of 1627 variants in HBA1, HBA2, and HBB. Through varying the decision threshold for each tool, we analyse their performance (a) as binary classifiers of pathogenicity, and (b) using different nonoverlapping pathogenic and benign thresholds for their optimal use in the ACMG/AMP framework.

ResultsCADD, Eigen-PC, and REVEL are the overall top performers, with the former reaching moderate strength level for pathogenic prediction. Eigen-PC and REVEL achieve the highest accuracies for missense variants, while CADD is also a reliable predictor of nonmissense variants. Moreover, SpliceAl is the top performing splicing predictor, reaching strong level of evidence, while GERP++ and phyloP are the most accurate conservation tools.

DiscussionThis study provides evidence about the optimal use of computational evidence in globin gene clusters under the ACMG/AMP framework.


title: DeepParcellation: a novel deep learning method for robust brain magnetic resonance imaging parcellation in older East Asians

abstract: Accurate parcellation of cortical regions is crucial for distinguishing morphometric changes in aged brains, particularly in degenerative brain diseases. Normal aging and neurodegeneration precipitate brain structural changes, leading to distinct tissue contrast and shape in people aged > 60 years. Manual parcellation by trained radiologists can yield a highly accurate outline of the brain; however, analyzing large datasets is laborious and expensive. Alternatively, newly-developed computational models can quickly and accurately conduct brain parcellation, although thus far only for the brains of Caucasian individuals. DeepParcellation, our novel deep learning model for 3D magnetic resonance imaging (MRI) parcellation, was trained on 5,035 brains of older East Asians (Gwangju Alzheimers & Related Dementia) and 2,535 brains of Caucasians. We trained full 3D models for N-way individual regions of interest using memory reduction techniques. Our method showed the highest similarity and robust reliability among age-ethnicity groups, especially when parcellating the brains of older East Asians.


title: Ketamine and its metabolite 2R,6R-hydroxynorketamine promote ocular dominance plasticity and release TRKB from inhibitory control without changing perineuronal nets enwrapping parvalbumin interneurons

abstract: Ketamine has been described as a fast-acting antidepressant, exerting effects in depressed patients and in preclinical models with a rapid onset of action. The typical antidepressant fluoxetine is known to induce plasticity in the adult rodent visual cortex, as assessed by a shift in ocular dominance, a classical model of brain plasticity, and a similar effect has been described for ketamine and its metabolite 2R,6R-hydroxynorketamine (R,R-HNK). Here, we demonstrate that ketamine (at 3 or 20 mg/kg) and R,R-HNK facilitated the shift in ocular dominance in monocularly deprived mice, after 3 injections, throughout the 8-days regimen. Notably, the comparison between the treatments indicates a higher effect size of R,R-HNK compared to ketamine. Treatment with ketamine or R,R-HNK failed to influence the levels of perineuronal nets (PNNs) surrounding parvalbumin-positive interneurons. However, we observed in vitro that both ketamine and R,R-HNK are able to disrupt the tropomyosin-related kinase B (TRKB) interaction with the protein tyrosine phosphatase sigma (PTP{sigma}), which upon binding to PNNs dephosphorylates TRKB. These results support a model where diverse drugs promote the reinstatement of juvenile-like plasticity by directly binding TRKB and releasing it from PTP{sigma} regulation, without necessarily affecting PNNs deposits.


title: Cataloging the potential functional diversity of Cacna1e splice variants using long-read sequencing

abstract:
Voltage gated calcium channels (VGCCs) regulate the influx of calcium ions in many cell types, but our lack of knowledge about the plethora of VGCC splice variants remains a gap in our understanding of calcium channel function. A recent advance in profiling gene splice variation is to use long-read RNA-sequencing technology. We sequenced Cacna1e transcripts from the rat thalamus using Oxford Nanopore sequencing, yielding the full structure of 2,110 Cacna1e splice variants. However, we observed that only 154 Cacna1e splice variants were likely to encode for a functional VGCC based on predicted amino acid sequences. We then computationally prioritized these 154 splice variants using expression and evolutionary conservation and found that four splice variants are candidate functionally distinct splice isoforms. Our work not only provides long-read sequencing of Cacna1e for the first time, but also the first computational evaluation of which Cacna1e splice variants are the best candidates for future follow-up.

SIGNIFICANCE STATEMENTVoltage gated calcium channels (Cacna1x genes) are implicated in many neurological disorders and their encoding genes are predicted to have complex patterns of alternative splicing. Previous approaches relied on short-read RNA-seq to characterize calcium channel splice variants. Here, we use long-read nanopore sequencing to establish a set of Cacna1e transcripts in the rat thalamus and use computational methods to prioritize four transcripts as functionally distinct splice isoforms. Our work to provide the field with prioritized transcripts will not only improve our understanding of Cacna1e function but its role in disease as well.


title: Myeloid hypoxia-inducible factor HIF1A provides cardio-protection during ischemia and reperfusion via induction of netrin-1

abstract: The transcription factor hypoxia-inducible factor HIF1A elicitics cardioprotection from ischemia and reperfusion injury. Here, we investigated tissue-specific pathways that are critical for HIF1A-elicited tissue protection. Initial studies showed that mice with induced global deletion of Hif1a (Hif1aloxP/loxP UbiquitinCre+) have exaggerated myocardial injury during in situ ischemia and reperfusion. Surprisingly, this phenotype was mirrored only in mice with myeloid-specific Hif1a deletion (Hif1aloxP/loxP LysM Cre+). In contrast, mice with myocardial specific (Hif1aloxP/loxP Myosin Cre+), or vascular Hif1a deletion (Hif1aloxP/loxP VEcadherin Cre+) experienced similar injury levels as controls. Subsequent studies using adoptive transfer of Hif1a-deficient polymorphonuclear neutrophils (PMNs) prior to myocardial injury demonstrated increased reperfusion injury. In contrast, adoptive transfer of PMNS treated ex-vivo with the HIF stabilizer dimethyloxalylglycine (DMOG) was associated with attenuated myocardial injury. Moreover, cardioprotection mediated by DMOG was abolished in Hif1aloxP/loxP LysM Cre+ mice, but not in Hif2aloxP/loxP LysM Cre+ mice. Finally, studies of PMN-dependent HIF1A target genes implicated the neuronal guidance molecule netrin-1 in mediating the cardioprotective effects of myeloid HIF1A. Taken together, the present studies identified a functional role for myeloid-expressed HIF1A in providing cardio-protection during ischemia and reperfusion injury, which - at least in part - is mediated by the induction of neuronal guidance molecule netrin-1 in neutrophils.


title: Autotaxin facilitates selective LPA receptor signaling

abstract:
Autotaxin (ATX; ENPP2) produces the lipid mediator lysophosphatidic acid (LPA) that signals through disparate EDG (LPA1-3) and P2Y (LPA4-6) G protein-coupled receptors. ATX/LPA promote several (patho)physiological processes, including in pulmonary fibrosis, thus serving as attractive drug targets. However, it remains unclear if clinical outcome depends on how different ATX inhibitors modulate the ATX/LPA signaling axis. Here, we show that inhibitors binding to the ATX "tunnel" specifically abrogate key aspects of ATX/LPA signaling. We find that the tunnel is essential for signaling efficacy and dictates cellular responses independent of ATX catalytic activity, with a preference for activation of P2Y LPA receptors. These responses are abrogated by tunnel-binding inhibitors, such as ziritaxestat, but not by inhibitors that exclusively target the active site, as shown in primary lung fibroblasts and a murine model of radiation-induced pulmonary fibrosis. Our results uncover a receptor-selective signaling mechanism for ATX, implying clinical benefit for tunnel-targeting ATX inhibitors.

HighlightsATX is a dual-function protein acting as an LPA-producing enzyme and LPA chaperone.

Structural integrity of the ATX tunnel is essential to mediate signaling functions.

ATX-bound LPA signals preferentially via P2Y family LPA receptors.

Occupancy of the ATX tunnel is crucial for ziritaxestat to exert inhibition in vivo.

category: cell biology


title: Plastic loss of motile cilia in the internal gills of 	Polypterus in response to high CO2 or terrestrial environments

abstract: The evolutionary transition of vertebrates from water to land during the Devonian period was accompanied by major changes in animal respiratory systems in terms of physiology and morphology. Indeed, the fossil record of the early tetrapods has revealed the existence of internal gills, which are vestigial fish-like traits used underwater. However, the fossil record provides only limited data to elucidate the process of the evolutionary transition of internal gills from fish to early tetrapods. This study investigated the internal gills of Polypterus senegalus, a basal ray-finned/amphibious fish which shows many ancestral features of stem Osteichthyes. Based on scanning electron microscopy observations and transcriptome analysis, the existence of motile cilia in the internal gills was revealed which may create a flow on the internal gill surface leading to efficient respiration. Interestingly, these cilia were observed to disappear after rearing in terrestrial or high CO2 environments, which mimics the environmental changes in the Devonian period. The cilia re-appeared after being returned to the original aquatic environment. The ability of plastic loss of internal gills in Polypterus revealed in this study may allow them to survive in fluctuating environments, such as shallow swamps. The ancestor of Osteichthyes is also expected to have possessed such plasticity in the internal gills, which may be one of the driving forces behind the transition of vertebrates from water to land.


title: Caenorhabditis elegans MES-3 is a highly divergent ortholog of the canonical PRC2 component SUZ12

abstract:
Polycomb Repressive Complex 2 (PRC2) catalyzes the mono-, di, and trimethylation of histone protein H3 on lysine 27 (H3K27). Trimethylation of H3K27 is strongly associated with transcriptionally silent chromatin and plays an important role in the regulation of cell identity and developmental gene expression1. The functional core of PRC2 is highly conserved in animals and consists of four subunits1 (Fig. 1a). Notably, one of these subunits, SUZ12, has not been identified in the genetic model Caenorhabditis elegans, whereas C. elegans PRC2 contains the lineage-specific protein MES-32,3 (Fig. 1a). Here, we demonstrate that MES-3 is in fact a highly divergent ortholog of SUZ12. Unbiased sensitive sequence similarity searches uncovered consistent but insignificant reciprocal best matches between MES-3 and SUZ12, suggesting that these proteins could share a common evolutionary history. We substantiate this hypothesis by directly comparing the predicted structures of SUZ12 and MES-3, which revealed shared protein folds and residues of key domains. Thus, in agreement with the observations in previous genetic and biochemical studies2,3, we here provide evidence that C. elegans, like other animals, contains a diverged yet evolutionary conserved core PRC2.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/487461v1_fig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1b0c78corg.highwire.dtl.DTLVardef@fcbd4corg.highwire.dtl.DTLVardef@efe3c1org.highwire.dtl.DTLVardef@151b328_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 1.C_FLOATNO MES-3 is a highly divergent ortholog of the canonical Polycomb Repressive Complex 2 component SUZ12. a. The Polycomb Repressive Complex 2 (PRC2) core components EZH2/1, EED, RBBP4/7, and SUZ12 are conserved in a broad range of metazoans; the presence of orthologs is indicated by filled boxes. Notably, based on sequence similarity searches, an ortholog of SUZ12 is absent in the nematode model species Caenorhabditis elegans, but present in other, closely related nematodes (Brugia malayi and Trichinella spiralis). C. elegans encodes the PRC2 core component MES-32,3 that lack obvious motifs or sequence similarity to SUZ122. b. Schematic representation of the composition of the core PRC2. The zinc finger binding (ZnB; red), WD-domain binding 1 (WDB1; blue), C2 domain (green), zinc finger (Zn; yellow), WD-domain binding 2 (WDB2; pink), and VEFS (orange) motifs or domains involved in SUZ12 protein-interactions are shown in the schematic as well as along the protein sequence4,5. Schematic representation of the protein sequence of MES-3 is shown, and regions of here uncovered sequence (c) and structural (e, f) similarity are highlighted. c. Protein sequence alignment between the N-terminal region of SUZ12 and MES-3, as identified by sensitive profile-vs-profile sequence similarity searches, covers part of the zinc finger binding (ZnB; red), WD-domain binding 1 (WDB1; blue), and C2 domain (green). The conserved RBBP4/7 binding epitope as well as Gly299 are highlighted8-10. Identical amino acids are shown in blue and biochemically similar amino acids are shown in turquoise. d. The predicted aligned error (in [A]; based on model 2 ptm) of the MES-3 structure is shown as a heatmap and reveals two separated globular regions in the N- and C-terminus, the former overlaps with the profile-vs-profile match (c) and corresponds to the C2 domain of SUZ12 (e; Fig. S1i; RMSD = 1.607) while the latter overlaps with the regions that structurally resemble the VEFS domain (f; Fig. S1j; RMSD = 3.676). The black arrows (e, f) highlight regions that differ considerably between SUZ12 and MES-3 (Fig. S1i, j). g. Sequence-independent structure alignment of the VEFS regions of SUZ12 and MES-3 reveals significantly structural similarity (Dali Z-score = 8.3; TM-score = 0.55), especially along the alpha helices in the C-terminus; a region previously shown to stimulate histone methyltransferase activity in SUZ129 (pos. 580 to 612) is highlighted by a black bar, and individual amino acids important for PRC2 assembly9 are shown by black arrows.

C_FIG


title: Structure of phage lambda Red-beta(177) annealase shows how it anneals DNA strands during single-strand annealing homologous DNA recombination

abstract:
The bacteriophage {lambda} red recombination system catalyzes the single-strand annealing homologous DNA recombination reaction, in which Red{beta} annealase protein plays a critical role. Using cryogenic electron microscopy, we were able to determine a structure of a C-terminally truncated Red{beta} with the residues 1-177 bound to two complementary 27mer oligonucleotides forming an annealing intermediate, to a final resolution of 3.3 [A]. This structure validates and rationalizes decades of experimental observations on the biochemistry of Red{beta}. Definition of the interaction surfaces between subunits explains not only the DNA binding properties of Red{beta}, but also its propensity to oligomerize into long helical filaments, which are also formed by almost all annealases and are known to be functionally important. In addition, this annealing intermediate structure provides a detailed picture of the hydrogen bonding network that positions the DNA strands in a planar orientation to facilitate base pairing. Residues 133-138, which are missing from our structure, form a flexible loop. Molecular dynamics simulations were used to model the range of motion of the flexible loop, which suggested that it has a crucial role in keeping the DNA strands in the DNA-binding groove long enough to allow homology searching. The combination of structural and computational observations has allowed us to propose a detailed mechanism for the action of Red{beta}. More than half a century after its discovery, our work shines a light not only on the structure and mechanisms of Red{beta}, but also of other proteins within the annealase superfamilies.

Significance StatementSingle-strand annealing homologous DNA recombination is a process that is conserved throughout evolution from bacteriophages to humans, highlighting its importance and indispensability. It is a process that repairs double-stranded DNA breaks and is particularly vital in dsDNA viruses. The Red{beta} protein from the bacteriophage lambda is the archetypal annealase protein, forming the basis of our knowledge about this class of proteins. Along with the exonuclease {lambda}Exo, these two proteins not only form the model system for single-strand annealing homologous recombination, but are also used in thousands of laboratories worldwide for performing genetic manipulations. After its discovery in 1966, we report the first structure of the DNA-binding and oligomerization domain of Red{beta}, providing details about the mechanism of homologous DNA annealing.


title: Myelination-dependent mitochondria clustering and calcium buffering in parvalbumin interneuron axons

abstract: Parvalbumin-expressing (PV+) interneurons are fast-spiking cells that exert critical control over local neuronal circuit activity and oscillations. While it is well established that axons of PV+ interneurons are myelinated its role remains poorly understood. One leading hypothesis is that myelin provides trophic support to sustain the metabolic demands of high-frequency spike generation of PV+ interneurons. Here we developed Cre-dependent AAV vectors enabling cell type-specific investigation of the distribution and functional properties of mitochondria using genetically encoded fluorescent probes. Patch-clamp recordings followed by single-cell reconstructions revealed that mitochondria cluster to myelinated segments of PV+ axons, which was lost upon experimental demyelination. Furthermore, two-photon imaging of action potential-evoked mitochondrial calcium (mt-Ca2+) responses revealed high mt-Ca2+ in unmyelinated segments and branch points compared to myelinated internodes. In contrast, demyelination caused uniform low mt-Ca2+ responses. Together, these results suggest that PV+ myelination regulates localization of mitochondria and activity-dependent local metabolic demands in axons.


title: Mouse brain-wide transgene expression by systemic injection of genetically engineered exosomes: CAP-Exosomes

abstract:
The bottleneck in drug discovery for central nervous system diseases is the absence of effective systemic drug delivery technology for delivering therapeutic drugs into the brain. Although some Adeno Associated Virus (AAV) serotype can cross blood brain barrier and deliver virus genome packaged therapeutic DNA (gene) or RNA molecules to brain cells along with other organs, several hurdles have emerged in the AAV9 vector gene transfer technology in both preclinical studies and clinical trials. In order to overcome some of the hurdles, we have developed a workflow to generate a novel brain targeted drug delivery system (DDS) that involves generation of genetically engineering exosomes by first selecting various functional AAV capsid specific peptides (collectively called CAP) known to be involved in brain targeted high expression gene delivery, and then expressing the CAP in frame with lysosome-associated membrane glycoprotein (Lamp2b) followed by expressing CAP-Lamp2b fusion protein on the surface of mesenchymal stem cell derived exosomes, generating CAP-exosomes. Intravenous injection of green fluorescent protein (GFP) gene loaded CAP-exosomes in mice transfer GFP gene throughout the CNS as measured by monitoring brain sections for GFP expression with confocal microscopy. GFP gene transfer efficiency is at least 20-fold greater than that of control Lamp2b-exosomes. GFP gene transduction to mouse liver was low. CAP-exosome has advantage over AAV-vector including, 1) no restriction in gene size to be delivered, 2) expected reduced production of neutralizing antibody, and 3) can be used separately for drug repurposing and/or in combination with therapeutic genes.

Graphical Abstract

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title: Multilevel selection analysis reveals diversity in selfish mitochondrial genome dynamics

abstract: Mutant copies of mitochondrial DNA (mtDNA) can behave as selfish entities, propagating within organisms at the expense of host fitness. We previously developed experiments to separately measure the within-host proliferation, and host fitness cost, of selfish mtDNA. By focusing on a single mutant genome, the uaDf5 variant, our previous work raises the question of generalizability: do selfish genomes uniformly overcome a heavy fitness cost by proliferating to high frequency, or do their population dynamics vary? By applying a standardized multilevel selection analysis across a collection of mitochondrial mutants, we uncover variation in cheating strategies. Mutants that impose a heavy fitness cost nevertheless persist by outcompeting wildtype mtDNA within hosts. Conversely, some mutants fail to outcompete wildtype mtDNA but nevertheless persist, maintaining a frequency range at which host fitness cost is negligible. These findings suggest that the population dynamics of mutant mtDNA depend on the loci affected.


title: Neuronal HLH-30/TFEB modulates muscle mitochondrial fragmentation to improve thermoresistance in C. elegans

abstract: Transcription factor EB (TFEB) is a conserved master transcriptional activator of autophagy and lysosomal genes that modulates organismal lifespan regulation and stress resistance. As neurons can coordinate organism-wide mechanisms, we investigated the role of neuronal TFEB in stress resistance and longevity. To this end, the C. elegans TFEB orthologue, hlh-30, was rescued panneuronally in hlh-30 loss of function mutants. While important in the long lifespan of daf-2 animals, neuronal hlh-30 was not sufficient to restore normal lifespan in short-lived hlh-30 mutants. However, neuronal HLH-30/TFEB rescue mediated robust improvements in the heat stress resistance of wild-type but not daf-2 animals. Notably, these mechanisms can be uncoupled, as neuronal HLH-30/TFEB regulates longevity and thermoresistance dependently and independently of DAF-16/FOXO respectively. Through transcriptomics profiling and functional analysis, we identified the uncharacterized gene W06A11.1 as a bona fide mediator of heat stress resistance via the induction of mitochondrial fragmentation in distal muscles. Neuron-to-muscle communication occurred through a modulation of neurotransmission. Taken together, this study uncovers a novel mechanism of heat stress protection mediated by neuronal HLH-30/TFEB.


title: Use of a novel knock-in allele of Pkd1 identifies nicotinamide nucleotide dehydrogenase as a mitochondrial binding partner of polycystin-1

abstract: The localization and function of Polycystin-1, the protein encoded by the gene most commonly mutated in autosomal dominant polycystic kidney disease, remains controversial. We have recently reported that its C-terminus is cleaved and traffics to the mitochondria rather than to the nucleus as had been previously described, and we found that absence of PC1 resulted in fragmented mitochondrial networks and increased mitochondrial membrane potential. Direct visualization of PC1 in mitochondria was only possible, however, after over-expression of recombinant, fluorescently labeled-PC1 in a cell culture system. To resolve the issue, we generated a new mouse model with three copies of the HA epitope and eGFP knocked-in frame into the endogenous mouse Pkd1 gene by CRISPR/Cas9. We show that the modified allele is fully functional but the eGFP-tagged protein cannot be detected without antibody amplification methods. We were, however, able to use nanobody-coupled beads and large quantities of tissue to isolate a PC1 interactome and verify nicotinamide nucleotide transhydrogenase (Nnt) as a mitochondrial partner, linking PC1 to regulation of reactive oxygen species levels in the mitochondria. Loss of Nnt function had no significant effect on renal cystic disease in Pkd1 mutants but treatment of young mice with early onset cystic disease with n-acetyl-cysteine (NAC) provided modest benefit only in the Nnt+/+ genetic background. These studies suggest that new methods and brighter tags will be required to track endogenous PC1, but this new mouse model will be a valuable resource for characterizing the protein interactome of endogenous PC1. The data also support our prior findings that the PC1 C-terminus localizes to mitochondria and regulates their function.


title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

abstract: Neural stem cells reside in a specialized neurogenic niche of the hippocampus termed the subgranular zone. Throughout life, they give rise to adult-born neurons in the dentate gyrus thereby contributing to learning and memory. Here, we report that neurons together with neural stem and precursor cells secrete the neurovascular protein epidermal growth factor-like protein 7 (EGFL7) to shape this niche. EGFL7 knock-out in vivo promoted adult neurogenesis generating neurons forming additional spines which permanently integrated into the neural circuit until old age. RNA-sequencing identified the cytokine VEGF-D as a major molecular driver of this process in vivo. In behavioral studies EGFL7 knock-out mice displayed stronger maintenance of memory suggesting longer-lasting spatial memory and improved memory consolidation in the hippocampus by modulation of pattern separation in young and aged mice. Taken together, EGFL7 is an upstream regulator of the VEGF-D in adult neurogenesis and a key regulator of learning and memory.


title: Abstinence-dependent effects of long-access cocaine self-administration on nucleus accumbens astrocytes are observed in male, but not female rats

abstract: Accumulating evidence indicates significant consequences for astrocytes associated with drug abuse. For example, reductions in structural features and synaptic colocalization of male rat nucleus accumbens (NAc) astrocytes are observed following short-access (ShA, 2 hours/day) self-administration and extinction from cocaine, methamphetamine, and heroin. However, it is unknown whether these observations extend to other rodent models of drug abuse, how enduring these effects may be, and whether similar effects are observed in female rats. Here we assess the effects of long-access (LgA, 6 hours/day) cocaine self-administration and abstinence on NAc astrocytes separately in male and female rats, a commonly used behavioral approach to investigate the incubation of cocaine craving. NAc astrocytes from male rats exhibit extensive ([~]40%) reductions in surface area, volume, and postsynaptic colocalization 45 days, but not 24 hours after the last self-administration session. In contrast, no effect of self-administration was observed in astrocytes from female rats. Moreover, no effect of LgA self-administration and abstinence was observed on NAc GLT-1 expression in female rats, an effect that has been well described in males. The results indicate striking and sexually dimorphic effects of abstinence subsequent to LgA self-administration on astrocytes. Taken together, these results indicate a pivotal role of prolonged abstinence in the effects of cocaine self-administration on NAc astrocytes, and extend a growing body of evidence regarding sex differences in the cellular consequences of drug self-administration in the brain.


title: A subtype of melanopsin ganglion cells encodes ground luminance

abstract: Visual input to the hypothalamus from intrinsically photosensitive retinal ganglion cells (ipRGCs) influences several functions including circadian entrainment, body temperature, and sleep. ipRGCs also project to nuclei such as the supraoptic nucleus (SON), which is involved in systemic fluid homeostasis, maternal behavior, and appetite. However, little is known about the SON-projecting ipRGCs or their relationship to well-characterized ipRGC subtypes. Using a GlyT2Cre mouse line, we identify a subtype of ipRGCs restricted to the dorsal retina that selectively project to the SON. These ipRGCs form a non-overlapping tiled mosaic that is limited to a dorsal region of the retina, forming a substrate for encoding ground luminance. Optogenetic activation of their axons demonstrates they release the neurotransmitter glutamate and that the SON is retinorecipient, receiving synaptic input from dorsal ipRGCs. Our results challenge the idea that ipRGC dendrites overlap to optimize photon capture and suggests non-image forming vision operates to sample local regions of the visual field.


title: Barcoding and its application for visualizing ecological dynamics

abstract: Time is perceived to be unidirectional and continuous in the philosophy of science. This continuity can play a crucial role in time series analysis as events are generally seen as an outcome of the past, or subject to events that occurred previously in time. In this study, we describe an ordinal approach to perceiving ecological time series - one that relies on pattern formation with both antecedent and future events. Our approach defines a limited set of structural shapes that can occur for past, present, and future time points. Such a library of all possible shapes can then be used for novel approaches to data visualization and time series analysis. We applied this method to simple ecological models and then to natural time series data for measles cases in London and the phytoplankter Pseudo-nitzschia spp. in Narragansett Bay, Rhode Island. Alternative perspectives on time series representation can strengthen our ability to identify important patterns in dynamics and effectively discriminate between similar time series. When used in conjunction with conventional line-plots, barcodes can be tailored to demonstrate the presence or absence of specific structural patterns or features. Our results show that data exploration without the assumption of time series continuity can yield important and novel insight into the behavior of ecological systems.


title: Global disparity of research allocation and the Aichi biodiversity conservation targets

abstract: Tracking progress towards global biodiversity conservation targets requires appropriate allocation of research and monitoring efforts. We conducted a global review of camera trap research on mammals as a proxy for biodiversity research and monitoring over the last two decades. We assessed how 3395 research locations from 2324 studies tracked priority regions for attaining the 2020 Aichi Biodiversity Targets. We used a geospatial distribution modelling approach to predict the spatial allocation of biodiversity research and to identify its key drivers. We show that conservation research in the past two decades has often failed to target areas important for conservation, and that 76.8% of the global research allocation can be attributed to country income, biome, mammal richness and accessibility. We predicted lowest probabilities of research allocation in low income countries. The Amazon and Congo Forest basins -- two highly biodiverse ecosystems facing unprecedented human alteration -- received inadequate research attention. Even in the most researched regions, an average of 51.4% of the research locations were outside the top 20% most important areas for the global biodiversity Aichi Targets. To support biodiversity conservation targets, policy and practice, more research and monitoring is required in regions with high importance for conservation.


title: The effects of environmental heterogeneity within cities on the evolution of clines

abstract:
O_LIThere is increasing evidence that environmental change associated with urbanization can drive rapid adaptation. However, most studies of urban adaptation have focused on coarse urban vs. rural comparisons or sampled along a single urban-rural environmental gradients, thereby ignoring the role that within-city environmental heterogeneity might play in adaptation to urban environments.
C_LIO_LIIn this study, we examined fine-scale variation in the presence of HCN--a potent anti-herbivore defense--and its two underlying genes (Ac and Li) between park green spaces and surrounding suburban habitats for five city parks in the Greater Toronto Area.
C_LIO_LIWe show that fine-scale urbanization has driven the formation of micro-clines in HCN on a scale of < 2 km, though the presence and strength of micro-clines varied across parks. Interestingly, these micro-clines were in the opposite direction to that predicted based on previously described patterns of HCN frequency change along urban-rural gradients.
C_LIO_LISynthesis: These results suggest larger scale, adaptive urban-rural clines occur across a complex matrix of environmental heterogeneity within cities that drives fine-scale adaptive microclines of varying strengths and directions.
C_LI


title: Genome structures resolve the early diversification of teleost fishes

abstract:
Accurate species phylogenies are a prerequisite for evolutionary research. Teleosts are by far the largest and the most diversified group of extant vertebrates, but relationships among the three oldest lineages of extant teleosts remain unresolved. Based on seven high-quality new genome assemblies in Elopomorpha (tarpons, eels), we revisited the topology of the deepest branches of the teleost phylogeny using independent gene sequence and chromosomal rearrangement phylogenomic approaches. These analyses converged to a single scenario that unambiguously places the Elopomorpha and Osteoglossomorpha (bony-tongues) in a monophyletic group sister to all other teleosts, i.e., the Clupeocephala lineage. This finding resolves over 50 years of controversy on the evolutionary relationships of these lineages and highlights the power of combining different levels of genome-wide information to solve complex phylogenies.

One-Sentence SummaryWhole-genome analyses place Elopomorpha (tarpons, eels) and Osteoglossomorpha (bony-tongues) as sister groups at the deepest branching of crown teleosts.


title: The GUN mutants: new weapons to unravel ascospore germination regulation in the model fungus Podospora anserina

abstract: In Podospora anserina as in many other ascomycetes, ascospore germination is a regulated process that requires breaking of dormancy. Despite its importance in survival and dispersal, ascospore germination in filamentous fungi has been poorly investigated and little is known about its regulation and genetic control. We have designed a positive genetic screen that led to the isolation of mutants showing uncontrolled germination, the GUN mutants. In this paper, we report on the characterization of GUN1SG. We show that GUN1SG is mutated in Pa_6_1340, the ortholog of Magnaporthe oryzae Pth2, which encodes a Carnitine-acetyltransferase (CAT) involved in the shuttling of acetyl-CoA between peroxisomes and mitochondria and which is required for appressorium-development. Bioinformatic analysis revealed that the mutated residue (I441) is highly conserved among the Fungi, and that the mutation has a deleterious impact on the protein function. We show that GUN1 is essential for ascospore germination and that the protein is localized both in mitochondria and in peroxisomes. Finally, epistasis studies allowed us to place GUN1 upstream of the PaMpk2 MAPK pathway and the PaNox2/PaPls1 complex in the regulation of ascospore germination. The identification of GUN1, the ortholog of Pth2, in ascospore germination, strengthens the idea of a common genetic regulation governing both appressorium development and melanized ascospore germination. In addition, we characterize the second CAT encoded in P. anserina genome, Pa_3_7660/GUP1, and we show that the function of both CATs is conserved in P. anserina.


title: RetroSnake: a Modular End-to-End Pipeline for Detection of Human Endogenous Retrovirus (HERV) Transposable Elements in Next Generation Sequencing (NGS) Data

abstract:
Human Endogenous Retroviruses (HERVs) integrated into the genome of vertebrates as a result of ancient exogenous infections and currently comprise [~]8% of our genome. The majority of these elements have accumulated mutations rendering them inactive. The most recently acquired members, HERV-K have potential to produce viral particles and have been linked to a wide range of diseases including cancer and neurodegeneration. Although a range of tools for HERV discovery exist, most of them lack wet-lab validation of their results and are not end-to-end as they do not cover all steps of the analysis. These factors greatly limit their use. Here we describe RetroSnake, an end-to-end, modular, computationally efficient and customisable pipeline for the discovery of HERVs in short-read NGS data. RetroSnake presents important advantages with respect to other available tools. For instance, it is the only pipeline based on an extensively wet-lab validated protocol, and it is the most complete transposable elements detection pipeline, producing annotated insertions presented as an interactive html file, easy enough to use by life scientists without substantial computational training.

Availability and implementationThe Pipeline and an extensive documentation are available at https://github.com/KHP-Informatics/RetroSnake

Contactalfredo.iacoangeli@kcl.ac.uk


title: Immobilised enzyme cascade for targeted glycosylation

abstract: Glycosylation is a critical post-translational modification of proteins, improving properties such as folding, half-life and functionality. However, glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. Here we describe a platform for sequential glycosylation reactions for tailored sugar structures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro. This novel proof-of-concept system is enabled by immobilised enzymes produced with a "one-step immobilisation/purification" method to express, biotinylate in vivo and immobilise glycosyltransferases. The immobilised enzymes are used in a reaction cascade mimicking a human-like N-linked glycosylation pathway where promiscuity naturally exists. The enzyme cascade is applied to free glycans, and a monomeric Fc domain expressed in glycoengineered Pichia pastoris, yielding near homogeneous glycoforms (>95% conversion). Finally, immobilised {beta}-1,4 galactosyltransferase is used to enhance the galactosylation profile of three different IgGs yielding 80.2 - 96.3 % terminal galactosylation. Enzyme recycling was further demonstrated for 7 cycles, with a combined reaction time greater than 140 hours. The novel SUGAR-TARGET platform is easy to implement, modular and reusable, and therefore can lead to the development of homogeneous glycan structures for functional and clinical evaluation. The use of immobilised enzymes enables the economical modification of cell-based material supporting applications at a large industrial scale.


title: Experimental identification of individual insect visual tracking delays in free flight and their effects on visual swarm patterns

abstract:
Insects are model systems for swarming robotic agents, yet engineered descriptions do not fully explain the mechanisms by which they provide onboard sensing and feedback to support such motions; in particular, the exact value and population distribution of visuomotor processing delays are not yet quantified, nor the effect of such delays on a visually-interconnected swarm. This study measures untethered insects performing a solo in-flight visual tracking task and applies system identification techniques to build an experimentally-consistent model of the visual tracking behaviors, and then integrates the measured experimental delay and its variation into a visually interconnected swarm model to develop theoretical and simulated solutions and stability limits. The experimental techniques include the development of a moving visual stimulus and real-time multi camera based tracking system called VISIONS1 providing the capability to recognize and simultaneously track both a visual stimulus (input) and an insect at a frame rate of 60-120 Hz. A frequency domain analysis of honeybee tracking trajectories is conducted via fast Fourier and Chirp Z transforms, identifying a coherent linear region and its model structure. The model output is compared in time and frequency domain simulations. The experimentally measured delays are then related to probability density functions, and both the measured delays and their distribution are incorporated as inter-agent interaction delays in a second order swarming dynamics model. Linear stability and bifurcation analysis on the long range asymptotic behavior is used to identify delay distributions leading to a family of solutions with stable and unstable swarm center of mass (barycenter) locations. Numerical simulations are used to verify these results with both continuous and modeled distributions.

The results of this experiment quantify a model structure and temporal lag (transport delay) in the closed loop dynamics, and show that this delay varies across 50 individuals from 5-110ms, with an average delay of 22ms and a standard deviation of 40ms. When analyzed within the swarm model, the measured delays support a diversity of solutions and indicate an unstable barycenter.