title: Coupling of pupil- and neuronal population dynamics reveals diverse influences of arousal on cortical processing

abstract: Fluctuations in arousal, controlled by subcortical neuromodulatory systems, continuously shape cortical state, with profound consequences for information processing. Yet, how arousal signals influence cortical population activity in detail has so far only been characterized for a few selected brain regions. Traditional accounts conceptualize arousal as a homogeneous modulator of neural population activity across the cerebral cortex. Recent insights, however, point to a higher specificity of arousal effects on different components of neural activity and across cortical regions. Here, we provide a comprehensive account of the relationships between fluctuations in arousal and neuronal population activity across the human brain. Exploiting the established link between pupil size and central arousal systems, we performed concurrent magnetoencephalographic (MEG) and pupillographic recordings in a large number of participants, pooled across three laboratories. We found a cascade of effects relative to the peak timing of spontaneous pupil dilations: Decreases in low-frequency (2-8 Hz) activity in temporal and lateral frontal cortex, followed by increased high-frequency (>64 Hz) activity in mid-frontal regions, followed by monotonic and inverted-U relationships with intermediate frequency-range activity (8-32 Hz) in occipito-parietal regions. Pupil-linked arousal also coincided with widespread changes in the structure of the aperiodic component of cortical population activity, indicative of changes in the excitation-inhibition balance in underlying microcircuits. Our results provide a novel basis for studying the arousal modulation of cognitive computations in cortical circuits.


title: SynapseJ: an automated, synapse identification macro for ImageJ

abstract: While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. These methods are applied to analysis of a presynaptic marker, bassoon, with two different postsynaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Using gephyrin as an inhibitory, postsynaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental neuron (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation.

category: neuroscience


title: Neural activity in human brainstem adapts to sound-level statistics

abstract: Optimal perception requires adaptation to sounds in the environment. Adaptation involves representing the acoustic stimulation history in neural response patterns, for example, by altering response magnitude or latency as sound-level context change. Neurons in the auditory brainstem of rodents are sensitive to acoustic stimulation history and sound-level context (often referred to as sensitivity to stimulus statistics), but the degree to which the human brainstem exhibits such neural adaptation is unclear. In six electroencephalography experiments with over 125 participants, we demonstrate that the response latency of the human brainstem is sensitive to the history of acoustic stimulation over a few tens of milliseconds. We further show that human brainstem responses adapt to sound-level context in, at least, the last 44 ms, but that neural sensitivity to sound-level context decreases when the time window over which acoustic stimuli need to be integrated becomes wider. Our study thus provides evidence of adaptation to sound-level context in the human brainstem and of the timescale over which sound-level information affects neural responses to sound. The research delivers an important link to studies on neural adaptation in non-human animals.


title: Sound level context modulates neural activity in the human brainstem


title: Ketamine and sleep modulate neural complexity dynamics in cats

abstract:
There is increasing evidence that level of consciousness can be captured by neural informational complexity: for instance, complexity, as measured by the Lempel Ziv (LZ) compression algorithm, decreases during anesthesia and non-rapid eye movement (NREM) sleep in humans and rats, when compared to LZ in awake and REM sleep. In contrast, LZ is higher in humans under the effect of psychedelics, including subanesthetic doses of ketamine. However, it is both unclear how this result would be modulated by varying ketamine doses, and whether it would extend to other species. Here we studied LZ with and without auditory stimulation during wakefulness and different sleep stages in 5 cats implanted with intracranial electrodes, as well as under subanesthetic doses of ketamine (5, 10, and 15 mg/kg i.m.). In line with previous results, LZ was lowest in NREM sleep, but similar in REM and wakefulness. Furthermore, we found an inverted U-shaped curve following different levels of ketamine doses in a subset of electrodes, primarily in prefrontal cortex. However, it is worth noting that the variability in the ketamine dose-response curve across cats and cortices was larger than that in the sleep-stage data, highlighting the differential local dynamics created by two different ways of modulating conscious state. These results replicate previous findings, both in humans and other species, demonstrating that neural complexity is highly sensitive to capture state changes between wake and sleep stages while adding a local cortical description. Finally, this study describes the differential effects of ketamine doses, replicating a rise in complexity for low doses, and further fall as doses approach anesthetic levels in a differential manner depending on the cortex.

In briefPrevious studies have shown that Lempel Ziv complexity (LZ) decreases during anesthesia and non-rapid eyes movement (NREM) sleep in humans and rats whereas it increases in REM sleep and under the effect of psychedelics. In this work we show that in the cat, LZ is lowest in NREM sleep, but similar in REM and wakefulness. We also found a ketamine inverted U-shape dose-response curve only in the auditory and prefrontal cortex, with a much larger variability in the ketamine across cats and cortices when compared to the sleep cycle.

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title: Using dual EEG to analyse event-locked changes in child-adult neural connectivity

abstract: Current approaches typically measure the connectivity between interacting physiological systems as a time-invariant property. This approach obscures crucial information about how connectivity between interacting systems is established and maintained. Here, we describe methods, and present computational algorithms, that will allow researchers to address this deficit. We focus on how two different approaches to measuring connectivity, namely concurrent (e.g., power correlations, phase locking) and sequential (e.g., Granger causality), can be applied to three aspects of the brain signal, namely amplitude, power, and phase. We guide the reader through worked examples using mainly simulated data on how to leverage these methods to measure changes in interbrain connectivity between adults and children/infants relative to events identified within continuous EEG data during a free-flowing naturalistic interaction. For each, we aim to provide a detailed explanation of the interpretation of the analysis and how they can be usefully used when studying early social interactions.


title: Macrophage inflammatory and regenerative response periodicity is programmed by cell cycle and chromatin state

abstract:
Cell cycle (CC) is a fundamental biological process with robust, cyclical gene expression programs to facilitate cell division. In the immune system, a productive immune response requires the expansion of pathogen-responsive cell types, but whether CC also confers unique gene expression programs that inform the subsequent immunological response remains unclear. Here we demonstrate that single macrophages adopt different plasticity states in CC, which is a major source of heterogeneity in response to polarizing cytokines. Specifically, macrophage plasticity to interferon gamma (IFNG) is substantially reduced, while interleukin 4 (IL-4) can induce S-G2/M-biased gene expression. Additionally, IL-4 polarization shifts the CC-phase distribution of the population towards G2/M phase, providing a mechanism for reduced IFNG-induced repolarization. Finally, we show that macrophages express tissue remodeling genes in the S-G2/M-phases of CC, that can be also detected in vivo during muscle regeneration. Therefore, macrophage inflammatory and regenerative responses are gated by CC in a cyclical phase-dependent manner.

HighlightsO_LISingle-cell chromatin maps reveal heterogeneous macrophage polarization states
C_LIO_LICell cycle coincides with heterogeneity and alters macrophage plasticity to polarizing cytokines
C_LIO_LIMacrophage polarization is a cell cycle phase-dependent immunological process
C_LIO_LIS-G2/M-biased gene expression is linked to tissue remodeling and detected in proliferating macrophages during muscle regeneration
C_LI

category: immunology


title: Prophages in the infant gut are largely induced, and may be functionally relevant to their hosts.

abstract:
BackgroundBacteriophages are the most abundant biological entity on the planet, and are key components of any ecosystem they are present in. The gut virome is increasingly being implicated in disease states although these studies largely focus on lytic phages in adults. Here we identify prophages from a large infant cohort and investigate their potential functions.

ResultsWe identified 10645 vOTUs from 662 metagenomes. No core virome was found: the most prevalent vOTU was identified in 70% of the samples. The most abundant and prevalent group of phages are a novel group closely related to Bacteroides phage Hanky p00. Functional annotation of this group revealed the presence of genes in the dDTP-L-rhamnose pathway, possibly involved in the production of capsular polysaccharides. We also found an abundance of diversity generating retroelements in the phages. Additionally, paired virome data allowed us to show that the majority of prophages are induced in at least one sample and that this is not affected by the use of antibiotics in the 4 weeks prior to sampling.

ConclusionsProphages in the infant gut are largely unique to the individual and not shared. Most of them appear to be induced and so may be key drivers in shaping the bacterial microbiome. The most abundant group of phages are novel, and possess elements that may allow them to maintain differentially susceptible subpopulations of their host bacterium; whilst also containing diversity generating retroelements that could expand their host range. Prophages are important components of the infant gut that may have far reaching influences on the composition and function of the microbiome.

category: microbiology


title: Analysis of rhizosphere bacterial diversity of Angelica dahurica var. formosana based on different experimental sites and varieties (strains)

abstract:
Only with good producing areas and good germplasm can good medicinal materials be produced. In this study, in order to explore the effects of soil environment and germplasm on yield and quality of Angelica dahurica var. formosana from microorganism level, Illumina MiSeq was used to study the rhizosphere bacterial diversity of A. dahurica var. formosana based on different origin and varieties (strains). The results showed that the bacterial community of A. dahurica var. formosana was stable and conserved to a certain extent, and the bacteria of Proteobacteria and Firmicutes might play an important role in improving the yield and quality of A. dahurica var. formosana. Soil variables were the key factors affecting the rhizosphere bacterial community composition of A. dahurica var. formosana. These results are of great significance for further understanding the growth promotion mechanism of beneficial rhizosphere bacteria, reducing fertilizer application, expanding planting area, improving soil environment, and improving yield and quality.

ImportanceRhizosphere microorganisms play an important role in plant growth and accumulation of secondary metabolites. In this study, it was found for the first time that the rhizosphere bacterial community of A. dahurica var. formosana was relatively stable and conserved which could adapt to different environmental changes and resist the interference of external factors to a certain extent, providing a theoretical basis for distant planting and promotion of planting. The bacterial taxa of Proteobacteria and Firmicutes might play an important role in improving the yield and quality of A. dahurica var. formosana. This provided a theoretical basis for the exploitation and utilization of rhizosphere microbial resources of A. dahurica var. formosana to artificially control the soil environment and provided a new idea for increasing yield and quality of A. dahurica var. formosana.


title: Comparative analysis of bile-salt degradation in Sphingobium sp. strain Chol11 and P. stutzeri Chol1 reveals functional diversity of β-proteobacterial steroid degradation enzymes and suggests a novel reaction sequence for side-chain degradation involving a hydroxylation step

abstract:
The reaction sequence for aerobic degradation of bile salts by environmental bacteria resembles degradation of other steroid compounds. Recent findings show that bacteria belonging to the Sphingomonadaceae use a pathway variant for bile-salt degradation. This study addresses this so-called {Delta}4,6 -variant by comparative analysis of unknown degradation steps in Sphingobium sp. strain Chol11 with known reactions found in Pseudomonas stutzeri Chol1. Investigations with strain Chol11 revealed an essential function of the acyl-CoA dehydrogenase Scd4AB for growth with bile salts. Growth of the scd4AB deletion mutant was restored with a metabolite containing a double bond within the side chain which was produced by the {Delta}22-acyl-CoA dehydrogenase Scd1AB from P. stutzeri Chol1. Expression of scd1AB in the scd4AB deletion mutant fully restored growth with bile salts while expression of scd4AB only enabled constricted growth in P. stutzeri Chol1 scd1A or scd1B deletion mutants. Strain Chol11 {Delta}scd4A accumulated hydroxylated steroid metabolites which were degraded and activated with coenzyme A by the wild type. Activities of five Rieske type monooxygenases of strain Chol11 were screened by heterologous expression and compared to the B-ring cleaving KshABChol1 from P. stutzeri Chol1. Three of the Chol11 enzymes catalyzed B-ring cleavage of only {Delta}4,6 -steroids while KshABChol1 was more versatile. Expression of a fourth KshA homolog, Nov2c228 led to production of metabolites with hydroxylations at an unknown position. These results indicate functional diversity of {beta}-proteobacterial enzymes for bile-salt degradation and suggest a novel side-chain degradation pathway involving an essential ACAD reaction and a steroid hydroxylation step.

ImportanceThis study highlights the biochemical diversity of bacterial degradation of steroid compounds in different aspects. First, it further elucidates an unexplored variant in the degradation of bile-salt side chains by Sphingomonads, a group of environmental bacteria that is well-known for their broad metabolic capabilities. Moreover, it adds a so-far unknown hydroxylation of steroids to the reactions Rieske monooxygenases can catalyze with steroids. Additionally, it analyzes the first proteobacterial ketosteroid-9-hydroxylase and shows that this enzyme is able to catalyze side reactions with non-native substrates.


title: Essential role of translation factor eIF5a in cytokine production and cell cycle regulation in primary CD8 T lymphocytes

abstract: Translational control adjusts protein production rapidly and facilitates local cellular responses to environmental conditions. Translation can be regulated through sequestration of mRNAs by regulatory proteins or RNAs, but also by the availability of ribosomes and translation factors which enable initiation and elongation of nascent polypeptides. Traditionally initiation of mRNA translation has been considered to be a major translational control point, however, control of peptide elongation can also play a role. Here we show that post-translational modification of the elongation factor, eIF5a, controls translation of subsets of proteins in naive T-cells upon activation. Sequencing of nascent polypeptides indicated that functional eIF5a was required for the production of proteins which regulate T-cell proliferation and effector function. Control of translation in multiple immune cell lineages is required to co-ordinate immune responses and these data illustrate that translational elongation can contribute to post-transcriptional regulons important for the control of inflammation.

category: molecular biology


title: Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number

abstract: Centrosomes act as the main microtubule organizing centre (MTOC) in metazoans. Centrosome number is tightly regulated by limiting centriole duplication to a single round per cell cycle. This control is achieved by multiple mechanisms, including the regulation of the protein kinase PLK4, the most upstream facilitator of centriole duplication. Altered centrosome numbers in mouse and human cells cause p53-dependent growth arrest through poorly defined mechanisms. Recent work has shown that the E3 ligase TRIM37 is required for cell cycle arrest in acentrosomal cells. To gain further insights into this process, we used a series of genome-wide CRISPR/Cas9 screens to identify factors important for growth arrest by treating multiple cell lines with centrinone B, a selective PLK4 inhibitor. We found that TRIM37 is a key mediator of growth arrest after partial or full PLK4 inhibition. Interestingly, PLK4 cellular mobility decreased in a dose-dependent manner after centrinone B treatment. In contrast to recent models, we found that growth arrest after PLK4 inhibition correlated with PLK4 activity, rather than with altered mitotic length or centrosome number. These data provide insights into the global response to changes in centrosome number and PLK4 activity and extend the role for TRIM37 in regulating the abundance, localization and function of centrosome proteins.

category: cell biology


title: Contractile ring constriction and septation in fission yeast are integrated mutually stabilizing processes

abstract: In common with other cellular machineries, the actomyosin contractile ring that divides cells during cytokinesis does not operate in isolation. Contractile rings in animal cells interact with contiguous actomyosin cortex, while ring constriction in many cell-walled organisms couples tightly to cell wall growth. In fission yeast, a septum grows in the wake of the constricting ring, ensuring cytokinesis leaves two daughter cells fully enclosed by cell wall. Here we mathematical modeled the integrated constriction-septation system in fission yeast, with a kinetic growth model evolving the 3D septum shape coupled to a molecularly explicit simulation of the contractile ring highly constrained by experimental data. Simulations revealed influences in both directions, stabilizing the ring-septum system as a whole. By providing a smooth circular anchoring surface for the ring, the inner septum leading edge stabilized ring organization and tension production; by mechanically regulating septum circularity and in-plane growth, ring tension stabilized septum growth and shape. Genetic or pharmacological perturbation of either subsystem destabilized this delicate balance, precipitating uncontrolled positive feedback with disastrous morphological and functional consequences. Thus, high curvature septum irregularities triggered bridging instabilities, in which contractile ring segments became unanchored. Bridging abolished the local tension-mediated septum shape regulation, exacerbating the irregularity in a mutually destabilizing runaway process. Our model explains a number of previously mysterious experimental observations, including unanchoring of ring segments observed in cells with mutations in the septum-growing {beta}-glucan synthases, and irregular septa in cells with mutations in the contractile ring myosin-II Myo2. Thus, the contractile ring and cell wall growth cellular machineries operate as a single integrated system, whose stability relies on mutual regulation by the two subsystems.


title: Characterisation of the transcriptional dynamics underpinning the function, fate, and migration of the mouse Anterior Visceral Endoderm

abstract: During early post-implantation development of the mouse embryo, the Anterior Visceral Endoderm (AVE) differs from surrounding visceral endoderm (VE) in its migratory behaviour and ability to restrict primitive streak formation to the opposite side of the egg cylinder. In order to characterise the molecular basis for the unique properties of the AVE, we combined single-cell RNA-sequencing of the VE prior to and during AVE migration, with high-resolution imaging, short-term lineage labelling, phosphoproteomics and pharmacological intervention. This revealed the transient nature of the AVE, the emergence of heterogeneities in AVE transcriptional states relative to position of cells, and its prominence in establishing gene expression asymmetries within the spatial constraints of the embryo. We identified a previously unknown requirement of Ephrin- and Semaphorin-signalling for AVE migration. These findings point to a tight coupling of transcriptional state and position in the AVE and reveal molecular heterogeneities underpinning its migratory behaviour and function.

category: developmental biology


title: Nde1 is Required for Heterochromatin Compaction and Stability in Neocortical Neurons

abstract: The NDE1 gene encodes a scaffold protein essential for brain development. While biallelic NDE1 loss of function (LOF) causes microcephaly with profound mental retardation, NDE1 missense mutations and copy number variations are associated with multiple neuropsychiatric disorders. However, the etiology of the diverse phenotypes resulting from NDE1 aberrations remains elusive. Here we show Nde1 controls neurogenesis through facilitating heterochromatin compaction via histone H4K20 trimethylation. This mechanism patterns diverse chromatin landscapes and stabilizes constitutive heterochromatin of neocortical neurons. We show NDE1 undergoes dynamic liquid-liquid phase separation, partitioning to the nucleus and interacting with pericentromeric and centromeric satellite repeats. Nde1 LOF results in nuclear architecture aberrations and DNA double strand breaks, as well as instability and derepression of pericentromeric satellite repeats in neocortical neurons. These findings uncover a pivotal role of NDE1/Nde1 in establishing and maintaining neuronal heterochromatin. They suggest that heterochromatin impairments underlie a wide range of brain dysfunction.

category: genetics


title: Precancer: Mutant clones in normal epithelium outcompete and eliminate esophageal micro-tumors

abstract: Human epithelial tissues accumulate cancer-driver mutations with age1-7, yet tumor formation remains rare. The positive selection of these mutations argues they alter the behavior and fitness of proliferating cells8-10. Hence, normal adult tissues become a patchwork of mutant clones competing for space and survival, with the fittest clones expanding by eliminating their less-competitive neighbors9-12. However, little is known about how such dynamic competition in normal epithelia impacts early tumorigenesis. Here we show that the majority of newly formed esophageal tumors are eliminated through competition with mutant clones in the surrounding normal epithelium. We followed the fate of microscopic tumors in a mouse model of esophageal carcinogenesis. Most neoplasms are rapidly lost despite no indication of tumor cell death, decreased proliferation, or an anti-tumor immune response. Deep-sequencing of 10-day and 1-year-old tumors shows evidence of genetic selection on the surviving neoplasms. Induction of highly competitive clones in transgenic mice increased tumor removal, while pharmacologically inhibiting clonal competition reduced tumor loss. The results are consistent with a model where survival of early neoplasms depends on their competitive fitness relative to that of mutant clones in the adjacent normal tissue. We have identified an unexpected anti-tumorigenic role for mutant clones in normal epithelium by purging early neoplasms through cell competition, thereby preserving tissue integrity.

category: cancer biology


title: Discovery and visualization of uncharacterized drug-protein adducts using mass spectrometry.

abstract: Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause life threatening adverse drug reactions. The masses of reactive metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics incapable of adduct identification. Here, we present Magnum, an open-mass search algorithm optimized for adduct identification, and Limelight, a web-based data processing package for analysis and visualization of data from all existing algorithms. Limelight incorporates tools for sample comparisons and xenobiotic-adduct discovery. We validate our tools with two drug/protein combinations and apply our workflow to identify novel xenobiotic-protein adducts in CYP3A4. Our new methods and software enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open-mass searching, which until now lacked comprehensive data visualization tools.

category: pharmacology and toxicology


title: Disrupted social memory ensembles in the ventral hippocampus underlie social amnesia in autism-associated Shank3 mutant mice

abstract: The ability to remember conspecifics is critical for adaptive cognitive functioning and social communication, and impairments of this ability are hallmarks of autism spectrum disorders (ASDs). Although hippocampal ventral CA1 (vCA1) neurons are known to store social memories, how their activities are coordinated remains unclear. Here we show that vCA1 social memory neurons, characterized by enhanced activity in response to memorized individuals, were preferentially reactivated during sharp-wave ripples (SPW-Rs). Spike sequences of these social replays reflected the temporal orders of neuronal activities within theta cycles during social experiences. In ASD model Shank3 knockout mice, the proportion of social memory neurons was reduced, and neuronal ensemble spike sequences during SPW-Rs were disrupted, which correlated with impaired discriminatory social behavior. These results suggest that SPW-R-mediated sequential reactivation of neuronal ensembles is a canonical mechanism for coordinating hippocampus-dependent social memories and its disruption underlies the pathophysiology of social memory defects associated with ASD.


title: Swarm-mediated phage transport disrupts a biofilm inherently protected from phage penetration

abstract: The treatment of chronic bacterial infections by phages has shown promise in combating antimicrobial resistance. A typical phage particle is at least an order of magnitude larger than an antibiotic molecule. Hence, phages diffuse slower than antibiotics, and can also get trapped in the polymeric mesh of biofilm matrix. By tracking fluorescently labeled lambda phages that do not infect Capnocytophaga gingivalis, a bacterium abundant in the human oral microbiota, we demonstrate active phage transportation by a C. gingivalis swarm. As a result, the rate of disruption of the prey of lambda phage i.e., an Escherichia coli colony, increases 10 times. C. gingivalis drills tunnels within a curli fiber containing E. coli biofilm and increase the efficiency of phage penetration. This provides evidence for a novel mechanism of phage-bacterial warfare.


title: Bacteria swarm-mediated phage transportation disrupts a biofilm inherently protected from phage penetration


title: Bacterial swarm-mediated phage transportation disrupts a biofilm inherently protected from phage penetration


title: Identification of antigenic epitopes on the F and G glycoproteins of bovine respiratory syncytial virus and in vitro assessment of their synthetic peptide vaccine potential

abstract: Globally, bovine respiratory disease (BRD) remains the principal reason for mortality of calves over one month of age despite the availability of various vaccines on the UK market. Bovine respiratory syncytial virus (BRSV) was first discovered in the 1970s and is now considered a principal pathogen implicated in the disease complex. Outbreaks occur annually and re-infections are common even in the presence of maternal antibodies. Difficulties have arisen from using both live-attenuated and inactivated vaccines and recent efforts have focused on the development of sub-unit vaccines that are suitable for use in neonatal calves with maternally-derived circulating antibodies. This study was undertaken to identify antigenic epitopes on two of the surface glycoproteins of BRSV, the fusion (F) and attachment (G) proteins, the major surface viral antigens, for inclusion into a novel subunit peptide vaccine. Sequencing and antigenicity prediction of the F and G genes of BRSV revealed 21 areas of potential antigenicity; of which genuine peptide/antisera binding occurred with 4 peptides. Identification of the antigenic components of a vaccine is an important first step in the development of novel BRSV vaccines and this data, therefore, provides the basis for the generation of such vaccines.


title: Phylogenetic network analysis revealed the recombinant origin of the SARS-CoV-2 VOC202012/01 (B.1.1.7) variant first discovered in U.K.

abstract:
The emergence of new variants of the SARS-CoV-2 virus poses serious problems to the control of the current COVID-19 pandemic. Understanding how the variants originate is critical for effective control of the spread of the virus and the global pandemic. The study of the virus evolution so far has been dominated by phylogenetic tree analysis, which however is inappropriate for a few important reasons. Here we used phylogenetic network approach to study the origin of the VOC202012/01 (Alpha) or so-called UK variant (PANGO Lineage B.1.1.7). The multiple network analyses using different methods consistently revealed that the VOC202012/01 variant was a result of recombination, in contrast to the common assumption that the variant evolved from step-wise mutations in a linear order. The study provides an example for the power and application of phylogenetic network analysis in studying virus evolution, which can be applied to study the evolutionary processes leading to the emergence of other variants of the SARS-CoV-2 virus as well as many other viruses.

SignificanceThe emergence of new variants of the SARS-CoV-2 virus, including the Alpha variant first found in U.K., poses serious challenges to the control of the current COVID-19 pandemic. Understanding how new variant originated is paramount to end the pandemic as effectively and quickly as possible. The dominant phylogenetic tree approach to study virus evolution has been inadequate and even misleading. Here we used a phylogenetic network approach to study the origin of the VOC202012/01 (Alpha) variant which was first reported in U.K. last year but has soon spread into many other countries, leading to dramatic increase in infection and death. Multiple analyses consistently revealed that the variant originated through recombination of pre-existing virus strains, highlighting an important but largely ignored mechanism in the evolution of the SARS-CoV-2 virus so far.


title: Diversity of Treponema denticola and other oral treponeme lineages in subjects with periodontitis and gingivitis

abstract: More than 75 species/species-level phylotypes of taxa belonging to the genus Treponema inhabit the human oral cavity. Treponema denticola is commonly associated with periodontal disease, but the etiological roles and ecological distributions of other oral treponemes remain more obscure. Here, we compared the clinical distributions of phylogroup 1 and 2 oral treponemes in subgingival plaque sampled from Chinese subjects with periodontitis (n=10) and gingivitis (n=8), via the sequence analysis of the highly-conserved pyrH  housekeeping gene. Two PCR primer sets that respectively targeted oral phylogroup 1 and 2 treponeme pyrH genes were used to construct plasmid clone-amplicon libraries for each subject, which were sequenced for bioinformatic analysis. 1,204 quality-filtered, full-length pyrH gene sequences were obtained from the cohort (median: 61.5; range: 59-83 of cloned pyrH sequences per subject), which were assigned to 34  pyrH genotypes (pyrH001-pyrH034; 97% sequence-identity cut-off). 18 pyrH genotypes (536 pyrH sequences) corresponded to phylogroup 1 treponeme taxa (including Treponema vincentii, Treponema medium). 16 pyrH genotypes (668 pyrH sequences) corresponded to T. denticola and other phylogroup 2 treponemes. Periodontitis subjects contained a greater diversity of phylogroup 2 pyrH genotypes, compared to gingivitis subjects (Mann-Whitney U-test). One T. denticola pyrH genotype (pyrH001) was highly prevalent: detected in 10/10 periodontitis and 6/8 gingivitis subjects. Several subjects harboured multiple T. denticola pyrH genotypes. Non-metric multidimensional scaling and PERMANOVA tests revealed no significant differences in overall pyrH genotype compositions between periodontitis and gingivitis subjects. In conclusion, our results clearly show that subjects with periodontitis and gingivitis commonly harbour highly-diverse oral treponeme communities.


title: A random priming amplification method for whole genome sequencing of SARS-CoV-2 and H1N1 influenza A virus.

abstract:
BackgroundNon-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions.

MethodsThe performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2-4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing.

ResultsThe SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of COVID that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection.

ConclusionsThe SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.


title: Cryo-EM structure of the transcription termination factor Rho from Mycobacterium tuberculosis reveals mechanism of resistance to bicyclomycin

abstract: The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis where rho gene inactivation leads to rapid death. Yet, the M. tuberculosis Rho [MtbRho] factor displays poor NTPase and helicase activities, and resistance to the natural Rho inhibitor bicyclomycin [BCM] that remain unexplained. Here, we address these unusual features by solving the cryo-EM structure of MtbRho at 3.3 [A] resolution, providing a new framework for future antibiotic development. The MtbRho hexamer is poised into a pre-catalytic, open-ringed state wherein specific contacts stabilize ATP in intersubunit ATPase pockets, thereby explaining the cofactor preference of MtbRho. We reveal a leucine-to-methionine substitution that creates a steric bulk in BCM binding cavities near the positions of ATP {gamma}-phosphates, and confers resistance to BCM at the expense of motor efficiency.


title: The developmental transcriptome of Parhyale hawaiensis: microRNAs and mRNAs show different expression dynamics during the maternal-zygotic transition

abstract: Parhyale hawaiensis has emerged as the crustacean model of choice due to its tractability, ease of imaging, sequenced genome, and development of CRISPR/Cas9 genome editing tools. However, transcriptomic datasets spanning embryonic development are lacking, and there is almost no annotation of non-protein-coding RNAs, including microRNAs. We have sequenced microRNAs, together with mRNAs and long non-coding RNAs, in Parhyale using paired size-selected RNA-seq libraries at seven time-points covering important transitions in embryonic development. Focussing on microRNAs, we annotate 175 loci in Parhyale, 85 of which have no known homologs. We use these data to annotate the microRNome of 37 crustacean genomes, and suggest a core crustacean microRNA set of around 61 sequence families. We examine the dynamic expression of microRNAs and mRNAs during the maternal-zygotic transition. Our data suggest that zygotic genome activation occurs in two waves in Parhyale with microRNAs transcribed almost exclusively in the second wave. Contrary to findings in other arthropods, we do not predict a general role for microRNAs in clearing maternal transcripts. These data significantly expand the available transcriptomics resources for Parhyale, and facilitate its use as a model organism for the study of small RNAs in processes ranging from embryonic development to regeneration.


title: Population structure and introgression among recently differentiated Drosophila melanogaster populations

abstract: Despite a century of genetic analysis, the evolutionary history underlying patterns of exceptional genetic and phenotypic variation in the model organism Drosophila melanogaster remains poorly understood. How genetic and phenotypic variation is partitioned across the range of D. melanogaster, particularly in its putative ancestral range in Subtropical Africa, remains unresolved. Here, we assess patterns of population genetic structure, admixture, mate preference, and genetic incompatibility across a global sample, including 174 new accessions from remote regions within Subtropical Africa. While almost all Out of Africa genomes correspond to a single genetic ancestry, different geographic regions within Africa contain multiple ancestries, with substantial cryptic diversity in Subtropical Africa. Admixture between distinct lineages is prevalent across the range, but admixture rates vary between lineages. Female mate choice within Subtropical Africa is highly polymorphic and behavioral types are not monophyletic. The genetic architecture of mate choice is highly polygenic, including loci associated with neurological development, behavior, olfactory perception, and learning. Finally, we discovered that many segregating putative incompatibilities likely evolved during or after expansion out of Africa. This work contributes to our understanding of the evolutionary history of a key model system, and provides insight into the distribution of polymorphic reproductive barriers.

category: evolutionary biology


title: Patterns of and processes shaping population structure and introgression among recently differentiated Drosophila melanogaster populations


title: Engineered chimeric T cell receptor fusion construct (TRuC)-expressing T cells prevent translational shutdown in SARS-CoV-2-infected cells

abstract: SARS-CoV-2, the causative agent of Covid-19, is known to evade the immune system by several mechanisms. This includes the shutdown of the host cellular protein synthesis, which abrogates the induction of antiviral interferon responses. The virus initiates the infection of susceptible cells by binding with its spike protein (S) to the host angiotensin-converting enzyme 2 (ACE2). Here we applied the T cell receptor fusion construct (TRuC) technology to engineer T cells against such infected cells. In our TRuCs an S-binding domain is fused to the CD3{varepsilon} component of the T cell receptor (TCR) complex, enabling recognition of S-containing cells in an HLA independent manner. This domain either consists of the S-binding part of ACE2 or a single-chain variable fragment of an anti-S antibody. We show that the TRuC T cells are activated by and kill cells that express S of SARS-CoV-2 and its alpha (B.1.1.7) and beta (B.1.351) variants at the cell surface. Treatment of SARS-CoV-2 infected cells with our engineered T cells did not lead to massive cytotoxicity towards the infected cells, but resulted in a complete rescue of the translational shutdown despite ongoing viral replication. Our data show that engineered TRuC T cell products might be used against SARS-CoV-2 by exposing infected cells to the host innate immune system.

category: bioengineering


title: SARS-CoV-2 Variants are Selecting for Spike Protein Mutations that Increase Protein Stability

abstract:
The emergence of SARS-CoV-2 in 2019 has caused severe disruption and a huge number of human deaths across the globe. As the pandemic spreads, a natural result is the emergence of variants with a variety of amino acid mutations. Variants of SARS-CoV-2 with mutations in their spike protein may result in an increased infectivity, increased lethality, or immune escape, and whilst many of these properties can be explained through changes to binding affinity or changes to post-translational modification, many mutations have no known biophysical impact on the structure of protein. The Gibbs free energy of a protein represents a measure of protein stability, with an increased stability resulting in a protein that is more thermodynamically stable, and more robust to changes in external environment.

Here we show that mutations in the spike proteins of SARS-CoV-2 are selecting for amino acid changes that result in a more stable protein than expected by chance. We calculate all possible mutations in the SARS-CoV-2 spike protein, and show that many variants are more stable than expected when compared to the background, indicating that protein stability is an important consideration for the understanding of SARS-CoV-2 evolution. Variants exhibit a range of stabilities, and we further suggest that some stabilising mutations may be acting as a "counterbalance" to destabilising mutations that have other properties, such as increasing binding site affinity for the human ACE2 receptor. We suggest that protein folding calculations offer a useful tool for early identification of advantageous mutations.

category: biophysics


title: Persistence of SARS CoV-2 S1 Protein in CD16+ Monocytes in Post-Acute Sequelae of COVID-19 (PASC) Up to 15 Months Post-Infection

abstract: The recent COVID-19 pandemic is a treatment challenge in the acute infection stage but the recognition of chronic COVID-19 symptoms termed post-acute sequelae SARS-CoV-2 infection (PASC) may affect up to 30% of all infected individuals. The underlying mechanism and source of this distinct immunologic condition three months or more after initial infection remains elusive. Here, we investigated the presence of SARS-CoV-2 S1 protein in 46 individuals. We analyzed T-cell, B-cell, and monocytic subsets in both severe COVID-19 patients and in patients with post-acute sequelae of COVID-19 (PASC). The levels of both intermediate (CD14+, CD16+) and non-classical monocyte (CD14Lo, CD16+) were significantly elevated in PASC patients up to 15 months post-acute infection compared to healthy controls (P=0.002 and P=0.01, respectively). A statistically significant number of non-classical monocytes contained SARS-CoV-2 S1 protein in both severe (P=0.004) and PASC patients (P=0.02) out to 15 months post-infection. Non-classical monocytes were sorted from PASC patients using flow cytometric sorting and the SARS-CoV-2 S1 protein was confirmed by mass spectrometry. Cells from 4 out of 11 severe COVID-19 patients and 1 out of 26 PASC patients contained ddPCR+ peripheral blood mononuclear cells, however, only fragmented SARS-CoV-2 RNA was found in PASC patients. No full length sequences were identified, and no sequences that could account for the observed S1 protein were identified in any patient. Non-classical monocytes are capable of causing inflammation throughout the body in response to fractalkine/CX3CL1 and RANTES/CCR5.


title: Drug repurposing based on a Quantum-Inspired method versus classical fingerprinting uncovers potential antivirals against SARS-CoV-2 including vitamin B12

abstract: The COVID-19 pandemic has accelerated the need to identify new therapeutics at pace, including through drug repurposing. We employed a Quadratic Unbounded Binary Optimization (QUBO) model, to search for compounds similar to Remdesivir (RDV), the only antiviral against SARS-CoV-2 currently approved for human use, using a quantum-inspired device. We modelled RDV and compounds present in the DrugBank database as graphs, established the optimal parameters in our algorithm and resolved the Maximum Weighted Independent Set problem within the conflict graph generated. We also employed a traditional Tanimoto fingerprint model. The two methods yielded different lists of compounds, with some overlap. While GS-6620 was the top compound predicted by both models, the QUBO model predicted BMS-986094 as second best. The Tanimoto model predicted different forms of cobalamin, also known as vitamin B12. We then determined the half maximal inhibitory concentration (IC50) values in cell culture models of SARS-CoV-2 infection and assessed cytotoxicity. Lastly, we demonstrated efficacy against several variants including SARS-CoV-2 Strain England 2 (England 02/2020/407073), B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). Our data reveal that BMS-986094 and different forms of vitamin B12 are effective at inhibiting replication of all these variants of SARS-CoV-2. While BMS-986094 can cause secondary effects in humans as established by phase II trials, these findings suggest that vitamin B12 deserves consideration as a SARS-CoV-2 antiviral, particularly given its extended use and lack of toxicity in humans, and its availability and affordability. Our screening method can be employed in future searches for novel pharmacologic inhibitors, thus providing an approach for accelerating drug deployment.


title: Multiplexed single-molecule analysis of human telomerase synthesizing DNA

abstract: Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we developed a real-time single-molecule RNA sequencing approach that visualizes telomerase catalysis and structural dynamics at single-nucleotide resolution using FRET and zero-mode waveguides. The method permits direct detection of dynamic steps and structural states throughout the telomerase catalytic cycle and can be generalized to other nucleic acid polymerase systems.


title: ROCK1 regulates insulin secretion from β-cells

abstract:
ObjectiveThe endocrine pancreatic {beta}-cells play a pivotal role in the maintenance of whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate {beta}-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homoeostasis.

MethodsMice lacking ROCK1 in pancreatic {beta}-cells (RIP-Cre; ROCK1loxP/loxP, {beta}-ROCK1-/-) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. Insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from {beta}-ROCK1-/- mice or {beta}-cell lines with knockdown of ROCK1 were also evaluated. Proximity ligation assay was performed to determine the physical interactions between PK and ROCK1.

ResultsMice with a deficiency of ROCK1 in pancreatic {beta}-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, {beta}-ROCK1-/- mice displayed progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS was markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium levels, ATP levels, and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown {beta}-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that ROCK1 binding to PK is greatly enhanced by glucose stimulation in {beta}-cells.

ConclusionsOur findings demonstrate that {beta}-cell ROCK1 is essential for glucose-stimulated insulin secretion and maintenance of glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling.

category: physiology


title: Use of hiPSC-derived cardiomyocytes to rule out proarrhythmic effects of drugs: the case of hydroxychloroquine in COVID-19

abstract:
In the early phases of the COVID-19 pandemic, drug repurposing was widely used to identify compounds that could improve the prognosis of symptomatic patients infected by SARS-CoV-2. Hydroxychloroquine (HCQ) was one of the first drugs used to treat COVID-19 patients due to its supposed capacity of inhibiting SARS-CoV-2 infection and replication in vitro.

While its efficacy is debated, HCQ has been associated with QT interval prolongation and potentially Torsades de Pointes, especially in patients predisposed to developing drug-induced Long QT Syndrome (LQTS) as silent carriers of variants associated with congenital LQTS. If confirmed, these effects represent a limitation to the at-home use of HCQ for COVID-19 infection as adequate ECG monitoring may be challenging.

We investigated the proarrhythmic profile of HCQ with Multi-Electrode Arrays after subchronic exposure of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from two healthy donors, one asymptomatic and two symptomatic LQTS patients.

We demonstrate that: I) HCQ induced a concentration-dependent Field Potential Duration (FPD) prolongation in vitro and triggered arrhythmias that halted the beating at high concentration. II) hiPSC-CMs from healthy or asymptomatic carriers tolerated higher concentrations of HCQ and showed lower susceptibility to HCQ-induced electrical abnormalities regardless of baseline FPD values. These findings agree with the clinical safety records of HCQ and demonstrated that hiPSC-CMs potentially discriminates symptomatic vs asymptomatic mutation carriers through pharmacological interventions. Disease-specific cohorts of hiPSC-CMs may be a valid preliminary addition to quickly assess drug safety in vulnerable populations, offering rapid preclinical results with valuable translational relevance for precision medicine.


title: Functional diversification gave rise to allelic specialization in a rice NLR immune receptor pair

abstract: Cooperation between receptors from the NLR superfamily is important for intracellular activation of immune responses. NLRs can function in pairs that, upon pathogen recognition, trigger hypersensitive cell death and stop pathogen invasion. Natural selection drives specialization of host immune receptors towards an optimal response, whilst keeping a tight regulation of immunity in the absence of pathogens. However, the molecular basis of co-adaptation and specialization between paired NLRs remains largely unknown. Here, we describe functional specialization in alleles of the rice NLR pair Pik that confers resistance to strains of the blast fungus Magnaporthe oryzae harbouring AVR-Pik effectors. We revealed that matching pairs of allelic Pik NLRs mount effective immune responses whereas mismatched pairs lead to autoimmune phenotypes, a hallmark of hybrid necrosis in both natural and domesticated plant populations. We further showed that allelic specialization is largely underpinned by a single amino acid polymorphism that determines preferential association between matching pairs of Pik NLRs. These results provide a framework for how functionally linked immune receptors undergo co-adaptation to provide an effective and regulated immune response against pathogens. Understanding the molecular constraints that shape paired NLR evolution has implications beyond plant immunity given that hybrid necrosis can drive reproductive isolation.

category: plant biology


title: PV-specific loss of the transcriptional coactivator PGC-1α slows down the evolution of epileptic activity in an acute ictogenic model.

abstract: The transcriptional coactivator, PGC-1 (peroxisome proliferator activated receptor gamma coactivator 1), plays a key role coordinating energy requirement within cells. Its importance is reflected in the growing number of psychiatric and neurological conditions that have been associated with reduced PGC-1 levels. In cortical networks, PGC-1 is required for the induction of parvalbumin (PV) expression in interneurons, and PGC-1 deficiency affects synchronous GABAergic release. It is unknown, however, how this affects cortical excitability. We show here that knocking down PGC-1 specifically in the PV-expressing cells (PGC-1PV-/-), blocks the activity-dependent regulation of the synaptic proteins, SYT2 and CPLX1. More surprisingly, this cell-class specific knock-out of PGC-1 appears to have a novel anti-epileptic effect, as assayed in brain slices bathed in 0 Mg2+ media. The rate of pre-ictal discharges developed approximately equivalently in wild-type and PGC-1PV-/- brain slices, but the intensity of these discharges was lower in PGC-1PV-/- slices, as evident from the reduced power in the gamma range and reduced firing rates in both PV interneurons and pyramidal cells during these discharges. Reflecting this reduced intensity in the pre-ictal discharges, the PGC-1PV-/- brain slices experienced many more discharges before transitioning into a seizure-like event. Consequently, there was a large increase in the latency to the first seizure-like event in brain slices lacking PGC-1 in PV interneurons. We conclude that knocking down PGC-1 limits the range of PV interneuron firing, and this slows the pathophysiological escalation during ictogenesis.


title: Developmental onset of a cerebellar-dependent forward model of movement in motor thalamus

abstract: To execute complex behavior with temporal precision, adult animals use internal models to predict the sensory consequences of self-generated movement. Here, taking advantage of the unique kinematic features of twitches--the brief, discrete movements of active sleep--we captured the developmental onset of a cerebellar-dependent internal model. Using rats at postnatal days (P) 12, P16, and P20, we compared neural activity in two thalamic structures: the ventral posterior (VP) and ventral lateral (VL) nuclei, both of which receive somatosensory input but only the latter of which receives cerebellar input. At all ages, twitch-related activity in VP lagged behind movement, consistent with sensory processing; similar activity was observed in VL through P16. At P20, however, VL activity precisely mimicked the twitch itself, a pattern of activity that depended on cerebellar input. Altogether, these findings implicate twitches in the development and refinement of internal models of movement.


title: Use of untargeted magnetic beads to capture Mycobacterium smegmatis and Mycobacterium avium paratuberculosis prior detection by mycobacteriophage D29 and Real-Time-PCR

abstract: Untargeted magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Untargeted magnetic beads recovered more M. smegmatis cells from PBS suspension than the typical centrifugation method; 96.31% of 1.68 x 104 CFU/mL viable M. smegmatis were recovered by beads and 0% by centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p<0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of higher sensitivity. Magnetic separation of MAP cells from milk, urine, and feces specimens were detected by F57 and IS900 sequences. Ct values demonstrated that beads captured no less than 109 CFU/mL from feces and no less than 104 CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 x 105 and 2 x 103 CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of Johnes disease and other mycobacterial diseases such as tuberculosis.


title: Efficient tagging and purification of endogenous proteins for structural studies by single particle cryo-EM

abstract: A major bottleneck in structural biology is producing biologically relevant samples at sufficient quantities. This is particularly true for large protein assemblies where conventional techniques of gene overexpression require substantial optimization, hampering structural studies and drug development efforts. Here we describe a method combining CRISPR/Cas gene editing and fluorescence cell sorting to rapidly tag and purify endogenous human proteins from cell lines, enabling structural analysis of native proteins that are properly folded and assembled. We applied this approach to study the human proteasome from HEK cells and rapidly determined structures of major proteasomal complexes. Structures of the PA28-20S complex reveal the native subunit stoichiometry of PA28 and a distinct functional state of the complex. The efficient strategy for tagging and extracting endogenous proteins described here will enable the structural study of many challenging targets and provide more biologically relevant samples for research and therapeutic development.


title: Xrn1 is a deNADding Enzyme Modulating Mitochondrial NAD Levels

abstract: The existence of non-canonical nicotinamide adenine diphosphate (NAD) 5-end capped RNAs is now well established. Nevertheless, the biological function of this nucleotide metabolite cap remains elusive. Here, we show that the yeast Saccharomyces cerevisiae cytoplasmic 5-end exoribonuclease Xrn1 is also a NAD cap decapping (deNADding) enzyme that releases intact NAD and subsequently degrades the RNA. The significance of Xrn1 deNADding is evident in a deNADding deficient Xrn1 mutant that still retains its 5-monophosphate exonuclease activity. This mutant reveals Xrn1 deNADding is necessary for normal growth on non-fermenting sugar and is involved in modulating mitochondrial NAD-capped RNA levels and in turn intramitochondrial NAD levels. Our findings uncover a functional role for mitochondrial NAD-capped RNAs as a reservoir to maintain overall NAD homeostasis. We propose NAD-capped RNAs function as a cistern for mitochondrial NAD with Xrn1 serving as a rheostat for NAD-capped RNAs.


title: AP-4 regulates neuronal lysosome composition, function and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network

abstract: The adaptor protein complex-4 or AP-4 is known to mediate autophagosome maturation through regulating sorting of transmembrane cargo such as ATG9A at the Golgi. There is a need to understand AP-4 function in neurons, as mutations in any of its four subunits cause a complex form of hereditary spastic paraplegia (HSP) with intellectual disability. While AP-4 has been implicated in regulating trafficking and distribution of cargo such as ATG9A and APP, little is known about its effect on neuronal lysosomal protein traffic, lysosome biogenesis and function. In this study, we demonstrate that in human iPSC-derived neurons AP-4 regulates lysosome composition, function and transport via regulating export of critical lysosomal receptors, including Sortilin 1, from the trans-Golgi network to endo-lysosomes. Additionally, loss of AP-4 causes endo-lysosomes to stall and build up in axonal swellings potentially through reduced recruitment of retrograde transport machinery to the organelle. These findings of axonal lysosome build-up are highly reminiscent of those observed in Alzheimers disease as well as in neurons modelling the most common form of HSP, caused by spastin mutations. Our findings implicate AP-4 as a critical regulator of neuronal lysosome biogenesis and altered lysosome function and axonal endo-lysosome transport as an underlying defect in AP-4 deficient HSP.


title: RNF168-mediated localization of BARD1 recruits the BRCA1-PALB2 complex to DNA damage

abstract: DNA damage prompts a diverse range of alterations to the chromatin landscape. The RNF168 E3 ubiquitin ligase catalyzes the mono-ubiquitination of histone H2A at lysine (K)13/15 (mUb-H2A), forming a binding module for DNA repair proteins. BRCA1 promotes homologous recombination (HR), in part, through its interaction with PALB2, and the formation of a larger BRCA1-PALB2-BRCA2-RAD51 (BRCA1-P) complex. The mechanism by which BRCA1-P is recruited to chromatin surrounding DNA breaks is unclear. In this study, we reveal that an RNF168-governed signaling pathway is responsible for localizing the BRCA1-P complex to DNA damage. Using mice harboring a Brca1CC (coiled coil) mutation that blocks the Brca1-Palb2 interaction, we uncovered an epistatic relationship between Rnf168- and Brca1CC alleles, which disrupted development, and reduced the efficiency of Palb2-Rad51 localization. Mechanistically, we show that RNF168-generated mUb-H2A recruits BARD1 through a BRCT domain ubiquitin-dependent recruitment motif (BUDR). Subsequently, BARD1-BRCA1 accumulate PALB2-RAD51 at DNA breaks via the CC domain-mediated BRCA1-PALB2 interaction. Together, these findings establish a series of molecular interactions that connect the DNA damage signaling and HR repair machinery.


title: ETV2 primes hematoendothelial gene enhancers prior to hematoendothelial fate commitment

abstract:
The lineage-determining transcription factor ETV2 is necessary and sufficient for hematoendothelial fate commitment. We investigated how ETV2-driven gene regulatory networks promote hematoendothelial fate commitment. We resolved the sequential determination steps of hematoendothelial versus cardiac differentiation from mouse embryonic stem cells. Etv2 was strongly induced and bound to the enhancers of hematoendothelial genes in a common cardiomyocyte-hematoendothelial mesoderm progenitor. However, only Etv2 itself and Tal1, not other ETV2-bound genes, were induced. Despite ETV2 genomic binding and Etv2 and Tal1 expression, cardiomyogenic fate potential was maintained. A second wave of ETV2-bound target genes was up-regulated during the transition from the common cardiomyocyte-hematoendothelial progenitor to the committed hematoendothelial population. A third wave of ETV-bound genes were subsequently expressed in the committed hematoendothelial population for sub-lineage differentiation. The shift from ETV2 binding to productive transcription, not ETV2 binding to target gene enhancers, drove hematoendothelial fate commitment. This work identifies mechanistic phases of ETV2-dependent gene expression that distinguish hematoendothelial specification, commitment, and differentiation.

HIGHLIGHTSO_LIThe hematoendothelial master TF ETV2 is expressed in a multipotent mesoderm progenitor.
C_LIO_LIETV2 binds to target genes in the mesoderm progenitor without target gene activation.
C_LIO_LIETV2 binding in the mesoderm progenitor does not diminish alternative cardiac fate potential.
C_LIO_LIETV2-bound target genes are activated upon hematoendothelial fate commitment.
C_LI

GRAPHICAL ABSTRACT

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title: Intrinsic and extrinsic regulation of human fetal bone marrow haematopoiesis and perturbations in Down syndrome

abstract: Throughout postnatal life, haematopoiesis in the bone marrow (BM) maintains blood and immune cell production. Haematopoiesis first emerges in human BM at 12 post conception weeks while fetal liver (FL) haematopoiesis is still expanding. Yet, almost nothing is known about how fetal BM evolves to meet the highly specialised needs of the fetus and newborn infant. Here, we detail the development of fetal BM including stroma using single cell RNA-sequencing. We find that the full blood and immune cell repertoire is established in fetal BM in a short time window of 6-7 weeks early in the second trimester. Fetal BM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell (DC) subsets emerging for the first time. B-lymphocyte expansion occurs, in contrast with erythroid predominance in FL at the same gestational age. We identify transcriptional and functional differences that underlie tissue-specific identity and cellular diversification in fetal BM and FL. Finally, we reveal selective disruption of B-lymphocyte, erythroid and myeloid development due to cell intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in the fetal BM from constitutional chromosome anomaly Down syndrome during this crucial developmental time window.


title: IFT20 is critical for early chondrogenesis during endochondral ossification

abstract: Ciliogenic components, such as the family of intraflagellar transport (IFT) proteins, are recognized to play key roles in endochondral ossification, a critical process to form most bones. However, it remains unclear how each IFT protein performs its unique function to regulate endochondral ossification. Here, we show that intraflagellar transport 20 (IFT20) is required for early chondrogenesis. Utilizing three osteo-chondrocyte lineage-specific Cre mice (Prx1-Cre, Col2-Cre and Aggrecan-CreERT2), we deleted Ift20 to examine its function. While chondrocyte-specific Ift20 deletion with Col2-Cre or Aggrecan-CreERT2 drivers did not cause overt skeletal defects, mesoderm-specific Ift20 deletion using Prx1-Cre (Ift20:Prx1-Cre) resulted in shortened limb outgrowth. Although primary cilia were not formed in Ift20:Prx1-Cre mice, ciliary Hedgehog signaling was only moderately affected. Interestingly, loss of Ift20 lead to upregulation of Fgf18 expression resulting in ERK1/2 activation and sustained Sox9 expression, thus preventing endochondral ossification. Inhibition of enhanced phospho-ERK1/2 activation partially rescued defective chondrogenesis in Ift20 mutant cells, supporting an important role for FGF signaling. Our findings demonstrate a novel mechanism of IFT20 in early chondrogenesis during endochondral ossification.


title: Simulation study evaluating the ability of two statistical approaches to identify variance quantitative trait loci Arabidopsis and maize

abstract: Genomic loci that control the variance of agronomically important traits are increasingly important due to the profusion of unpredictable environments arising from climate change. The ability to identify such variance quantitative trait loci (vQTL) in association studies will be critical for future breeding efforts. Two statistical approaches that have already been used to detect vQTL are the Brown-Forsythe test (BFT) and the double generalized linear model (DGLM). To ensure that they are deployed to variance genome-wide association studies as effectively as possible, it is critical to study the factors that influence their ability to identify vQTL. We used genome-wide marker data in maize (Zea mays L.) and Arabidopsis thaliana to simulate traits controlled by variance quantitative trait nucleotides (vQTNs) and then quantified true and false positive detection rates of the BFT and DGLM. We observed that the DGLM yielded similar or higher true positive vQTN detection rates than the BFT, regardless of the effect size or minor allele frequency (MAF) of the vQTNs. Low true positive detection rates were noted for QTNs with low MAFs (~0.10), especially when tested on subsets of n = 500 individuals. We recommend that larger data sets than those used in our study (i.e., n > 2,532) be considered to overcome these low observed true positive detection rates. Such an undertaking should maximize the potential of the BFT and DGLM to highlight which vQTLs should be considered for further study.


title: Impact of SNP microarray analysis of compromised DNA on kinship classification success in the context of investigative genetic genealogy

abstract:
Single nucleotide polymorphism (SNP) data generated with microarray technologies have been used to solve murder cases via investigative leads obtained from identifying relatives of the unknown perpetrator included in accessible genomic databases, referred to as investigative genetic genealogy (IGG). However, SNP microarrays were developed for relatively high input DNA quantity and quality, while SNP microarray data from compromised DNA typically obtainable from crime scene stains are largely missing. By applying the Illumina Global Screening Array (GSA) to 264 DNA samples with systematically altered quantity and quality, we empirically tested the impact of SNP microarray analysis of deprecated DNA on kinship classification success, as relevant in IGG. Reference data from manufacturer-recommended input DNA quality and quantity were used to estimate genotype accuracy in the compromised DNA samples and for simulating data of different degree relatives. Although stepwise decrease of input DNA amount from 200 nanogram to 6.25 picogram led to decreased SNP call rates and increased genotyping errors, kinship classification success did not decrease down to 250 picogram for siblings and 1st cousins, 1 nanogram for 2nd cousins, while at 25 picogram and below kinship classification success was zero. Stepwise decrease of input DNA quality via increased DNA fragmentation resulted in the decrease of genotyping accuracy as well as kinship classification success, which went down to zero at the average DNA fragment size of 150 base pairs. Combining decreased DNA quantity and quality in mock casework and skeletal samples further highlighted possibilities and limitations. Overall, GSA analysis achieved maximal kinship classification success from 800-200 times lower input DNA quantities than manufacturer-recommended, although DNA quality plays a key role too, while compromised DNA produced false negative kinship classifications rather than false positive ones.

Author SummaryInvestigative genetic genealogy (IGG), i.e., identifying unknown perpetrators of crime via genomic database-tracing of their relatives by means of microarray-based single nucleotide polymorphism (SNP) data, is a recently emerging field. However, SNP microarrays were developed for much higher DNA quantity and quality than typically available from crime scenes, while SNP microarray data on quality and quantity compromised DNA are largely missing. As first attempt to investigate how SNP microarray analysis of quantity and quality compromised DNA impacts kinship classification success in the context of IGG, we performed systematic SNP microarray analyses on DNA samples below the manufacturer-recommended quantity and quality as well as on mock casework samples and on skeletal remains. In addition to IGG, our results are also relevant for any SNP microarray analysis of compromised DNA, such as for the DNA prediction of appearance and biogeographic ancestry in forensics and anthropology and for other purposes.


title: Functional divergence of the bag of marbles gene in the Drosophila melanogaster species group

abstract: In Drosophila melanogaster, a key germline stem cell (GSC) differentiation factor, bag of marbles (bam) shows rapid bursts of amino acid fixations between sibling species D. melanogaster and D. simulans, but not in the outgroup species D. ananassae. Here, we test the null hypothesis that bams differentiation function is conserved between D. melanogaster and four additional Drosophila species in the melanogaster species group spanning approximately 30 million years of divergence. Surprisingly, we demonstrate that bam is not necessary for oogenesis or spermatogenesis in D. teissieri nor is bam necessary for spermatogenesis in D. ananassae. Remarkably bam function may change on a relatively short time scale. We further report tests of neutral sequence evolution at bam in additional species of Drosophila and find a positive, but not perfect, correlation between evidence for positive selection at bam and its essential role in GSC regulation and fertility for both males and females. Further characterization of bam function in more divergent lineages will be necessary to distinguish between bams critical gametogenesis role being newly derived in D. melanogaster, D. simulans, D. yakuba, and D. ananassae females or it being basal to the genus and subsequently lost in numerous lineages.


title: Hybrid assembly using ultra-long reads resolves repeats and completes the genome sequence of a laboratory strain of Staphylococcus aureus subsp. aureus RN4220

abstract: Staphylococcus aureus RN4220 has been extensively used by staphylococcal researchers as an intermediate strain for genetic manipulation due to its ability to accept foreign DNA. Despite its wide use in laboratories, its complete genome is not available. In this study, we used the hybrid genome assembly approach using the minION long reads and Illumina short reads to sequence the complete genome of S. aureus RN4220. The comparative analysis of the annotated complete genome showed the presence of 39 genes fragmented in the previous assembly, many of which were located near the repeat regions. Using RNA-Seq reads, we showed that a higher number of reads could be mapped to the complete genome than the draft genome and the gene expression profile obtained using the complete genome also differs from that obtained from the draft genome. Furthermore, by comparative transcriptomic analysis, we showed the correlation between expression levels of staphyloxanthin biosynthetic genes and the production of yellow pigment. This study highlighted the importance of long reads in completing the microbial genomes, especially those possessing repetitive elements.

category: genomics


title: Mitogen Kinase Kinase (MKK7) controls cytokine production in vitro and in vivo in mice

abstract: Mitogen kinase kinase 4 (MKK4) and Mitogen kinase kinase 7 (MKK7) are members of the MAP2K family which can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both, c-Jun N-terminal Kinase (JNK) and p38 MAPK, whereas MKK7 only activates JNK in response to different stimuli. The stimuli as well as cell type determine the choice of MAP2K member that mediates the response. In a variety of cell types, the MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK7 and MKK4 contributes to innate immune response in macrophages as well as during inflammation in vivo. To address this question and elucidate the role of MKK7 and MKK4 in macrophage and in vivo, we developed MKK7- and MKK4-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for LPS induced cytokine production and migration which appears to be a major contributor to the inflammatory response in vivo. Whereas MKK4 plays a significant but minor role in cytokine production in vivo.

category: biochemistry


title: Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing

abstract: Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at unprecedented resolution. Using our platform, we have corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Our transcriptomic analysis revealed that this neuronal bias is mainly due to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. Such ability to parallelize the characterization of AAV tropism and simultaneously measure the transcriptional response of transduction will facilitate the advancement of safe and precise gene delivery vehicles.


title: Single Molecule Studies Reveal Regulatory Interactions between Master Kinases PDK1, AKT1 and PKCalpha

abstract:
Leukocyte migration is controlled by a leading edge chemosensory pathway that generates the regulatory lipid PIP3, a growth signal, thereby driving leading edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP3 also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1/PKB and PKC are key components of a plasma membrane-based PIP3 and Ca2+ signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP3, while PKC is recruited to the membrane by Ca2+. All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKC and other AGC kinases by phosphorylation at key sites. PDK1 is known to form PDK1:AKT1 and PDK1:PKC heterodimers stabilized by a PIF interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here we present the first, to our knowledge, single molecule studies of full length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full length PKC. The findings show that membrane-bound PDK1:AKT1 and PDK1:PKC heterodimers form under physiological conditions, and are stabilized by PIF interaction. PKC exhibits 8-fold higher PDK1 affinity than AKT1, thus PKC competitively displaces AKT1 from PDK1:AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1:AKT1 heterodimer, while PKC acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide new insights into molecular and regulatory interactions of the three master kinases on their target membrane, and suggest that the recently described tumor suppressor activity of PKC may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP3-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis.

STATEMENT OF SIGNIFICANCEThis work investigates three master kinases that play central roles in guiding white blood cell migration to sites of infection, inflammation or tissue damage. More broadly, the same kinases help regulate production of a cell growth signal, and may trigger cancer when dysregulated. Using powerful single molecule methods, the work detects and analyzes the interactions between the three purified kinases on their target membrane surface. The findings reveal functionally important differences between pairwise binding affinities of different binding partners. Additional studies reveal that the highest affinity kinase can disrupt and inhibit the activated complex formed by association of the other two kinases. Such inhibition is proposed to help prevent cancer by limiting growth signal production by the activated complex.


title: Single cell multi-omics analysis of chromothriptic medulloblastoma highlights genomic and transcriptomic consequences of genome instability

abstract:
Chromothripsis is a form of genome instability, whereby a presumably single catastrophic event generates extensive genomic rearrangements of one or few chromosome(s). However, little is known about the heterogeneity of chromothripsis across different clones from the same tumor, as well as changes in response to treatment. We analyzed single-cell genomic and transcriptomic alterations linked with chromothripsis in human p53-deficient medulloblastoma (n=7). We reconstructed the order of somatic events, identified early alterations likely linked to chromothripsis and depicted the contribution of chromothripsis to malignancy. We characterized subclonal variation of chromothripsis and its effects on double-minute chromosomes, cancer drivers and putatively druggable targets. Furthermore, we highlighted the causative role and the fitness consequences of specific rearrangements in neural progenitors.

Graphical Abstract

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title: FMRP regulates experience-dependent maturation of callosal synaptic connections and bilateral cortical synchrony

abstract: Reduced structural and functional interhemispheric connectivity correlates with the severity of Autism Spectrum Disorder (ASD) behaviors in humans. Little is known of how ASD-risk genes regulate callosal connectivity. Here we show that Fmr1, whose loss-of-function leads to Fragile X Syndrome (FXS), cell autonomously promotes maturation of callosal excitatory synapses between somatosensory barrel cortices in mice. Postnatal, cell-autonomous deletion of Fmr1 in postsynaptic Layer (L) 2/3 or L5 neurons results in a selective weakening of AMPA receptor- (R), but not NMDA receptor-, mediated callosal synaptic function, indicative of immature synapses. Sensory deprivation by contralateral whisker trimming normalizes callosal input strength, suggesting that experience-driven activity of postsynaptic Fmr1 KO L2/3 neurons weakens callosal synapses. In contrast to callosal inputs, synapses originating from local L4 and L2/3 circuits are normal, revealing an input-specific role for postsynaptic Fmr1 in regulation of synaptic connectivity within local and callosal neocortical circuits. These results suggest direct cell autonomous and postnatal roles for FMRP in development of specific cortical circuits and suggest a synaptic basis for long-range functional underconnectivity observed in FXS patients.


title: Experience-dependent weakening of callosal synaptic connections in the absence of postsynaptic FMRP


title: Sex Differences in Cerebral Edema after Experimental Traumatic Brain Injury

abstract: Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide. Cerebral edema following TBI is known to play a critical role in injury severity and prognosis. In the current study we used multimodal magnetic resonance imaging (MRI) to assess cerebral edema 24 hours after unilateral contusive TBI in male and female rats. We then directly quantified brain water content in the same subjects ex vivo. We found that both males and females had similarly elevated T2 values after TBI compared with sham controls. Apparent diffusion coefficient (ADC) was more variable than T2 and did not show significant injury effects in males or females. Brain water was elevated in male TBI rats compared with sham controls, but there was no difference between female TBI and sham groups. Notably, MRI biomarkers of edema were more closely correlated with brain water in male rats; female rats did not show any relationship between brain water and T2 or ADC. These observations raise questions about the interpretation of radiological findings traditionally interpreted as edema in female TBI patients. A better understanding of sex differences and similarities in the pathophysiology of post-traumatic edema is needed to help improve patient management and the development of effective treatment strategies for men and women.


title: Comparing Imaging Biomarkers of Cerebral Edema after TBI in Young Adult Male and Female Rats


title: A metabolically stable PET tracer for imaging synaptic vesicle protein 2A: Synthesis and preclinical characterization of SDM-16

abstract:
PurposeTo investigate the synaptic vesicle glycoprotein 2A (SV2A) expression in the whole central nervous system and peripheral tissues, a metabolically stable SV2A radiotracer is desirable to minimize a potential confounding effect of radiometabolites. The aim of this study was to develop and evaluate a metabolically stable SV2A radiotracer, [18F]SDM-16, in nonhuman primate brains.

MethodsThe racemic SDM-16 (4-(3,5-difluorophenyl)-1-((2-methyl-1H-imidazol-1-yl)methyl)pyrrolidin-2-one) was synthesized and assayed for in vitro SV2A binding affinity. We synthesized the enantiopure [18F]SDM-16 using the corresponding arylstannane precursor. Nonhuman primate brain PET was performed on a FOCUS 220 system. Arterial blood was drawn for metabolite analysis and construction of plasma input function. Regional time-activity curves (TACs) were evaluated with the one-tissue compartment (1TC) model to obtain the volume of distribution (VT). Binding potential (BPND) was calculated using either the nondisplaceable volume of distribution (VND) or the centrum semiovale (CS) as the reference region.

ResultsRacemic SDM-16 was synthesized in 3 steps with 44% overall yield and has high affinity (Ki = 3.7 nM) to human SV2A. [18F]SDM-16 was prepared in greater than 99% radiochemical and enantiomeric purity. This radiotracer displayed high specific binding in brain and was metabolically more stable than other SV2A PET tracers. The plasma free fraction (fP) of [18F]SDM-16 was 69%, which was higher than those of [11C]UCB-J (46%), [18F]SynVesT-1 (43%), [18F]SynVesT-2 (41%), and [18F]UCB-H (43%). The TACs were well described with the 1TC. The averaged test-retest variability (TRV) was -9{+/-}8%, and averaged absolute TRV (aTRV) was 10{+/-}7% for all analyzed brain regions.

ConclusionWe have successfully synthesized a metabolically stable and high affinity SV2A PET tracer, [18F]SDM-16, which showed high specific and reversible binding in the NHP brain. [18F]SDM-16 may have potential application in the visualization and quantification of SV2A beyond the brain.


title: Enhanced FcγRIII/CD16 activation by discrete ligands as independent correlates of disease severity in COVID-19 patients

abstract:
A dysregulated immune response with high levels of SARS-CoV-2 specific IgG antibodies characterizes patients with severe or critical COVID-19. Although a robust IgG response is traditionally considered to be protective, excessive triggering of activating Fc-gamma-receptors (Fc{gamma}Rs) could be detrimental and cause immunopathology. Here, we document that patients who develop soluble circulating IgG immune complexes (sICs) during infection are subject to enhanced immunopathology driven by Fc{gamma}R activation. Utilizing cell-based reporter systems we provide evidence that sICs are predominantly formed prior to a specific humoral response against SARS-CoV-2. sIC formation, together with increased afucosylation of SARS-CoV-2 specific IgG eventually leads to an enhanced CD16 (Fc{gamma}RIII) activation of immune cells reaching activation levels comparable active systemic lupus erythematosus (SLE) disease. Our data suggest a vicious cycle of escalating immunopathology driven by an early formation of sICs in predisposed patients. These findings reconcile the seemingly paradoxical findings of high antiviral IgG responses and systemic immune dysregulation in severe COVID-19.

Clinical implicationsThe identification of sICs as drivers of an escalating immunopathology in predisposed patients opens new avenues regarding intervention strategies to alleviate critical COVID-19 progression.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/449893v4_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@b4616corg.highwire.dtl.DTLVardef@682d1aorg.highwire.dtl.DTLVardef@16946cborg.highwire.dtl.DTLVardef@a6ef7d_HPS_FORMAT_FIGEXP  M_FIG C_FIG A vicious cycle of immunopathology in COVID-19 patients is driven by soluble multimeric immune complexes (sICs). SARS-CoV-2 infection triggers sIC formation in prone individuals. Activation of Fc{gamma}RIII/CD16 expressing immune cells by sICs precedes a humoral response to SARS-CoV2 infection. sICs and infection add to IgG afucosylation, further enhancing Fc{gamma}RIII/CD16 activation by opsonized targets. High inflammation induces further sIC mediated immune cell activation ultimately leading to an escalating immunopathology.


title: Circulating immune complexes drive immunopathology in COVID-19


title: Circulating multimeric immune complexes drive immunopathology in COVID-19


title: Coordinated Action of Multiple Transporters in the Acquisition of Essential Cationic Amino Acids by the Intracellular Parasite Toxoplasma gondii

abstract:
Intracellular parasites of the phylum Apicomplexa are dependent on the scavenging of essential amino acids from their hosts. We previously identified a large family of apicomplexan-specific plasma membrane-localized amino acid transporters, the ApiATs, and showed that the Toxoplasma gondii transporter TgApiAT1 functions in the selective uptake of arginine. TgApiAT1 is essential for parasite virulence, but dispensable for parasite growth in medium containing high concentrations of arginine, indicating the presence of at least one other arginine transporter. Here we identify TgApiAT6-1 as the second arginine transporter. Using a combination of parasite assays and heterologous characterisation of TgApiAT6-1 in Xenopus laevis oocytes, we demonstrate that TgApiAT6-1 is a general cationic amino acid transporter that mediates both the high-affinity uptake of lysine and the low-affinity uptake of arginine. TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. gondii and is essential for parasite proliferation. We demonstrate that the uptake of cationic amino acids by TgApiAT6-1 is  trans-stimulated by cationic and neutral amino acids and is likely promoted by an inwardly negative membrane potential. These findings demonstrate that T. gondii has evolved overlapping transport mechanisms for the uptake of essential cationic amino acids, and we draw together our findings into a comprehensive model that highlights the finely-tuned, regulated processes that mediate cationic amino acid scavenging by these intracellular parasites.

Author summaryThe causative agent of toxoplasmosis, Toxoplasma gondii, is a versatile intracellular parasite that can proliferate within nucleated cells of virtually all warm-blooded organisms. In order to survive, T. gondii must scavenge the cationic amino acids lysine and arginine from their hosts. In a previous study, we demonstrated that a plasma membrane-localized protein called TgApiAT1 facilitates the uptake of arginine into the parasite. We found that parasites lacking TgApiAT1 could proliferate when cultured in medium containing high concentrations of arginine, suggesting the existence of an additional uptake pathway for arginine. In the present study, we demonstrate that this second uptake pathway is mediated by TgApiAT6-1, a protein belonging to the same solute transporter family as TgApiAT1. We show that TgApiAT6-1 is the major lysine transporter of the parasite, and that it is critical for parasite proliferation. Furthermore, we demonstrate that TgApiAT6-1 can transport arginine into parasites in conditions where arginine concentrations are high and lysine concentrations are comparatively lower. These data support a model for the finely-tuned acquisition of essential cationic amino acids that involves multiple transporters, and which likely contributes to these parasites being able to survive and proliferate within a wide variety of host cell types.


title: A steroid hormone regulates growth in response to oxygen availability

abstract: In almost all animals, physiologically low oxygen (hypoxia) during development slows growth and reduces adult body size1-3. The developmental mechanisms that determine growth under hypoxic conditions are, however, poorly understood. One hypothesis is that the effect of hypoxia on growth and final body size is a non-adaptive consequence of the cell-autonomous effects of hypoxia on cellular metabolism. Alternatively, the effect may be an adaptive coordinated response mediated through systemic physiological mechanisms. Here we show that the growth and body size response to moderate hypoxia (10% O2) in Drosophila melanogaster is systemically regulated via the steroid hormone ecdysone, acting partially through the insulin-binding protein Imp-L2. Ecdysone is necessary to reduce growth in response to hypoxia: hypoxic growth suppression is ameliorated when ecdysone synthesis is inhibited. This hypoxia-suppression of growth is mediated by the insulin/IGF-signaling (IIS) pathway. Hypoxia reduces systemic IIS activity and the hypoxic growth-response is eliminated in larvae with suppressed IIS. Further, loss of Imp-L2, an ecdysone-response gene that suppresses systemic IIS, significantly reduces the negative effect of hypoxia on final body size. Collectively, these data indicate that growth suppression in hypoxic Drosophila larvae is accomplished by systemic endocrine mechanisms rather than direct suppression of tissue aerobic metabolism.


title: Revisiting the STRmixlikelihood ratio probability interval coverage considering multiple factors

abstract:
In previously reported work a method for applying a lower bound to the variation induced by the Monte Carlo effect was trialled. This is implemented in the widely used probabilistic genotyping system, STRmix. The approach did not give the desired 99% coverage.

However, the method for assigning the lower bound to the MCMC variability is only one of a number of layers of conservativism applied in a typical application. We tested all but one of these sources of variability collectively and term the result the near global coverage. The near global coverage for all tested samples was greater than 99.5% for inclusionary average LRs of known donors. This suggests that when included in the probability interval method the other layers of conservativism are more than adequate to compensate for the intermittent underperformance of the MCMC variability component. Running for extended MCMC accepts was also shown to result in improved precision.