title: The Neisseria meningitidis iron acquisition protein HpuA moonlights as an adhesin and inhibits host cell migration

abstract:
Neisseria meningitidis can cause meningococcal disease, a rapidly developing and potentially fatal infection. Despite this, it normally resides as a commensal in the nasopharynx of healthy individuals. The mechanisms by which meningococci access deeper tissues remain unknown. Epidemiological data suggest that mucosal disruptions increase the risk of meningococcal disease. We previously investigated whether meningococci inhibit host cell wound repair, enhancing invasive disease risk. Here, using genome sequencing and a collection of closely related household isolates that differ in their ability to inhibit host wound repair, we identify the responsible meningococcal factor. This protein, HpuA, has previously been characterized as part of a bipartite heme acquisition transporter. We constructed mutants to demonstrate that HpuA, but not HpuB, inhibits wound repair, acts as an adhesin for epithelial cells, and promotes cellular invasion. We showed this was not due to iron starvation resulting from the bacteria, differences in growth rate, or manipulation of host haptoglobin. Heterologous expression of HpuA in E. coli mediated adherence to 16HBE cells in an HpuA-dependent manner and conferred an aggregative phenotype onto E. coli, suggesting that HpuA may play a role in the formation of microcolonies on host cells. We also demonstrated that iron supplementation of meningococci restored the inhibition of wound repair in strains lacking HpuA (NZCM112, {Delta}hpuA mutant) to levels seen with the wild type. This was also seen with unrelated carriage strains previously shown not to inhibit wound repair. Iron supplementation also increased adherence and invasion of meningococci for strains lacking HpuA, while not affecting those that expressed HpuA. These findings suggest there may be a second meningococcal protein that inhibits wound repair. Together, these results suggest that HpuA is an important meningococcal virulence factor with multiple moonlighting functions, mediating adherence, invasion, inhibition of wound repair, and bacterial aggregation.

Author SummaryNeisseria meningitidis causes meningococcal disease, a potentially fatal and rapidly developing illness that most often occurs in children. Despite this, the bacteria are frequently carried harmlessly as part of the normal airway microflora in healthy people, only rarely causing invasive disease, which involves replication in the bloodstream or central nervous system. It remains unknown precisely how the bacteria reach the deeper tissues from the airways, though some epidemiological evidence suggests that wounds or disruptions to the airways may increase risk. Here, we show that a N. meningitidis protein, HpuA, moonlights from its usual job of acquiring nutrients from the host, to enable the bacteria to adhere to and invade host cells, as well as inhibiting wound closure. Furthermore, we also show that meningococci that lack HpuA acquire the ability to inhibit wound repair when they are supplemented with iron, suggesting that there are additional meningococcal proteins to be discovered that may inhibit wound repair.


title: Active site mutations in the bacterial actin homolog FtsA impair direct interactions with the divisome in Escherichia coli

abstract: During cell division in Escherichia coli, the highly conserved tubulin homolog FtsZ polymerizes and assembles into a ring-like structure, called the Z-ring, at the site of septation early in the division pathway. For recruitment to the membrane surface, FtsZ polymers directly interact with membrane-associated proteins. In E. coli, membrane recruitment and tethering of FtsZ are predominantly carried out by FtsA. FtsA shares structural homology with actin and, like actin, hydrolyzes ATP. Yeast actin detects nucleotide occupancy through a sensor region adjacent to the nucleotide binding site and adopts distinct conformations in monomeric and filamentous actin. Accordingly, bacterial actin homologs also display considerable conformational flexibility across different nucleotide-bound states and adopt a polymerized conformation. Here, we show that a cluster of amino acid residues in the central region of FtsA and proximal to the nucleotide binding site are critical for FtsA function in vitro and in vivo. Each of these residues are important for ATP hydrolysis, phospholipid (PL) binding, ATP-dependent vesicle remodeling, and recruitment to the divisome in vivo, to varying degrees. Notably, we observed that Ser 84 and Glu 14 are essential for ATP-dependent vesicle remodeling and magnesium-dependent membrane release of FtsA from vesicles in vitro, and these defects likely underlie the loss of function by FtsA(E14R) and FtsA(S84L) in vivo. Finally, we demonstrate that FtsA(A188V), which is associated with temperature-sensitive growth in vivo, is defective for rapid ATP hydrolysis and ATP-dependent remodeling of PL vesicles in vitro. Together, our results show that loss of nucleotide-dependent activities by FtsA, such as ATP hydrolysis, ATP-dependent PL vesicle remodeling, and membrane release, lead to failed Z-ring assembly and division defects in cells.

category: microbiology


title: Herpes Simplex Virus Reactivation Induced Ex Vivo Involves a DLK-Dependent but Histone Demethylase-Independent Wave of Lytic Gene Expression

abstract: Herpes Simplex virus-1 (HSV-1) maintains a lifelong latent infection in neurons and periodically reactivates, resulting in the production of infectious virus. The exact cellular pathways that induce reactivation are not understood. In primary neuronal models of HSV latency, the cellular protein Dual Leucine Zipper kinase (DLK) has been found to initiate a wave of viral gene expression known as Phase I. Phase I occurs independently of both viral DNA replication and the activities of histone demethylase enzymes required to remove repressive heterochromatin modifications associated with the viral genome. Here we investigated whether Phase-I like gene expression occurs in ganglia reactivated from infected mice. Using the combined trigger of explant-induced axotomy and inhibition of PI3K signaling, we found that HSV lytic gene expression was induced rapidly from both sensory and sympathetic neurons. Ex vivo reactivation involved a wave of viral late gene expression that occurred independently of viral genome synthesis and histone demethylase activity, and preceded the detection of infectious virus. Importantly, we found that DLK was required for the initial induction of lytic gene expression. These data confirm the essential role of DLK in inducing HSV-1 gene expression from the heterochromatin associated genome and further demonstrate that HSV-1 gene expression during reactivation occurs via mechanisms that are distinct from lytic replication.


title: Reduced polymerase pausing compensates for increased chromatin accessibility in the aging liver

abstract: Regulation of gene expression is tightly linked to the organization of the mammalian genome. With age, chromatin alterations occur on all levels of genome organization, accompanied by changes in the gene expression profile. However, little is known about the changes on the level of transcriptional regulation with age. Here, we used a multi-omic approach and integrated ATAC-, RNA- and NET-seq to identify age-related changes in the chromatin landscape of murine liver and to investigate how these are linked to transcriptional regulation. We provide the first systematic inventory of the connection between aging, chromatin accessibility and transcriptional regulation in a whole tissue. We observe that aging in murine liver is accompanied by an increase in chromatin accessibility at promoter regions of protein-coding genes. Yet, although promoter accessibility is a requirement for transcription, the increased accessibility does not result in enhanced transcriptional output. Instead, aging is accompanied by a decrease of promoter-proximal pausing of RNA polymerase II (Pol II). We propose that these changes in transcriptional regulation are due to a reduced stability of the pausing complex and may represent a mechanism to compensate for the age-related increase in chromatin accessibility in order to prevent aberrant transcription.

category: molecular biology


title: Comparative transcriptomics reveal highly conserved regional programs between porcine and human colonic enteric nervous system

abstract: The porcine gut is increasingly regarded as a useful translational model. The enteric nerve system (ENS) in the colon coordinates diverse functions. However, knowledge of the molecular profiling of porcine ENS and its similarity to that of human is limited. We identified the distinct transcriptional programs associated with functional characteristics between inner submucosal and myenteric ganglia (ISG, MG) in porcine proximal and distal colon (p-pC, p-dC) using bulk RNA sequencing (RNA-seq) and single-cell RNA-seq. Comparative transcriptomics of MG in corresponding colonic regions of porcine and human revealed highly conserved programs existing in p-pC and p-dC, which explained >90% of their transcriptomic responses to vagal nerve stimulation (VNS), suggesting that p-pC and p-dC could serve as predictors in translational studies. The conserved programs specific for inflammatory modulation were displayed in porcine with VNS. This study provides a valuable transcriptomic resource for understanding of human colonic functions and neuromodulation using porcine model.


title: The BAF A12T mutation reduces BAF affinity to lamin A/C, preventing its recruitment to nuclear ruptures in Nestor Guillermo Progeria Syndrome cells.

abstract: The premature aging disorder Nestor Guillermo Progeria Syndrome (NGPS) is caused by a homozygous Alanine to Threonine mutation at position 12 (A12T) in Barrier-to- Autointegration Factor (BAF). BAF is a small essential protein that binds to DNA and nuclear envelope proteins. It contributes to important cellular processes including transcription regulation and nuclear envelope reformation after mitosis. More recently, BAF was identified as an important factor for nuclear envelope repair upon rupture in interphase. However, the mechanism by which the BAF A12T mutation causes NGPS has remained unclear. To investigate the effects of this mutation on nuclear envelope integrity, we used NGPS-derived patient cells and engineered an isogenic cell line by reversing the BAF A12T homozygous mutation using CRISPR/Cas9. Using a combination of cellular models, structural data and in vitro assays, we identified that the A12T mutation reduces the affinity of BAF for lamin A/C by tenfold. As a result, BAF A12T is unable to recruit lamin A/C to sites of nuclear envelope rupture. This leads to persistent lamin A/C gaps at sites of ruptures, and contributes to nuclear fragility in NGPS patient cells, which show increased frequency of nuclear envelope re- rupturing. Overexpression of wild-type BAF in a NGPS context rescues lamin A/C recruitment to sites of nuclear rupture, which could explain why the heterozygous A12T mutation does not cause premature aging.

category: cell biology


title: The BAF A12T mutation associated with premature aging impedes lamin A/C recruitment to sites of nuclear rupture, contributing to nuclear envelope fragility.


title: Selective binding and transport of protocadherin 15 isoforms by stereocilia unconventional myosins in a heterologous expression system

abstract: During hair cell development, the mechanoelectrical transduction (MET) apparatus is assembled at the stereocilia tips, where it coexists with the stereocilia actin regulatory machinery. While the myosin-based tipward transport of actin regulatory proteins is well studied, isoform complexity and built-in redundancies in the MET apparatus have limited our understanding of how MET components are transported. We used a heterologous expression system to elucidate the myosin selective transport of isoforms of protocadherin 15 (PCDH15), the protein that mechanically gates the MET apparatus. We show that MYO7A selectively transports the CD3 isoform while MYO3A and MYO3B transports the CD2 isoform. Furthermore, MYO15A showed an insignificant role in the transport of PCDH15, and none of the myosins tested transport PCDH15-CD1. Our data suggest an important role for MYO3A, MYO3B, and MYO7A in the MET apparatus formation and highlight the intricate nature of MET and actin regulation during development and functional maturation of the stereocilia bundle.


title: Horizontal gene transfer increases microbiome stability

abstract: Genes encoding resistance to stressors, such as antibiotics or environmental pollutants, are widespread across microbiomes, often encoded on mobile genetic elements. Yet despite their prevalence, the impact of resistance genes and their mobility upon the dynamics of microbial communities remains largely unknown. Here we develop eco-evolutionary theory to explore how resistance genes alter the stability of diverse microbiomes. We show that adding resistance genes to a microbiome typically increases its overall stability, particularly for mobile resistance genes with high transfer rates that efficiently spread resistance throughout the community. However, the impact of resistance genes upon the stability of individual taxa varies depending upon the mobility of the resistance gene and the network of ecological interactions within the community. Non-mobile resistance genes can benefit susceptible taxa in cooperative communities, yet damage those in competitive communities. Moreover, whilst the transfer of mobile resistance genes generally increases the stability of previously susceptible recipient taxa to perturbation, it can, counterintuitively, decrease the stability of the originally resistant donor species. We confirmed these theoretical predictions experimentally using competitive soil microcosm communities. Here the stability of a susceptible microbial community to perturbation was increased by adding mobile resistance genes encoded on conjugative plasmids but was decreased when these same genes were encoded on the chromosome. Together these findings highlight the importance of horizontal gene transfer in driving the eco-evolutionary dynamics of diverse microbiomes.

category: evolutionary biology


title: A Deep Survival EWAS approach estimating risk profile based on pre-diagnostic DNA methylation: an application to Breast Cancer time to diagnosis

abstract:
Previous studies for cancer biomarker discovery based on pre-diagnostic blood DNA methylation profiles, either ignore the explicit modeling of the time to diagnosis (TTD) as in a survival analysis setting, or provide inconsistent results. This lack of consistency is likely due to the limitations of standard EWAS approaches, that model the effect of DNAm at CpG sites on TTD independently. In this work, we argue that a global approach to estimate CpG sites effect profile is needed, and we claim that such approach should capture the complex (potentially non-linear) relationships interplaying between sites. To prove our concept, we develop a new Deep Learning-based approach assessing the relevance of individual CpG Islands (i.e., assigning a weight to each site) in determining TTD while modeling their combined effect in a survival analysis scenario. The algorithm combines a tailored sampling procedure with DNAm sites agglomeration, deep non-linear survival modeling and SHapley Additive exPlanations (SHAP) values estimation to aid robustness of the derived effects profile. The proposed approach deal with the common complexities arising from epidemiological studies, such as small sample size, noise, and low signal-to-noise ratio of blood-derived DNAm. We apply our approach to a prospective case-control study on breast cancer nested in the EPIC Italy cohort and we perform weighted gene-set enrichment analyses to demonstrate the biological meaningfulness of the obtained results. We compared the results of Deep Survival EWAS with those of a traditional EWAS approach, demonstrating that our method performs better than the standard approach in identifying biologically relevant pathways.

Author summaryBlood-derived DNAm profiles could be exploited as new biomarkers for cancer risk stratification and possibly, early detection. This is of particular interest since blood is a convenient tissue to assay for constitutional methylation and its collection is non-invasive. Exploiting pre-diagnostic blood DNAm data opens the further opportunity to investigate the association of DNAm at baseline on cancer risk, modeling the relationship between sites methylation and the Time to Diagnosis. Previous studies mostly provide inconsistent results likely due to the limitations of standard EWAS approaches, that model the effect of DNAm at CpG sites on TTD independently. In this work we argue that an approach to estimate single CpG sites effect while modeling their combined effect on the survival outcome is needed, and we claim that such approach should capture the complex (potentially non-linear) relationships interplaying between sites. We prove this concept by developing a novel approach to analyze a prospective case-control study on breast cancer nested in the EPIC Italy cohort. A weighted gene set enrichment analysis confirms that our approach outperforms standard EWAS in identifying biologically meaningful pathways.

category: genomics


title: Effect of Ionic Strength on the Assembly of Simian Vacuolating Virus Capsid Protein Around Poly(Styrene Sulfonate)

abstract: Virus-like particles (VLPs) are noninfectious nanocapsules that can be used for drug delivery or vaccine applications. VLPs can be assembled from virus capsid proteins around a condensing agent like RNA, DNA, or a charged polymer. Electrostatic interactions play an important role in the assembly reaction. VLPs assemble from many copies of capsid protein, with combinatorial intermediates, and therefore the mechanism of the reaction is poorly understood. In this paper, we determined the effect of ionic strength on the assembly of Simian Vacuolating Virus 40 (SV40)-like particles. We mixed poly(styrene sulfonate) with SV40 capsid protein pentamers at different ionic strengths. We then characterized the assembly product by solution small-angle X-ray scattering (SAXS) and cryo-TEM. To analyze the data, we performed Brownian dynamics simulations using a coarse-grained model that revealed incomplete, asymmetric VLP structures that were consistent with the experimental data. We found that close to physiological ionic strength, T=1 VLPs coexisted with VP1 pentamers. At lower or higher ionic strengths, incomplete particles coexisted with pentamers and T=1 particles. Including the simulation, structures were essential to explaining the SAXS data.

category: biophysics


title: Role of Delta-Notch signalling molecules on cell-cell adhesion in determining heterogeneous chemical and cell morphological patterning

abstract: Cell mechanics and motility are responsible for collective motion of cells that result in overall deformation of epithelial tissues. On the other hand, contact-dependent cell-cell signalling is responsible for generating a large variety of intricate, self-organized, spatial patterns of the signalling molecules. Moreover, it is becoming increasingly clear that the combined mechanochemical patterns of cell shape/size and signalling molecules in the tissues, for example, in cancerous and sensory epithelium, are governed by mechanochemical coupling between chemical signalling and cell mechanics. However, a clear quantitative picture of how these two aspects of tissue dynamics, i.e., signalling and mechanics, lead to pattern and form is still emerging. Although, a number of recent experiments demonstrate that cell mechanics, cell motility, and cell-cell signalling are tightly coupled in many morphogenetic processes, relatively few modeling efforts have focused on an integrated approach. We extend the vertex model of an epithelial monolayer to account for contact-dependent signalling between adjacent cells and between non-adjacent neighbors through long protrusional contacts with a feedback mechanism wherein the adhesive strength between adjacent cells is controlled by the expression of the signalling molecules in those cells. Local changes in cell-cell adhesion lead to changes in cell shape and size, which in turn drives changes in the levels of signalling molecules. Our simulations show that even this elementary two-way coupling of chemical signalling and cell mechanics is capable of giving rise to a rich variety of mechanochemical patterns in epithelial tissues. In particular, under certain parametric conditions, bimodal distributions in cell size and shape are obtained, which resemble experimental observations in cancerous and sensory tissues.


title: NSD2 E1099K drives relapse in pediatric acute lymphoblastic leukemia by disrupting 3D chromatin organization

abstract: The NSD2 p.E1099K (EK) mutation has been shown to be enriched in patients with relapsed ALL and found to play a role in clonal fitness dependent on the underlying genetic/epigenetic landscape of the cells. To uncover 3D chromatin architecture-related mechanisms underlying drug resistance, we systematically integrated Hi-C, ATAC-seq, RNA-seq and ChIP-seq data from three B-ALL cell lines heterozygous for NSD2 EK (RS4;11, RCH-ACV, SEM) and assessed changes upon knockdown. NSD2 knockdown revealed widespread remodeling of the 3D genome, specifically in terms of compartmentalization. Systematic integration of these datasets revealed significant switches in A/B compartments with a strong bias towards B compartments upon knockdown, suggesting that NSD2 EK plays a prominent role in maintaining A compartments through enrichment of H3K36me2 epigenetic marks. In contrast, we identified few changes in intra-TAD activity suggesting that the NSD2 EK impacts transcriptional changes through a remarkable dependence on compartmentalization. Furthermore, EK-mediated reorganization of compartments highlights the existence of a common core of compacting loci shared across the three cell lines that explain previously described phenotypes as well as serve as targets for therapeutic intervention. This study offers a novel mechanism by which NSD2 EK drives clonal evolution and drug resistance.

category: cancer biology


title: Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment

abstract: Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in approximately 45% of patients. Unfortunately, TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. We previously demonstrated mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual TNBC cells surviving NACT. We sought to investigate the mechanism underlying this enhanced reliance on mitochondrial metabolism. Mitochondria are morphologically plastic organelles that cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. The functional impact of mitochondrial structure on metabolic output is highly context dependent and not understood in TNBC. Several chemotherapy agents are conventionally used for neoadjuvant treatment of TNBC patients. Upon comparing mitochondrial effects of commonly used chemotherapies, we found that DNA-damaging agents increased mitochondrial elongation, mitochondrial content, flux of glucose through the TCA cycle, and OXPHOS, whereas taxanes instead decreased mitochondrial elongation and OXPHOS. Additionally, short protein isoform levels of the mitochondrial inner membrane fusion protein optic atrophy 1 (OPA1) were associated with those observations. Further, we observed heightened OXPHOS, OPA1 protein levels, and mitochondrial elongation in a patient-derived xenograft (PDX) model of residual TNBC. Pharmacologic or genetic disruption of mitochondrial fusion and fission resulted in decreased or increased OXPHOS, respectively, revealing that longer mitochondria favor oxphos in TNBC cells. Using TNBC cell lines and an in vivo PDX model of residual TNBC, we found that sequential treatment with DNA-damaging chemotherapy, thus inducing mitochondrial fusion and OXPHOS, followed by MYLS22, a specific inhibitor of OPA1, was able to suppress mitochondrial fusion and OXPHOS and significantly inhibited residual tumor regrowth. Taken together, our findings suggest that TNBC mitochondria can optimize OXPHOS through modulation of mitochondrial structure. This may provide an opportunity to overcome mitochondrial adaptations of chemoresistant TNBC.


title: Cortical compensation for afferent loss in older adults: Associations with GABA and speech recognition in noise

abstract:
Age-related deficits in auditory nerve (AN) function reduce afferent input to the auditory cortex. The extent to which the auditory cortex in older adults compensates for this loss of afferent input, also known as central gain, and the mechanisms underlying this compensation are not well understood. We took a neural systems approach to estimate central gain, measuring AN and cortical evoked responses within 50 older and 27 younger adults. Amplitudes were significantly smaller for older than for younger adults for AN responses but not for cortical responses. We used the relationship between AN and cortical response amplitudes in younger adults to predict cortical response amplitudes for older adults from their AN responses. Central gain in older adults was thus defined as the difference between their observed cortical responses and those predicted from the parameter estimates of younger adults. More central gain was associated with decreased cortical levels of GABA measured with 1H-MRS and poorer speech recognition in noise (SIN). Effects of central gain and GABA on SIN occur in addition to, and independent from, effects attributed to elevated hearing thresholds. Our results are consistent with animal models of central gain and suggest that reduced AN afferent input in some older adults may result in changes in cortical encoding and inhibitory neurotransmission, which contribute to reduced SIN. An advancement in our understanding of the changes that occur throughout the auditory system in response to the gradual loss of input with increasing age may provide potential therapeutic targets for intervention.

SignificanceAge-related hearing loss is one of the most common chronic conditions of aging, yet little is known about how the cortex compensates for this loss of sensory input. We measured AN and cortical responses to the same stimulus in younger and older adults. In older adults we found an increase in cortical activity following concomitant declines in afferent input that are consistent with central gain. Increased central gain was associated with lower levels of cortical GABA, an inhibitory neurotransmitter, which predicted poorer speech recognition in noise. The results suggest that the cortex in older adults can compensate for attenuated sensory input by reducing inhibition to amplify the cortical response, but this amplification may lead to poorer speech recognition in noise.

category: neuroscience


title: Event-related causality in Stereo-EEG discriminates syntactic processing of noun phrases and verb phrases

abstract: Syntax involves complex neurobiological mechanisms, which are difficult to disentangle for multiple reasons. Using a protocol able to separate syntactic information from sound information we investigated the neural causal connections evoked by the processing of homophonous phrases, either verb phrases (VP) or noun phrases (NP). We used event-related causality (ERC) from stereo-electroencephalographic (SEEG) recordings in 10 epileptic patients in multiple cortical areas, including language areas and their homologous in the non-dominant hemisphere. We identified the different networks involved in the processing of these syntactic operations (faster in the dominant hemisphere) showing that VPs engage a wider cortical network. We also present a proof-of-concept for the decoding of the syntactic category of a perceived phrase based on causality measures. Our findings help unravel the neural correlates of syntactic elaboration and show how a decoding based on multiple cortical areas could contribute to the development of speech prostheses for speech impairment mitigation.


title: Human neutrophil development and functionality are enabled in a humanized mouse model

abstract:
Mice with a functional human immune system serve as an invaluable tool to study the development and function of human immune system in vivo. A major technological limitation of all current humanized mouse models is the lack of mature and functional human neutrophils in circulation and tissues. To overcome this, we generated a humanized mouse model named MISTRGGR, in which the mouse granulocyte colony stimulating factor (G-CSF) was replaced with human G-CSF and the mouse G-CSF receptor gene was deleted (G-CSFR) in existing MISTRG mice. By targeting the GCSF cytokine-receptor axis, we dramatically improved the reconstitution of mature circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. Moreover, these functional human neutrophils in MISTRGGR are recruited upon inflammatory and infectious challenges and help reduce bacterial burden. MISTRGGR mice represent a unique mouse model that finally permits the study of human neutrophils in health and disease.

Key PointsO_LITargeting the GCSF cytokine-receptor axis dramatically improves circulating and tissue-infiltrating human neutrophils in MISTRGGR mice.
C_LIO_LIHuman neutrophils generated in MISTRGGR mice are functional and are able to respond robustly to inflammatory and infectious stimuli.
C_LI

category: immunology


title: A Combined Human Gastruloid Model of Cardiogenesis and Neurogenesis

abstract: Multi-lineage development from gastruloids is enabling unprecedented opportunities to model and study human embryonic processes and is expected to accelerate ex vivo strategies in organ development. Reproducing human cardiogenesis with neurogenesis in a multi-lineage context remains challenging, requiring spatiotemporal input of paracrine and mechanical cues. Here we extend elongating multi-lineage organized (EMLO) gastruloids to include cardiogenesis (EMLOC) and describe interconnected neuro-cardiac lineages in a single gastruloid model. Contractile EMLOCs recapitulate numerous interlinked developmental features including heart tube formation and specialization, cardiomyocyte differentiation and remodeling phases, epicardium, ventricular wall morphogenesis, chamber-like structures and formation of a putative outflow tract. The EMLOC cardiac region, which originates anterior to gut tube primordium, is progressively populated by neurons in a spatial pattern mirroring the known distribution of neurons in the innervated human heart. This human EMLOC model represents the first multi-lineage advancement for the study of coincident neurogenesis and cardiogenesis.

category: developmental biology


title: Brevican, Neurocan, Tenascin-C and Tenascin-R Act as Important Regulators of the Interplay between Perineuronal Nets, Synaptic Integrity, Inhibitory Interneurons and Otx2

abstract: Fast-spiking parvalbumin interneurons are critical for the function of mature cortical inhibitory circuits. Most of these neurons are enwrapped by a specialized extracellular matrix structure (ECM) called perineuronal net (PNN), which can regulate their synaptic input. In this study, we investigated the relationship between PNNs, parvalbumin interneurons and synaptic distribution on these cells in the adult primary visual cortex (V1) of quadruple knockout mice deficient for the ECM molecules brevican, neurocan, tenascin-C and tenascin-R. We used super-resolution structured illumination microscopy (SIM) to analyze PNN structure and associated synapses. Additionally, we examined parvalbumin and calretinin interneuron populations. We observed a reduction in the number of PNN-enwrapped cells and a clear disorganization of the PNN structure in the quadruple knockout V1. This was accompanied by an imbalance of inhibitory and excitatory synapses with a reduction of inhibitory and an increase of excitatory synaptic elements along the PNNs. Also, the number of parvalbumin interneurons was reduced in the quadruple knockout, while calretinin interneurons, which do not wear PNNs did not display differences in number. Interestingly, we found the transcription factor Otx2 homeoprotein positive cell population also reduced. Otx2 is crucial for parvalbumin and PNN maturation and a positive feedback loop between these parameters has been described. Collectively, these data indicate an important role of brevican, neurocan, tenascin-C and tenascin-R in regulating the interplay between PNNs, inhibitory interneurons, synaptic distribution as well as Otx2 in the V1.


title: Monkeys exhibit human-like gaze biases in economic decisions

abstract: In economic decision-making individuals choose between items on the basis of their perceived value. For both humans and nonhuman primates, these decisions are often carried out while shifting gaze between the available options. Recent studies in humans suggest that these shifts in gaze actively influence choice, manifesting as a bias in favor of the items that are viewed first, viewed last, or viewed for the overall longest duration in a given trial. This suggests the existence of a mechanism that links gaze behavior to the neural computations underlying value-based choices. In order to identify this mechanism, it is first necessary to develop and validate a suitable animal model of this behavior. To this end, we have created a novel value-based choice task for macaque monkeys that captures the essential features of the human paradigms in which gaze biases have been observed. Using this task, we identified gaze biases in the monkeys that were both qualitatively and quantitatively similar to those in humans. In addition, the monkeys gaze biases were well-explained using a sequential sampling model framework previously used to describe gaze biases in humans--the first time this framework has been used to explore value-based choice mechanisms in nonhuman primates. Together, these findings suggest a common mechanism that can explain gaze-related choice biases across species, and open the way for mechanistic studies to identify the neural origins of this behavior.


title: Expanding the pulse-reserve paradigm to microorganisms on the basis of differential reserve management strategies

abstract: The pulse-reserve paradigm (PRP) is central in dryland ecology, although traits of microorganisms were not explicitly considered in its inception. We asked if the PRP could be reframed to encompass organisms both large and small. We used a synthetic review of recent advances in arid land microbial autoecology combined with a mathematically explicit theoretical model. Preserving the PRPs original core of adaptations by reserve building, the model considers differential organismal strategies to manage these reserves. It proposes a gradient of organisms according to their reserve strategies, from nimble responders (NIRs) to torpid responders (TORs). It predicts how organismal fitness depends on pulse regimes and reserve strategies thus explaining organismal diversification and distribution. After accounting for scaling phenomena and redefining the microscale meaning of aridity, it becomes patent that the PRP is applicable to microbes, and that this modified PRP represents an inclusive theoretical framework working across life-forms.

category: ecology


title: FIT-GWA: A new method for the genetic analysis of small gene effects, high precision in phenotype measurements and small sample sizes.

abstract: Small gene effects involved in complex traits remains difficult to analyse using current genome-wide association methods (GWAS) due to the number of individuals required to return meaningful association(s), a.k.a. study power. Inspired by physics fields theory we provide a different method called Fields Informational Theory for Genome-Wide Associations (FIT-GWA). Contrary to GWAS, FIT-GWA that the phenotype is measured precisely enough and/or the number of individuals in the population is too small, to permit categories. To extract information FIT-GWA use the difference in the cumulated sum of gene microstates between two configurations: (i) when the individuals are taken at random without information on phenotype values and, (ii) when individuals are ranked as a function of their phenotype value. Such difference can be accounted through the emergence of  phenotypic fields. We demonstrate that FIT-GWA recovers GWAS, i.e., Fishers theory, when the phenotypic fields are linear. However, unlike GWAS, FIT-GWA permits to demonstrate how the variance of microstate distribution density functions are also involved in genotype-phenotype associations. Using genotype-phenotype simulations based on Fishers theory we illustrate the application and power of the method with a small sample size of 1000 individuals.

category: genetics


title: EagleImp-Web: A Fast and Secure Genotype Phasing and Imputation Web Service using Field-Programmable Gate Arrays

abstract: Imputation web servers have been developed that allow phasing and imputation of genome-wide data without the need for own computing resources. However, their terms of use, data sharing with third parties, the security architecture of the web service, and the algorithms and parameter settings used are only partially disclosed. We developed EagleImp-Web, a fast, secure and convenient web service for phasing and imputation of genome-wide data. The web service uses technical improvements in phasing and imputation algorithms and a field-programmable gate array (FPGA) accelerator design to reduce computation time without loss of phasing and imputation quality. Other key features include no exposure of user information and input/output data to third parties, high data security and fast secure download, user authentication through 2-factor authentication, full control in managing user accounts, and full transparency of algorithms and their settings. EagleImp-Web provides simple and convenient functionalities for monitoring running jobs and selecting parameter settings and output information. Due to the speed advantage over a purely CPU-based implementation, EagleImp-Web offers the user the ability to choose a more resource-intensive parameter setting in exchange for computation time to further improve phasing and imputation quality. EagleImp-Web is freely availabe at https://hybridcomputing.ikmb.uni-kiel.de.

category: bioinformatics


title: Development and optimization of a Barley stripe mosaic virus (BSMV)-mediated gene editing system to improve Fusarium head blight (FHB) resistance in wheat

abstract: Fusarium head blight (FHB) is a devastating disease in wheat that causes million dollars of wheat yield losses annually in the U.S.A. Recently we demonstrated that wheat carry FHB susceptible genes and knocking out the susceptible gene increased FHB resistance, which suggests that manipulating susceptible genes using gene editing may open a new avenue to create new sources of FHB resistance. However, wheat genome editing uses gene transformation to deliver CRISPR/Cas9 and gRNA into plants, and most wheat genotypes are not suitable for transformation due to low rates of callus induction and regeneration, therefore it cannot be used in practical wheat breeding. In this study, we developed a new Barley stripe mosaic virus (BSMV)-mediated gRNA delivery system that does not need the gene transformation and tissue culture and it can be used in any genotypes for gene function validation and editing. We used this system edited the susceptible allele of Fhb1, a major FHB resistance gene. We demonstrated that the edited trait is heritable in different genetic backgrounds and knocking out Fhb1 susceptible allele improved FHB resistance in wheat. We also modified system to improve editing efficiency by using floral dip agroinfiltration and adding RNA mobility sequences to the gRNA in the viral vectors. We believe this work will facilitate wheat FHB resistance research and gene editing in cereal crops and will benefit cereal crop researchers and breeders worldwide.

category: plant biology


title: Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole

abstract: Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, mechanisms of autophagosome maturation, i.e., delivery and fusion with the vacuole, remain largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1s function is evolutionarily conserved in plants as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by connecting autophagosomes with the ESCRT-I component VPS23, leading to the formation of amphisomes. Disrupting the VPS23-CFS1 interaction affects autophagic flux and renders plants sensitive to starvation stress. Altogether, our results reveal a deeply conserved mechanism of vacuolar delivery in plants that is mediated by amphisomes.


title: A novel missense mutation in the proprotein convertase gene furinb causes hepatic cystogenesis during liver development in zebrafish

abstract: Hepatic cysts are fluid-filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that develops hepatic cysts during larval stages. Cyst formation in these mutants is not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time-lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed an excessive and disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole-genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene which encodes a proprotein convertase. The mutation alters Furinb localization and causes endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4-phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant livers also exhibited increased mTOR signaling and treatment with the mTOR inhibitor rapamycin partially blocked cyst formation by reducing ER stress. Our study has established a novel vertebrate model for studying hepatic cystogenesis and illustrated the role of ER stress in the disease pathogenesis.


title: ATR kinase inhibition induces thymineless death in proliferating CD8+ T cells

abstract: ATR kinase is a central regulator of the DNA damage response (DDR) and cell cycle checkpoints. However, little is known about the role of ATR in the cell cycle and the impact of DDR inhibitors in immune cells in the absence of DNA damage. We previously showed that the ATR inhibitor AZD6738 (ATRi) combines with radiation to generate delayed, CD8+ T cell-dependent antitumor responses in mouse models of cancer. Here, we show that ATRi induces untimely CDK1 activity during S phase in CD8+ T cells and this induces origin firing and simultaneous degradation of dNTP synthesis and salvage enzymes. These pleiotropic effects of ATRi in proliferating CD8+ T cells induce deoxyuridine contamination in genomic DNA, R loops, RNA-DNA polymerase collisions, and death. Remarkably, thymidine significantly rescues ATRi-induced CD8+ T cell death. Our data identifies critical considerations for the design of clinical ATRi regimens with genotoxic chemo- and radiation and immunotherapies.


title: An inosine triphosphate pyrophosphatase safeguards nucleic acids from aberrant purine nucleotides and prevents a constitutive salicylic acid response

abstract:
Inosine is deaminated adenosine. Inosine is enzymatically introduced in some plant tRNAs but not in other RNAs or DNA. Nonetheless, our data show that RNA and DNA from Arabidopsis thaliana contain (deoxy)inosine, probably derived from non-enzymatic adenosine deamination in nucleic acids and usage of (deoxy)inosine triphosphate (ITP / dITP) during nucleic acid synthesis. We identified a plant INOSINE TRIPHOSPHATE PYROPHOSPHATASE (ITPA) which is conserved in many organisms. Arabidopsis ITPA dephosphorylates deaminated nucleoside di- and triphosphates to the respective monophosphates. ITPA mutation causes inosine di- and triphosphate accumulation in vivo and an elevated (deoxy)inosine content in DNA and RNA. Cadmium-induced oxidative stress, known to foster deamination, leads to more ITP in the wildtype and especially in itpa mutants suggesting that ITP originates from ATP deamination. By contrast, erroneous IMP phosphorylation by AMP kinases seems not to be a major ITP source in vivo although these enzymes phosphorylate IMP in vitro. Mutation of ITPA causes salicylic acid (SA) accumulation, early senescence and upregulation of transcripts associated with immunity and senescence. ITPA is part of a molecular protection system, preventing accumulation of (d)ITP, its usage for nucleic acid synthesis, and probably nucleic acid stress leading to SA accumulation, stress gene induction and early senescence.

One-sentence summaryITPA dephosphorylates deaminated purine nucleotides that arise from oxidative stress and thus contributes to nucleic acid integrity.


title: FtsH Protease Inactivation Allows Accumulation of Aberrant Photosystem II in a Chlamydomonas Rubredoxin Mutant

abstract: The assembly of photosystem II (PSII) requires the participation of assembly proteins that facilitate the step-wise association of its protein and pigment components into a functional complex capable of oxidizing water and reducing plastoquinone. We previously identified one such factor, the membrane-bound rubredoxin RBD1, but its precise role remains unknown in part due to the inability of the 2pac mutant strain of Chlamydomonas reinhardtii, which lacks RBD1, to accumulate PSII. Here, we show that decreased PSII accumulation in 2pac is due to increased proteolytic degradation. Inactivating the thylakoid membrane FtsH protease in the 2pac mutant background led to an increase in the abundance of PSII subunits and their integration into higher molecular weight complexes, including PSII dimers, capable of sustaining photoautotrophic growth. Dark- and low light-grown 2pac ftsh1-1 both accumulated a 23-kD fragment of the D1 protein, a marker typically associated with structural changes resulting from photodamage or photoinhibition. We introduced a HIS-tagged version of the PsbH protein into the 2pac ftsh1-1 background to purify and examine PSII. We found no detectable changes with respect to cofactor composition relative to the wild-type, leading to us to propose a model in which RBD1 promotes the proper folding of D1, possibly via delivery or reduction of the non-heme iron during PSII assembly. Our results demonstrate that introduction of the ftsh1-1 mutation into mutants defective in the biogenesis of thylakoid membrane complexes can allow for the accumulation and study of aberrant complexes that would otherwise be degraded due to their high protease sensitivity.


title: iPSC-based modeling of THD recapitulates disease phenotypes and reveals neuronal malformation

abstract: Tyrosine hydroxylase deficiency (THD) is a rare genetic disorder leading to dopaminergic depletion and early-onset parkinsonism. Affected children present with either a severe form that does not respond to L-Dopa treatment (THD-B), or a milder L-Dopa responsive form (THD-A). We generated induced pluripotent stem cells (iPSCs) from THD patients that were differentiated into dopaminergic neurons (DAn) and compared with control-DAn from healthy individuals and gene-corrected isogenic controls. Consistent with patients, THD iPSC-DAn displayed lower levels of DA metabolites and reduced TH expression, when compared to controls. Moreover, THD iPSC-DAn showed abnormal morphology, including reduced total neurite length and either an abnormal TH proximodistal gradient (THDA), or neurite arborization defects (THDB). Treatment of THD-iPSC-DAn with L-Dopa rescued the neuronal defects and disease phenotype only in THDA-DAn. Interestingly, L-Dopa treatment at the stage of neuronal precursors could prevent the alterations in THDB-iPSC-DAn, thus suggesting the existence of a critical developmental window in THD. Our iPSC-based model recapitulates THD disease phenotypes and response to treatment, representing a promising tool for investigating pathogenic mechanisms, drug screening, and personalized management.


title: Transcranial photobiomodulation enhances visual working memory capacity in humans

abstract: Transcranial photobiomodulation (tPBM) is a novel and noninvasive intervention, which has shown promise for improving cognitive performances. Whether tPBM can modulate brain activity and thereby enhance working memory (WM) capacity in humans remains unclear. In this study, we delivered double-blind and sham-control tPBM with different wavelengths to the prefrontal cortex (PFC) in 90 healthy participants and conducted four electroencephalography (EEG) experiments to investigate whether individual visual working memory capacity and related neural response could be modulated. We found that 1064 nm tPBM applied to the right PFC has both a substantial impact on visual working memory capacity and occipitoparietal contralateral delay activity (CDA), no matter orientation or color feature of the memorized objects. Importantly, the CDA set-size effect during the retention mediated the effect between 1064 nm tPBM and subsequent WM performance. However, these behavioral benefits and the corresponding changes of CDA set-size effect were absent with tPBM at 852 nm wavelength or with the stimulation on the left PFC. Our findings provide converging evidence that 1064 nm tPBM applied on the right PFC can improve visual working memory capacity, as well as explain the individuals electrophysiological changes about behavioral benefits.


title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

abstract: Traditionally, peripheral sensory neurons hold the monopole of transducing external stimuli. Current research moves epidermal keratinocytes into focus as sensors and transmitters of nociceptive and non-nociceptive sensations, tightly interacting with intraepidermal nerve fibers at the neuro-cutaneous unit. In animal models, epidermal cells establish close contacts and ensheath sensory neurites. However, ultrastructural morphological and mechanistic data examining the human keratinocyte-nociceptor interface are sparse. We investigated this exact interface in human skin applying super-resolution array tomography, expansion microscopy, and structured illumination microscopy. We show keratinocyte ensheathment of nociceptors and connexin 43 plaques at keratinocyte-nociceptor contact sites in healthy native skin. We further derived a fully human co-culture system, modeling ensheathment and connexin 43 plaques in vitro. Unraveling human intraepidermal nerve fiber ensheathment and interaction sites marks a milestone in research at the neuro-cutaneous unit. These findings are mind-changers on the way to decipher the mechanisms of cutaneous nociception.


title: Dissociable default-mode subnetworks subserve childhood attention and cognitive flexibility: evidence from deep learning and stereotaxic electroencephalography

abstract:
BackgroundCognitive flexibility encompasses the ability to efficiently shift focus and forms a critical component of goal-directed attention. The neural substrates of this process are incompletely understood in part due to difficulties in sampling the involved circuitry.

MethodsStereotactic intracranial recordings that permit direct resolution of local-field potentials from otherwise inaccessible structures were employed to study moment-to-moment attentional activity in children with epilepsy during the performance of an attentional set-shifting task. A combined deep learning and model-agnostic feature explanation approach was used to analyze these data and decode attentionally-relevant neural features. Connectomic profiling of highly predictive attentional nodes was further employed to examine task-related engagement of large-scale functional networks.

ResultsThrough this approach, we show that beta/gamma power within executive control, salience, and default mode networks accurately predicts single-trial attentional performance. Connectomic profiling reveals that key attentional nodes exclusively recruit dorsal default mode subsystems during attentional shifts.

ConclusionsThe identification of distinct substreams within the default mode system supports a key role for this network in cognitive flexibility and attention in children. Furthermore, convergence of our results onto consistent functional networks despite significant inter-subject variability in electrode implantations supports a broader role for deep learning applied to intracranial electrodes in the study of human attention.

FundingNo funds supported this specific investigation. Awards and grants supporting authors include: Canadian Institutes of Health Research (CIHR) Vanier Scholarship (NMW, HY); CIHR Frederick Banting and Charles Best Canada Graduate Scholarship Doctoral Award (SMW); CIHR Canada Graduate Scholarship Masters Award (ONA); and a CIHR project grant (GMI).


title: IRE1 RNase controls CD95-mediated cell death

abstract: Unfolded Protein Response (UPR) and Death Receptor (DR) signalling are cellular stress pathways frequently activated towards pro-tumoral cellular outputs in cancer. Experimental evidence has highlighted functional links between the UPR and signalling by the DR TRAIL-R1/2. Herein, we demonstrate that the UPR sensor IRE1 controls the expression of the DR CD95/Fas, and its cell death-inducing capacity. Whereas CD95 is not a general determinant of ER stress-induced cell death, IRE1 RNase activity inhibition increased CD95 expression and exacerbated CD95L-induced cell death in glioblastoma (GB) and Triple-Negative Breast Cancer (TNBC) cell lines. In accordance, CD95 mRNA was identified as a target of the Regulated IRE1-Dependent Decay of RNA (RIDD). Moreover, CD95 expression is elevated in TNBC and GB human tumours exhibiting low RIDD activity. Surprisingly, CD95 expression is also reduced in XBP1s-low human tumour samples. We show that IRE1 RNase inhibition led to CD95 expression attenuation and reduced CD95-mediated hepatic toxicity in mice. In addition, overexpression of XBP1s sensitized GB and TNBC cells to CD95L-induced cell death. Overall, these results demonstrate the tight IRE1-mediated control of CD95-dependent cell death signals in a dual manner through both RIDD and XBP1s, and they identify a pharmacologically actionable link between IRE1 and CD95 signalling.


title: Blood cells communicate remotely via an NMDA receptor-mediated glutamatergic chain-reaction

abstract: Neutrophils are white blood cells that are critical to the acute inflammatory and adaptive immune responses. While their diverse interactions with tissue cells and pathogens have been intensely studied, little is known about how they communicate among themselves. Here we employ up-to-date neuroscience techniques to find that electrical, mechanical, or ligand stimulation of an individual neutrophil triggers Ca2+ mobilisation in nearby cells by engaging their NMDA receptors. Glutamate imaging reveals that the activated cells in turn release glutamate thus triggering a chain reaction that generates long-range signal exchange among the neutrophil assembly. These observations uncover a regenerative-wave mechanism - NMDA receptor dependent glutamate-induced glutamate release - that enables remote communication, or a signal-spreading  detonation - among blood cells.


title: Skeletal muscle-derived extracellular vesicles are altered with chronic contractile activity

abstract: Extracellular vesicles (EVs) are small lipid membrane-bound structures that are secreted by all cells, and play a central role in cellular communication. EVs are released from skeletal muscle during exercise, but the effects of contractile activity on skeletal muscle-derived EVs (Skm-EVs) are poorly understood due to the challenges in distinguishing Skm-EVs derived from exercising muscle in vivo. To specifically characterize Skm-EVs, C2C12 myoblasts were differentiated into myotubes, and electrically paced (3h/day x 4days @14V, C-PACE EM, IonOptix) to mimic chronic exercise in vitro. EVs were isolated from conditioned media from control and stimulated myotubes using differential ultracentrifugation. Isolated EVs were characterized biophysically (size, zeta potential, yield, protein markers and by transmission electron microscopy; TEM). Chronic stimulation increased markers of mitochondrial biogenesis such as MitoTracker Red staining, cytochrome c oxidase activity and expression of cytochrome c (p<0.05, N=7-8) in stimulated vs. non-stimulated myotubes. The average size of EVs from chronically stimulated myotubes (CS-EVs, 132 nm) was 26% smaller than control (CON-EVs, 178 nm) (p<0.05, N=8). Size distribution analysis revealed that CS-EVs were enriched with 100-150 nm sized small EVs, while CON-EVs were largely composed of 200-250 nm sized vesicles (p<0.05, main interaction effect, N=8). TEM confirmed the presence of round-shaped vesicles of about 30-100 nm with an intact lipid bilayer in CON-EVs and CS-EVs. Zeta potential was 27% lower in CS-EVs vs. CON-EVs (p<0.05, N=8), and total EV protein yield remained unchanged between groups. Protein-based EV characterization showed that both CON-EVs/CS-EVs were CD81+ but CD63-, and expressed hallmark cytosolic proteins recovered in EVs: Tsg101, Flotillin-1, HSP70, and Alix. CD81 and HSP70 expression increased in CS-EVs vs. CON-EVs (p<0.05, N=9). We evaluated if chronic stimulation affected whole cell expression of transmembrane and cytosolic proteins used in EV origin/purity analysis. CD63 and ApoA1 were reduced with chronic stimulation in myotube lysates (p<0.05, N=7), whereas Tsg101, CD81, Flotillin-1 and HSP70 levels remained constant. Taken together, our study revealed that chronic stimulation triggers the release of more stable, smaller sized EVs, enriched with transmembrane and cytosolic protein markers of small EVs, and are CD81+/CD63-indicating the origin of these EVs might be ectosomal rather than endosomal. The upstream signaling cascades that regulate biogenesis of EVs with chronic stimulation, and whether skeletal muscle-EVs are released through endosomal or ectosomal pathways remains to be elucidated. Our findings support the role of chronic contractile activity in the modulation of EV biophysical characteristics. Whether this affects biological cargo recruitment into CS-EVs, and their subsequent biological activity remains to be established.


title: Human cytomegalovirus vMIA inhibits MAVS oligomerization at peroxisomes in an MFF-dependent manner

abstract:
Upon intracellular recognition of viral RNA, RIG-I-like proteins interact with MAVS at peroxisomes and mitochondria, inducing its oligomerization and the downstream production of direct antiviral effectors. The human cytomegalovirus (HCMV) is able to specifically evade this antiviral response, via its antiapoptotic protein vMIA. Besides suppressing the programmed cell death of infected cells, vMIA inhibits the antiviral signalling at mitochondria by inducing the organelles fragmentation, consequently hindering the interaction between MAVS and the endoplasmic reticulum protein STING. Here, we demonstrate that vMIA interferes with the peroxisomal antiviral signalling via a distinct mechanism that is independent of the organelles morphology and does not affect STING. vMIA interacts with MAVS at peroxisomes and inhibits its oligomerization, restraining downstream signalling, in an MFF-dependent manner. This study also demonstrates that vMIA is totally dependent on the organelles fission machinery to induce peroxisomal fragmentation, while this dependency is not observed at mitochondria. Furthermore, although we demonstrate that vMIA is also able to inhibit MAVS oligomerization at mitochondria, our results indicate that this process, such as the whole vMIA-mediated inhibition of the mitochondrial antiviral response, is independent of MFF. These observed differences in the mechanisms of action of vMIA towards both organelles, likely reflect their intrinsic differences and roles throughout the viral infection.

This study uncovers specific molecular mechanisms that may be further explored as targets for antiviral therapy and highlights the relevance of peroxisomes as platforms for antiviral signalling against HCMV.


title: Long-Term Culture of Patient-Derived Cardiac Organoids Recapitulated Duchenne Muscular Dystrophy Cardiomyopathy and Disease Progression

abstract: Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disease which to-date incurable. The major cause of death is dilated cardiomyopathy, however the pathogenesis is unclear as existing cellular and animal models do not fully recapitulate the human disease phenotypes. In this study, we generated cardiac organoids from patient-derived pluripotent stem cells (DMD-CO) and isogenic-corrected controls (DMD-Iso-CO) and studied if DMD-related cardiomyopathy and disease progression occur in the organoids upon long-term culture (up to 93 days). Histological analysis showed that DMD-CO lacks initial proliferative capacity, displayed a progressive loss of -sarcoglycan localization and high stress in endoplasmic reticulum. Additionally, the cardiomyocyte deteriorated over time, and fibrosis and adipogenesis were observed in DMD-CO. RNA sequencing analysis confirmed a distinct transcriptomic profile in DMD-CO which were associated with functional enrichment in hypertrophy/dilated cardiomyopathy, arrhythmia, adipogenesis and fibrosis pathways. Moreover, five miRNAs were identified to be crucial in this dysregulated gene network. In conclusion, we generated patient-derived cardiac organoid model that displayed DMD-related cardiomyopathy and disease progression phenotypes in long-term culture. We envision the feasibility to develop a more complex, realistic and reliable in vitro 3D human cardiac-mimics to study DMD-related cardiomyopathies.


title: Mouse models of COVID-19 recapitulate inflammatory pathways rather than gene expression

abstract:
BACKGROUNDHow well mouse models recapitulate the transcriptional profiles seen in humans remains debatable, with both conservation and diversity identified in various settings. The K18-hACE2 mouse model has been widely used for evaluation of new interventions for COVID-19.

METHODHerein we use RNA-Seq data and bioinformatics approaches to compare the transcriptional responses in the SARS-CoV-2 infected lungs of K18-hACE2 mice with those seen in humans.

RESULTSOverlap in differentially expressed genes was generally poor ({approx}20-30%), even when multiple studies were combined. The overlap was not substantially improved when a second mouse model was examined wherein hACE was expressed from the mouse ACE2 promoter. In contrast, analyses of immune signatures and inflammatory pathways illustrated highly significant concordances between the species.

CONCLUSIONAs immunity and immunopathology are the focus of most studies, these hACE2 transgenic mouse models can thus be viewed as representative and relevant models of COVID-19.


title: Dictionary learning for integrative, multimodal, and scalable single-cell analysis

abstract:
Mapping single-cell sequencing profiles to comprehensive reference datasets represents a powerful alternative to unsupervised analysis. Reference datasets, however, are predominantly constructed from single-cell RNA-seq data, and cannot be used to annotate datasets that do not measure gene expression. Here we introduce  bridge integration, a method to harmonize singlecell datasets across modalities by leveraging a multi-omic dataset as a molecular bridge. Each cell in the multi-omic dataset comprises an element in a  dictionary, which can be used to reconstruct unimodal datasets and transform them into a shared space. We demonstrate that our procedure can accurately harmonize transcriptomic data with independent single cell measurements of chromatin accessibility, histone modifications, DNA methylation, and protein levels. Moreover, we demonstrate how dictionary learning can be combined with sketching techniques to substantially improve computational scalability, and harmonize 8.6 million human immune cell profiles from sequencing and mass cytometry experiments. Our approach aims to broaden the utility of single-cell reference datasets and facilitate comparisons across diverse molecular modalities.

AvailabilityInstallation instructions, documentations, and vignettes are available at http://www.satijalab.org/seurat


title: The structure of Vibrio cholerae FeoC reveals conservation of the helix-turn-helix motif but not the cluster-binding domain

abstract: Most pathogenic bacteria require ferrous iron (Fe2+) in order to sustain infection within hosts. The ferrous iron transport (Feo) system is the most highly-conserved prokaryotic transporter of Fe2+, but its mechanism remains to be fully characterized. Most Feo systems are composed of two proteins: FeoA, a soluble SH3-like accessory protein and FeoB, a membrane protein that translocates Fe2+ across a lipid bilayer. Some bacterial feo operons encode FeoC, a third soluble, winged-helix protein that remains enigmatic in function. We previously demonstrated that select FeoC proteins bind O2-sensitive [4Fe-4S] clusters via Cys residues, leading to the proposal that some FeoCs could sense O2 to regulate Fe2+ transport. However, not all FeoCs conserve these Cys residues, and FeoC from the causative agent of cholera (Vibrio cholerae) notably lacks any Cys residues, precluding cluster binding. In this work, we determined the NMR structure of VcFeoC, which is monomeric and conserves the helix-turn-helix domain seen in other FeoCs. In contrast, however, the structure of VcFeoC reveals a truncated winged {beta}-sheet in which the cluster-binding domain is notably absent. To test the interactions of VcFeoC with VcFeoB, we used NMR to demonstrate that these proteins interact in a 1:1 stoichiometry with a Kd of ca. 25 M. Finally, using homology modeling, we predicted the structure of VcNFeoB and used docking to identify an interaction site with VcFeoC, which is confirmed by NMR spectroscopy. These findings provide the first atomic-level structure of VcFeoC and contribute to a better understanding of its role vis-a-vis FeoB.

category: biochemistry


title: Natural Variation Meets Synthetic Biology: Promiscuous Trichome Expressed Acyltransferases from Nicotiana acuminata

abstract:
Acylsugars are defensive, trichome-synthesized sugar esters produced in plants across the Solanaceae (nightshade) family. Although assembled from simple metabolites and synthesized by a relatively short core biosynthetic pathway, tremendous within- and across-species acylsugar structural variation is documented across the family. To advance our understanding of the diversity and the synthesis of acylsugars within the Nicotiana genus, trichome extracts were profiled across the genus coupled with transcriptomics-guided enzyme discovery and in vivo and in vitro analysis. Differences in the types of sugar cores, numbers of acylations, and acyl chain structures contributed to over 300 unique annotated acylsugars throughout Nicotiana. Placement of acyl chain length into a phylogenetic context revealed that an unsaturated acyl chain type was detected in a few closely-related species. A comparative transcriptomics approach identified trichome-enriched Nicotiana acuminata acylsugar biosynthetic candidate enzymes. > 25 acylsugar variants could be produced in a single enzyme assay with four acylsugar acyltransferases (NacASAT1-4) together with structurally diverse acyl-CoAs and sucrose. Liquid chromatography coupled with mass spectrometry screening of in vitro products revealed the ability of these enzymes to make acylsugars not present in Nicotiana plant extracts. In vitro acylsugar production also provided insights into acyltransferase acyl donor promiscuity and acyl acceptor specificity as well as regiospecificity of some ASATs. This study suggests that promiscuous Nicotiana acyltransferases can be used as synthetic biology tools to produce novel and potentially useful metabolites.

ONE SENTENCE SUMMARYAnalysis of Nicotiana glandular trichome metabolites and BAHD acyltransferases revealed diverse sucrose and glucose based acylesters.


title: A general theory of specific binding: insights from a genetic, mechano-chemical protein model

abstract:
Proteins need to selectively interact with specific targets among a multitude of similar molecules in the cell. Despite a firm physical understanding of binding interactions, we lack a general theory of how proteins evolve high specificity. Here, we present a model that combines chemistry, mechanics and genetics, and explains how their interplay governs the evolution of specific protein-ligand interactions. The model shows that there are many routes to achieving discrimination - by varying degrees of flexibility and shape/chemistry complementarity - but the key ingredient is precision. Harder discrimination tasks require more collective and precise coaction of structure, forces and movements. Proteins can achieve this through correlated mutations extending far from a binding site, which fine tune the localized interaction with the ligand. Thus, the solution of more complicated tasks is aided by increasing the protein, and proteins become more evolvable and robust when they are larger than the bare minimum required for discrimination. Our model makes testable, specific predictions about the role of flexibility in discrimination, and how to independently tune affinity and specificity. Thus, the proposed theory of molecular discrimination addresses the natural question "why are proteins so big?" A possible answer is that molecular discrimination is often a hard task best performed by adding more layers to the protein.

Significance statementProteins excel at discriminating between molecules. Otherwise, cells would not be able to accurately translate mRNA into protein sequences, chemical signals would get mixed up, and enzymes would catalyze unwanted interactions. To understand how proteins can achieve this, we develop a model of protein binding to study evolution of molecular discrimination through the interplay of shape, flexibility, chemical binding and entropy. The model reveals that mutations far from the binding site can fine-tune binding interactions, and larger proteins are better discriminators. Proteins thus solve the discrimination problem by being large so that they have many ways of fine-tuning binding.


title: Structural basis for receptor selectivity and inverse agonism in S1P5 receptors

abstract: The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PR) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 [A] resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric subpocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, the new S1P5-inverse agonist structure sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR-family.


title: Cryo-EM structure of the Rhodobaca bogoriensis RC-LH1-PufX dimeric complex at 2.9 angstrom

abstract: The reaction centre-light harvesting 1 (RC-LH1) complex is essential for converting light into proton motive force in photosynthetic bacteria. RC-LH1 is a monomer in most purple bacteria, but in Rhodobacter species, it is a dimer. Its assembly depends on an accessory polypeptide (PufX) and, ultimately, on photosynthetic growth. To date, knowledge on the RC-LH1-PufX structure, where the dimer has two incomplete  C-shaped antenna rings surrounding an RC, is mainly limited to the model organism Rhodobacter sphaeroides. Here we present a cryo-electron microscopy structure at 2.9 [A] from Rhodobaca bogorensis strain LBB1. RCs are surrounded by 30 antennas and incorporate protein Y and PufX. RCs are stably connected by PufX, which self-interacts, electrostatically attracts cytochrome c2 (cyt c2) and forms extensive networks with co-factors. This structure underlines coordinated energy transfer in a combinatorial manner, providing a basis to describe bacterial photosynthesis within a dimeric photosynthetic apparatus.


title: Cryo-EM structure of the Rhodobaca bogoriensis RC-LH1-PufX dimeric complex at 2.9 A


title: Distinct execution modes of a biochemical necroptosis model explain cell type-specific responses and variability to cell-death cues

abstract: Necroptosis is a form of regulated cell death that has been associated with degenerative disorders, autoimmune processes, inflammatory diseases, and cancer. To better understand the biochemical mechanisms of necroptosis cell death regulation, we constructed a detailed biochemical model of tumor necrosis factor (TNF)-induced necroptosis based on known molecular interactions. Intracellular protein levels, used as model inputs, were quantified using label-free mass spectrometry, and the model was calibrated using Bayesian parameter inference to experimental protein time course data from a well-established necroptosis-executing cell line. The calibrated model accurately reproduced the dynamics of phosphorylated mixed lineage kinase domain-like protein (pMLKL), an established necroptosis reporter. A dynamical systems analysis identified four distinct modes of necroptosis signal execution, which can be distinguished based on rate constant values and the roles of the deubiquitinating enzymes A20 and CYLD in the regulation of RIP1 ubiquitination. In one case, A20 and CYLD both contribute to RIP1 deubiquitination, in another RIP1 deubiquitination is driven exclusively by CYLD, and in two modes either A20 or CYLD acts as the driver with the other enzyme, counterintuitively, inhibiting necroptosis. We also performed sensitivity analyses of initial protein concentrations and rate constants and identified potential targets for modulating necroptosis sensitivity among the biochemical events involved in RIP1 ubiquitination regulation and the decision between complex II degradation and necrosome formation. We conclude by associating numerous contrasting and, in some cases, counterintuitive experimental results reported in the literature with one or more of the model-predicted modes of necroptosis execution. Overall, we demonstrate that a consensus pathway model of TNF-induced necroptosis can provide insights into unresolved controversies regarding the molecular mechanisms driving necroptosis execution for various cell types and experimental conditions.

category: systems biology


title: Resident T cells orchestrate adipose tissue remodeling in a site peripheral to infection

abstract: Infection with helminth parasites can affect adiposity, but underlying mechanisms that regulate this process are unclear. We found that fat content of mesenteric adipose tissue (mAT) declined in mice during infection with gut-restricted parasitic worms. This was associated with the accumulation of metabolically activated, immunostimulatory cytokine- and extracellular matrix-secreting multipotent stromal cells, which had potential to differentiate into preadipocytes. Concomitantly, mAT became infiltrated with Th2 lymphocytes that took up long-term residence and responded to signals from stromal cells by producing stromal cell-stimulating cytokines, including Amphiregulin. Signals delivered by Amphiregulin to stromal cells were required for immunity to infection. Our findings reveal intricate intercellular communication between Th2 cells and adipocyte progenitors and link immunity to intestinal infection to T cell-dependent effects on the adipocyte lineage.


title: The chromatin landscape of Th17 cells reveals mechanisms of diversification of regulatory and pro-inflammatory states

abstract: Th17 cells are a heterogenous cell population consisting of non-pathogenic Th17 cells (npTh17) that contribute to tissue homeostasis and pathogenic Th17 cells (pTh17) that are potent mediators of tissue inflammation. To reveal regulatory mechanisms underlying Th17 heterogeneity, we performed combined ATAC-seq and RNA-seq and discovered substantial differences in the chromatin landscape of npTh17 and pTh17 cells both in vitro and in vivo. Compared to other CD4+ T cell subsets, npTh17 cells share accessible chromatin programs with Tregs, and pTh17 cells have an intermediate profile spanning features of npTh17 cells and Th1 cells. Integrating single-cell ATAC-seq and single-cell RNA-seq, we inferred self-reinforcing and mutually exclusive regulatory networks controlling the different cell states and predicted transcription factors (TFs) shaping the chromatin landscape of Th17 cell pathogenicity. We validated one novel TF, BACH2, which promotes immunomodulatory npTh17 programs and restrains pro-inflammatory Th1-like programs in Th17 cells and showed genetic evidence for protective variants in the human BACH2 locus associated with multiple sclerosis. Our work uncovered mechanisms that regulate Th17 heterogeneity, revealed shared regulatory programs with other CD4+ T cell subsets, and identified novel drivers of Th17 pathogenicity as potential targets to mitigate autoimmunity.


title: IgG1 responses following SARS-CoV-2 infection are polyclonal and highly personalized, whereby each donor and each clone displays a distinct pattern of cross-reactivity against SARS-CoV-2 variants

abstract: Using a recently introduced efficient mass spectrometry-based approach we monitored individual donors IgG1 clonal responses in molecular detail, examining SARS-CoV-2 spike-protein-specific IgG1 repertoires. We monitored the plasma clonal IgG1 profiles of 8 donors (4 male and 4 female) who had recently experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we charted the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer as antigen. We observed that shortly after infection in between <0.1% to almost 10% of all IgG1 antibody molecules present in plasma did bind to the spike protein. Each donor displayed a unique plasma IgG1 repertoire, but also each donor displayed a unique and polyclonal antibody response against the SARS-CoV-2 spike-protein variants. Our analyses revealed that certain clones exhibit (alike) binding affinity towards all four tested spike-protein variants, whereas other clones displayed strong unique mutant-specific affinity. We conclude that each infected person generates a unique polyclonal response following infection, whereby some of these clones can bind multiple viral variants, whereas other clones do not display such cross-reactivity. In general, by assessing IgG1 repertoires following infection it becomes possible to identify and select fully matured human plasma antibodies that target specific antigens, and display either high specificity or cross-reactivity versus mutated versions of the antigen, which will aid in selecting antibodies that may be developed into biotherapeutics.


title: Shifting mutational constraints in the SARS-CoV-2 receptor-binding domain during viral evolution

abstract: SARS-CoV-2 has evolved variants with substitutions in the spike receptor-binding domain (RBD) that impact its affinity for ACE2 receptor and recognition by antibodies. These substitutions could also shape future evolution by modulating the effects of mutations at other sites--a phenomenon called epistasis. To investigate this possibility, we performed deep mutational scans to measure the effects on ACE2 binding of all single amino-acid mutations in the Wuhan-Hu-1, Alpha, Beta, Delta, and Eta variant RBDs. Some substitutions, most prominently N501Y, cause epistatic shifts in the effects of mutations at other sites, thereby shaping subsequent evolutionary change. These epistatic shifts occur despite high conservation of the overall RBD structure. Our data shed light on RBD sequence-function relationships and facilitate interpretation of ongoing SARS-CoV-2 evolution.


title: Conservation and evolution of the sporulation gene set in diverse members of the Firmicutes

abstract:
The current classification of the phylum Firmicutes (new name, Bacillota) features eight distinct classes, six of which include known spore-forming bacteria. In Bacillus subtilis, sporulation involves up to 500 genes, many of which do not have orthologs in other bacilli and/or clostridia. Previous studies identified about 60 sporulation genes of B. subtilis that were shared by all spore-forming members of the Firmicutes. These genes are referred to as the sporulation core or signature although many of these are found also in genomes of non-spore-formers. Using an expanded set of 180 firmicute genomes from 160 genera, including 76 spore-forming species, we investigated the conservation of the sporulation genes, in particular, seeking to identify lineages that lack some of the genes from the conserved sporulation core. The results of this analysis confirmed that many small acid-soluble spore proteins (SASPs), spore coat proteins, and germination proteins, which were previously characterized in bacilli, are missing in spore-forming members of Clostridia and other classes of Firmicutes. A particularly dramatic loss of sporulation genes was observed in the spore-forming members of the families Planococcaceae and Erysipelotrichaceae. Fifteen species from diverse lineages were found to carry skin (sigK-interrupting) elements of different sizes that all encoded SpoIVCA-like recombinases but did not share any other genes. Phylogenetic trees built from concatenated alignments of sporulation proteins and ribosomal proteins showed similar topology, indicating an early origin and subsequent vertical inheritance of the sporulation genes.

IMPORTANCEMany members of the phylum Firmicutes (Bacillota) are capable of producing endospores, which enhance the survival of important Gram-positive pathogens that cause such diseases as anthrax, botulism, colitis, gas gangrene, and tetanus. We show that the core set of sporulation genes, defined previously through genome comparisons of several bacilli and clostridia, is conserved in a wide variety of spore-formers from several distinct lineages of Firmicutes. We also detect widespread loss of sporulation genes in many organisms, particularly within families Planococcaceae and Erysipelotrichaceae. Members of these families, such as Lysinobacillus sphaericus and Clostridium innocuum, could be excellent model organisms for studying sporulation mechanisms, such as engulfment, formation of the spore coat, and spore germination.


title: Global distribution and diversity of prevalent sewage water plasmidomes

abstract:
Sewage water from around the world contains an abundance of short plasmids, a number of which harbor antimicrobial resistance genes (ARGs). The global dynamics of plasmid-derived antimicrobial resistance and functions is only starting to be unveiled. Here, we utilized a previously created dataset of 159,332 assumed small plasmids from 24 different globally collected sewage samples. We investigated the detailed phylogeny as well as the interplay between their protein domains, ARGs, and predicted bacterial host genera to help understand the global sewage plasmidome dynamics.

A total of 58,429 circular elements carried genes encoding for plasmid-related features, and MASH distance analyses showed a very high degree of diversity. A single very diverse cluster of 520 predicted Acinetobacter plasmids was predominant among the European sewage water.

Based on functional domain network analysis, we identified three groups of plasmids, mainly replication and mobilization domains. However, these backbone domains were not exclusive to any given group. Acinetobacter was the dominant host genus among theta-replicating plasmids at these size ranges. They contained a reservoir of the macrolide resistance gene pair msr(E) and mph(E). Macrolide resistance genes were the most common resistance genes in sewage plasmidomes and found in the largest number of unique plasmids.

While msr(E) and mph(E) were limited to Acinetobacter, erm(B) was disseminated among a range of Firmicutes plasmids, including Staphylococcus and Streptococcus, highlighting a potential reservoir of antibiotics resistance for these pathogens from around the globe.

ImportanceAntimicrobial resistance is a global threat to human health inhibiting our ability to treat infectious diseases. This study utilizes sewage water plasmidomes to identify plasmid-derived features, and highlights antimicrobial resistance genes, particularly macrolide resistance to be abundant in sewage water plasmidomes in Firmicutes and Acinetobacter hosts. The emergence of macrolide resistance in these bacteria hints that macrolide selective pressure exists in sewage water, and that the resident bacteria readily can acquire macrolide resistance via small plasmids.


title: Early events in reovirus infection influence induction of innate immune response

abstract:
Mammalian orthoreovirus (reovirus) is a double-stranded (ds)RNA virus which encapsidates its 10 genome segments within a double-layered viral particle. Reovirus infection triggers an antiviral response in host cells which serves to limit viral replication. This antiviral response is initiated by recognition of the incoming viral genome by host sensors present in the cytoplasm. However, how host sensors gain access to the reovirus genome is unclear as this dsRNA is protected by the viral particle proteins throughout infection. To initiate infection, reovirus particles are endocytosed and the outer viral particle layer is disassembled through the action of host proteases. This disassembly event is required for viral escape into the cytoplasm to begin replication. We show that even after disassembly is complete, endosomal proteases are required to induce an immune response to reovirus. Additionally, counter to dogma, our data demonstrate at least some viral dsRNA genome is exposed and detectable during entry. We hypothesize that some proportion of reovirus particles remain trapped within endosomes, allowing for the breakdown of these particles and release of their genome. We show that rapidly uncoating mutants escape the endosome more rapidly and induce a diminished immune response. Further, we show that particles entering through dynamin-independent pathways evade detection by host sensors. Overall, our data provide new insight into how genomes from entering reovirus particles are detected by host cells.

IMPORTANCEViruses must infect host cells to replicate, often killing the host cell in the process. However, hosts can activate defenses to limit viral replication and protect the organism. To trigger these host defenses to viral infections, host cells must first recognize they are infected. Mammalian orthoreovirus (reovirus) is a model system used to study host-virus interactions. This study identifies aspects of host and virus biology which determine the capacity of host cells to detect infection. Notably, entry of reovirus into host cells plays a critical role in determining the magnitude of immune response triggered during infection. Mutants of reovirus which can enter cells more rapidly are better at avoiding detection by the host. Additionally, reovirus can enter cells through multiple routes. Entry through some of these routes also helps reovirus evade detection.


title: Transcriptomic landscapes of SARS-CoV-2-infected and bystander lung cells reveal a selective upregulation of NF-κB-dependent coding and non-coding proviral transcripts

abstract: Detailed knowledge of cellular networks that are modulated by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to understand viral replication and host response. So far, transcriptomic analyses of interactions between SARS-CoV-2 and cells were performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, both leading to inaccurate or low-resolution gene expression interpretations. Moreover, they generally focused on annotated messenger RNAs (mRNAs), ignoring other transcripts, such as long non-coding RNAs (lncRNAs) and unannotated RNAs. Here, we performed deep polyA+ transcriptome analyses of lung epithelial A549 cells infected with SARS-CoV-2, which were sorted based on the expression of the viral protein spike (S). To increase the sequencing depth and improve the robustness of the analysis, the samples were depleted of viral transcripts. Infection caused a massive reduction in mRNAs and lncRNAs, including transcripts coding for antiviral innate immune proteins, such as interferons (IFNs). This absence of IFN response probably explains the poor transcriptomic response of bystander cells co-cultured with spike positive (S+) ones. NF-{kappa}B and inflammatory response were among the pathways that escaped the global shutoff in S+ cells. In agreement with the RNA-seq analysis, inflammatory cytokines, but not IFNs, were produced and secreted by infected cells. Functional investigations revealed the proviral function of the NF-kB subunit p105/p50 and some of its known target genes, including IL32 and IL8, as well as the lncRNA ADIRF-AS1, which we identified as a novel NF-kB target gene. Thus, analyzing the polyA+ transcriptome of sorted populations of infected lung cells allowed unprecedented identification of cellular functions that are directly affected by infection and the recovery of coding and non-coding genes that contribute to SARS-CoV-2 replication.


title: DLK-dependent biphasic reactivation of herpes simplex virus latency established in the absence of antivirals

abstract:
Understanding the molecular mechanism of herpes simplex virus type 1 (HSV-1) latent infection and reactivation in neurons requires the use of model systems. However, establishing a quiescent infection in cultured neurons is problematic as infectious virus released from any productively infected neuron can superinfect the cultures. Here, we describe a new reporter virus, HSV-1 Stayput-GFP, that is defective for cell-to-cell spread and can be used to model latency and reactivation at the single neuron level. Importantly, quiescent infection of neurons with Stayput-GFP can be established without the use of a viral DNA replication inhibitor. The establishment of a quiescent state requires a longer time frame than previous models of HSV latency using DNA replication inhibitors. This results in a decreased ability of the virus to reactivate, and the use of multiple reactivation triggers is required. Using this system, we demonstrate that an initial Phase I wave of lytic gene expression occurs independently of histone demethylase activity and viral DNA replication but is dependent on the neuronal cell stress protein, DLK. Progression into the later, Phase II wave of reactivation, characterized by detectable late viral protein synthesis and a requirement for histone demethylase activity, is also observed with the Stayput-GFP model system. These data demonstrate that the two waves of viral gene expression following HSV-1 reactivation are independent of secondary infection and occur when latent infections are established in the absence of a viral DNA replication inhibitor.

ImportanceHerpes simplex virus-1 (HSV-1) enters a latent infection in neurons and periodically reactivates. Reactivation manifests as a variety of clinical symptoms. Studying latency and reactivation in vitro is invaluable, allowing the molecular mechanisms behind both processes to be targeted by therapeutics that reduce the clinical consequences. Here, we describe a novel in vitro model system using a cell-to-cell spread defective HSV-1, known as Stayput-GFP, which allows for the study of latency and reactivation at the single neuron level. We anticipate this new model system will be an incredibly valuable tool for studying the establishment and reactivation of HSV-1 latent infection in vitro. Using this model, we find that initial reactivation events are dependent on cellular stress kinase DLK, but independent of histone demethylase activity and viral DNA replication. Our data therefore demonstrate the essential role of DLK in mediating a wave of lytic gene expression unique to reactivation.


title: A structural dynamic explanation for observed escape of SARS-CoV-2 BA.2 variant mutation S371L/F

abstract: The SARS-CoV-2 Omicron sub-variants BA.1 and BA.2 have become the dominant variants worldwide due to enhanced transmissibility and immune evasion. In response to the rise of BA.1 and BA.2, two recent studies by Liu et al. and Iketani et al. provide a detailed analysis of loss of therapeutic antibody potency through evaluation of escape by pseudotyped viruses harboring BA.1 and BA.2 receptor binding domain (RBD) point mutations. Surprisingly, Liu et al. and Iketani et al. observed a profoundly broad escape effect for the individual mutations S371L and S371F. This result cannot be explained by known escape mechanisms of the SARS-CoV-2 RBD, and conflicts with existing computational and experimental escape measurements for S371 mutations performed on monomeric RBD. Through an examination of these conflicting datasets and a structural analysis of the antibodies assayed by Liu et al. and Iketani et al., we propose a mechanism to explain S371L/F escape according to a perturbation of spike trimer conformational dynamics that has not yet been described for any SARS-CoV-2 escape mutation. The proposed mechanism is relevant to Omicron and future variant surveillance as well as therapeutic antibody design.


title: Adenosine A2A Receptor (A2AR) agonists improve survival in K28-hACE2 mice following SARS CoV-2 infection

abstract:
Effective and available therapies for the treatment of COVID-19 disease are limited. Apadenoson is a highly potent selective anti-inflammatory adenosine A2A receptor (A2AR) agonist and potential treatment option for COVID-19 patients. Apadenoson, when administered after infection with SARS CoV-2, was found to decrease weight loss, improve clinical symptoms, reduce levels of a several proinflammatory cytokines and chemokines in bronchial lavage (BAL) fluid, and promote increased survival in K18hACE2 transgenic mice. Of note, administering apadenoson after, but not prior to Covid-19 infection, caused a rapid decrease in lung viral burden. The work presented provides the foundation for further examination of these drugs as a therapy option for COVID-19.

SummaryApadenoson therapy improves COVID-19 outcome


title: Discovery of a novel coronavirus in Swedish bank voles (Myodes glareolus)

abstract: We identified a novel Betacoronavirus from bank voles (Myodes glareolus) in Grimso, Sweden. Repeated detection over three years and an overall prevalence of 3.4% suggests the virus commonly occurs in bank voles. Furthermore, phylogenetic analyses indicate the virus belongs to a highly divergent Embecovirus lineage predominantly associated with bank voles.


title: Zebrafish her3 knockout impacts developmental and cancer-related gene signatures

abstract:
HES3 is a basic helix-loop-helix transcription factor that regulates neural stem cell renewal during development. HES3 overexpression is predictive of reduced overall survival in patients with fusion-positive rhabdomyosarcoma, a pediatric cancer that resembles immature and undifferentiated skeletal muscle. However, the mechanisms of HES3 cooperation in fusion-positive rhabdomyosarcoma are unclear and are likely related to her3/HES3s role in neurogenesis. To investigate HES3s function during development, we generated a zebrafish CRISPR/Cas9 knockout of her3, the zebrafish ortholog of HES3. Loss of her3 is not embryonic lethal and adults exhibit expected Mendelian ratios. Embryonic her3 zebrafish mutants are significantly smaller than wildtype and a subset present with lens defects as adults. Transcriptomic analysis of her3 mutant embryos indicates that genes involved in organ development, such as pctp and grinab, are significantly downregulated. Further, differentially expressed genes in her3 knockout embryos are enriched for HOX and SOX10 motifs. Several cancer-related gene pathways are impacted, including the inhibition of matrix metalloproteinases. Altogether, this new model is a powerful system to study her3/HES3-mediated neural development and its misappropriation in cancer contexts.

Summary StatementHere, we generate and characterize a zebrafish her3/HES3 knockout to elucidate the functional role of her3/HES3, a transcriptional repressor, in neural development and tumorigenic processes.