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Background Isolation of neurons from the adult mouse CNS is important in order to study their gene expression during development or the course of different diseases. New methods Here we present two different methods for the enrichment or isolation of neurons from adult mouse CNS. These methods: are either based on flow cytometry sorting of eYFP expressing neurons, or by depletion of non-neuronal cells by sorting with magnetic-beads. Results Enrichment by FACS sorting of eYFP positive neurons results in a population of 62.4% NeuN positive living neurons. qPCR data shows a 3–5fold upregulation of neuronal markers. The isolation of neurons based on depletion of non-neuronal cells using the Miltenyi Neuron Isolation Kit, reaches a purity of up to 86.5%. qPCR data of these isolated neurons shows an increase in neuronal markers and an absence of glial markers, proving pure neuronal RNA isolation. Comparison with existing methods Former data related to neuronal gene expression are mainly based on histology, which does not allow for high-throughput transcriptome analysis to examine differential gene expression. Conclusion These protocols can be used to study cell type specific gene expression of neurons to unravel their function in the process of damage to the CNS.

In the central nervous system, the type I transmembrane glycoprotein NG2 (nerve-glia antigen 2) is only expressed by pericytes and oligodendrocyte precursor cells (OPCs). Therefore, OPCs are also termed NG2 glia. Their fate during development has been investigated systematically in several genetically modified mouse models. Consensus exists that postnatal NG2 glia are restricted to the oligodendrocyte (OL) lineage, while, at least in the forebrain, embryonic NG2 glia could also generate astrocytes. In addition, experimental evidence for a neurogenic potential of NG2 glia in the early embryonic brain (before E16.5) has been provided. However, this observation is still controversial. Here, we took advantage of reliable transgene expression in NG2-EYFP and NG2-CreERT2 knock-in mice to study the fate of early embryonic NG2 glia. While pericytes were the main cells with robust NG2 gene activity at E12.5, only a few OPCs expressed NG2 at this early stage of embryogenesis. Subsequently, this proportion of OPCs increased from 3% (E12.5) to 11% and 25% at E14.5 and E17.5, respectively. When Cre DNA recombinase activity was induced at E12.5 and E14.5 and pups were analyzed at postnatal day 0 (P0) and P10, the vast majority of recombined cells, besides pericytes, belonged to the OL lineage cells, with few astrocytes in the ventral forebrain. In other brain regions such as brain stem, cerebellum, and olfactory bulb only OL lineage cells were detected. Therefore, we conclude that NG2 glia from early embryonic brain are restricted to a gliogenic fate and do not differentiate into neurons after birth.

The electrographic hallmark of childhood absence epilepsy (CAE) and other idiopathic forms of epilepsy are 2.5–4 Hz spike and wave discharges (SWDs) originating from abnormal electrical oscillations of the cortico-thalamo-cortical network. SWDs are generally associated with sudden and brief non-convulsive epileptic events mostly generating impairment of consciousness and correlating with attention and learning as well as cognitive deficits. To date, SWDs are known to arise from locally restricted imbalances of excitation and inhibition in the deep layers of the primary somatosensory cortex. SWDs propagate to the mostly GABAergic nucleus reticularis thalami (NRT) and the somatosensory thalamic nuclei that project back to the cortex, leading to the typical generalized spike and wave oscillations. Given their shared anatomical basis, SWDs have been originally considered the pathological transition of 11–16 Hz bursts of neural oscillatory activity (the so-called sleep spindles) occurring during Non-Rapid Eye Movement (NREM) sleep, but more recent research revealed fundamental functional differences between sleep spindles and SWDs, suggesting the latter could be more closely related to the slow (+ clearance and neurotransmitter uptake from the extracellular space and the synaptic cleft, (iii) gap junction mechanical and functional coupling as well as hemichannel function, (iv) gliotransmission, (v) astroglial Ca2+ signaling and downstream effectors, (vi) reactive astrogliosis and cytokine release.

In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.

Senescent cells, which arise due to damage-associated signals, are apoptosis-resistant and can express a pro-inflammatory, tissue-destructive senescence-associated secretory phenotype (SASP). We recently reported that a component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface protein, S1, can amplify the SASP of senescent cultured human cells and that a related mouse β-coronavirus, mouse hepatitis virus (MHV), increases SASP factors and senescent cell burden in infected mice. Here, we show that SARS-CoV-2 induces senescence in human non-senescent cells and exacerbates the SASP in human senescent cells through Toll-like receptor-3 (TLR-3). TLR-3, which senses viral RNA, was increased in human senescent compared to non-senescent cells. Notably, genetically or pharmacologically inhibiting TLR-3 prevented senescence induction and SASP amplification by SARS-CoV-2 or Spike pseudotyped virus. While an artificial TLR-3 agonist alone was not sufficient to induce senescence, it amplified the SASP in senescent human cells. Consistent with these findings, lung p16INK4a+ senescent cell burden was higher in patients who died from acute SARS-CoV-2 infection than other causes. Our results suggest that induction of cellular senescence and SASP amplification through TLR-3 contribute to SARS-CoV-2 morbidity, indicating that clinical trials of senolytics and/or SASP/TLR-3 inhibitors for alleviating acute and long-term SARS-CoV-2 sequelae are warranted.

Horse mussels Modiolus modiolus can occur in dense aggregations and form areas of ecological and biological significance. In the Bay of Fundy, Canada, aggregations of horse mussels are associated with flow parallel bedforms, and this area is under consideration for designation as a sensitive benthic area which would provide protective measures. Basic demographic information is required to inform the development of effective conservation and management strategies and although general life-history characteristics of M. modiolus are known, detailed quantitative demographic information on this population is limited. The objective of this study was to characterize the population structure of horse mussels in the Bay of Fundy and assess change in key demographic characteristics since the last study in this area in 1997/1998. There have been significant changes in the population since 1998: the 2017 population contains larger, older, mature individuals, with significantly more females; 35% of the current population is over 20 yr of age. Direct evidence that this population has been impacted by bottom-contact fishing gear was also observed. Consistent with M. modiolus populations worldwide, this population demonstrates life-history traits (e.g. slow growth rates, late age of maturity, long lifespan) that make it sensitive and susceptible to disturbance. Coupled with the knowledge that this population overlaps with significant fishing activity, this study supports the assumption that this population is vulnerable to bottom-contact fishing and that recovery from adverse impacts would be slow and uncertain.

Additional treatment options for coronavirus disease (COVID-19) are urgently needed, particularly for populations at high risk of severe disease. This cross-sectional, retrospective study characterized the outcomes of 43 patients with nosocomial severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection with and without treatment using monoclonal SARS-CoV-2 spike antibodies (bamlanivimab or casirivimab/imdevimab). Our results indicate that treatment with monoclonal antibodies results in a significant decrease in disease progression and mortality when used for asymptomatic patients with early SARS-CoV-2 infection. Supplementary Information The online version contains supplementary material available at 10.1007/s15010-021-01657-y.

In photosynthetic thylakoid membranes the proton motive force (pmf) not only drives ATP synthesis, in addition it is central to controlling and regulating energy conversion. As a consequence, dynamic fine-tuning of the two pmf components, electrical (Δψ) and chemical (ΔpH), is an essential element for adjusting photosynthetic light reactions to changing environmental conditions. Good evidence exists that the Δψ/ΔpH partitioning is controlled by thylakoid potassium and chloride ion transporters and channels. However, a detailed mechanistic understanding of how these thylakoid ion transporter/channels control pmf partitioning is lacking. Here, we combined functional measurements on potassium and chloride ion transporter and channel loss-of-function mutants with extended mathematical simulations of photosynthetic light reactions in thylakoid membranes to obtain detailed kinetic insights into the complex interrelationship between membrane energization and ion fluxes across thylakoid membranes. The data reveal that potassium and chloride fluxes in the thylakoid lumen determined by the K+/H+ antiporter KEA3 and the voltage-gated Cl- channel VCCN1/Best1 have distinct kinetic responses that lead to characteristic and light-intensity-dependent Δψ/ΔpH oscillations. These oscillations fine-tune photoprotective mechanisms and electron transport which are particularly important during the first minutes of illumination and under fluctuating light conditions. By employing the predictive power of the model, we unravelled the functional consequences of changes in KEA3 and VCCN1 abundance and regulatory/enzymatic parameters on membrane energization and photoprotection.

Cells and tissues form the bewildering diversity of crustacean larval organ systems which are necessary for these organisms to autonomously survive in the plankton. For the developmental biologist, decapod crustaceans provide the fascinating opportunity to analyse how the adult organism unfolds from organ Anlagen compressed into a miniature larva in the sub-millimetre range. This publication is the second part of our survey of methods to study organogenesis in decapod crustacean larvae. In a companion paper, we have already described the techniques for culturing larvae in the laboratory and dissecting and chemically fixing their tissues for histological analyses. Here, we review various classical and more modern imaging techniques suitable for analyses of eidonomy, anatomy, and morphogenetic changes within decapod larval development, and protocols including many tips and tricks for successful research are provided. The methods cover reflected-light-based methods, autofluorescence-based imaging, scanning electron microscopy, usage of specific fluorescence markers, classical histology (paraffin, semithin and ultrathin sectioning combined with light and electron microscopy), X-ray microscopy (µCT), immunohistochemistry and usage of in vivo markers. For each method, we report our personal experience and give estimations of the method’s research possibilities, the effort needed, costs and provide an outlook for future directions of research.

The Coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To facilitate its own replication and avoid immune control, SARS-CoV-2 manipulates its target cells. Our results revealed that the SARS-CoV-2 non-structural protein 1 (Nsp1) plays a key role in viral immune evasion. It blocks the mRNA tunnel of the cellular ribosome, resulting in a shutdown of translation and strong attenuation of the host’s antiviral immune response.

GABA is the main inhibitory neurotransmitter in the CNS acting at two distinct types of receptor: ligand-gated ionotropic GABAA receptors and G protein-coupled metabotropic GABAB receptors, thus mediating fast and slow inhibition of excitability at central synapses. GABAergic signal transmission has been intensively studied in neurons in contrast to oligodendrocytes and their precursors (OPCs), although the latter express both types of GABA receptor. Recent studies focusing on interneuron myelination and interneuron-OPC synapses have shed light on the importance of GABA signaling in the oligodendrocyte lineage. In this review, we start with a short summary on GABA itself and neuronal GABAergic signaling. Then, we elaborate on the physiological role of GABA receptors within the oligodendrocyte lineage and conclude with a description of these receptors as putative targets in treatments of CNS diseases.

Astrocytes are an important component of the multipartite synapse and crucial for proper neuronal network function. Although small GTPases of the Rho family are powerful regulators of cellular morphology, the signaling modules of Rho-mediated pathways in astrocytes remain enigmatic. Here we demonstrated that the serotonin receptor 4 (5-HT4 R) is expressed in hippocampal astrocytes, both in vitro and in vivo. Through fluorescence microscopy, we established that 5-HT4 R activation triggered RhoA activity via Gα13 -mediated signaling, which boosted filamentous actin assembly, leading to morphological changes in hippocampal astrocytes. We investigated the effects of these 5-HT4 R-mediated changes in mixed cultures and in acute slices, in which 5-HT4 R was expressed exclusively in astrocytes. In both systems, 5-HT4 R-RhoA signaling changed glutamatergic synaptic transmission: It increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in mixed cultures and reduced the paired-pulse-ratio (PPR) of field excitatory postsynaptic potentials (fEPSPs) in acute slices. Overall, our present findings demonstrate that astrocytic 5-HT4 R-Gα13 -RhoA signaling is a previously unrecognized molecular pathway involved in the functional regulation of excitatory synaptic circuits.

Background Various tools are obtainable for the detection of Pneumocystis jirovecii, among them qPCR promising highest sensitivity. A novel molecular method is commercially available, the loop-mediated isothermal amplification (LAMP) assay. Objectives We compared the performance of the LAMP eazyplex® Pneumocystis jirovecii with the RealStar Pneumocystis jirovecii PCR 1.0 qPCR. Material/methods Overall, 162 lower respiratory tract specimens from 146 critically ill patients were investigated. LAMP assay and qPCR were carried out according to the manufacturer's recommendations. Positive results of the LAMP were described as time to positivity (TTP). The limit of detection (LOD) of the LAMP was analysed using 10-fold serial dilutions of a high positive P jirovecii respiratory sample. For each serial dilution, TTP of the LAMP was plotted against cycle threshold (Ct) values of the qPCR. Results The LOD of the LAMP was determined to be approximately 4 × 103 copies/mL. While the LAMP revealed 28 (17%) positive signals from 20 patients, by using qPCR 41 (25%) positive samples from 28 patients were identified. Overall agreement with qPCR was 92%. Five false-negative, one false-positive and nine invalid results were detected by the LAMP. Positive and negative predictive values were 96% each, and sensitivity and specificity were 84% and 99%, respectively. There was a low correlation between the TTP and the fungal load. Conclusion The LAMP is a time-saving and easy-to-perform method. It can be used as an alternative diagnostic method. However, for quantification purposes the qPCR is still the gold standard.

To avoid innate sensing and immune control, human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here, we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2-RanGAP1*SUMO1-Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein, unleashing end degradation of viral cDNA. Concomitantly, Vpu inhibits RAD52-mediated homologous repair of viral cDNA, preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs.

Genome editing tools such as zinc-finger nucleases provide novel strategies for genetic manipulation in plants. Unlike agrobacterium-mediated or direct gene transfer, which introduce genes randomly into the genome and thereby potentially resulting in high variation of gene expression, the targeted gene addition provides predictable integration of DNA sequences into a specified location of the plant genome. We investigated whether various independent cell lines that all contain a transgene placed in the same genomic locus by zinc-finger nuclease-mediated homologous recombination (HR) would yield a more reproducible and homogeneous level of expression compared to integration events generated via agrobacterium-mediated transformation at random sites. The variance of gene expression of targeted HR events and random integration events was analyzed in Nicotiana tabacum L cv. Bright Yellow 2 (BY-2) suspension cells by measuring protein amount produced from the transgene by flow cytometry, thus providing the first report on positional effects of marker gene expression in a quickly proliferating plant suspension cell line. Marker protein levels of targeted HR and single-copy random events covered a similar range; however, the uniformity of protein expression in a given cell line was significantly higher in targeted events than in lines with randomly inserted transgene; the same is true for the overall viability of protoplasts from HR lines. In conclusion, using targeted insertion into a qualified locus of a well-characterized line leads to more reliable results than random insertion into the genome.

Significance Lentiviruses encode accessory proteins to manipulate their host cells in order to efficiently replicate and evade antiviral defenses. Interestingly, most lentiviral Nefs down-regulate CD3 from the surface of infected T cells to perturb immune responses. However, for reasons that are incompletely understood, HIV-1 and its simian immunodeficiency virus ancestors lack this function. Here, we report that engineering HIV-1 for Nef-mediated down-regulation of CD3 reduces Env-dependent HIV-1 infectivity, resulting in less efficient cell-to-cell spread and replication. Our data suggest that HIV-1 may have evolved to lose the CD3 down-modulation function of Nef in order to allow T cell activation and to boost viral replication, possibly at the cost of less effective immune evasion and increased pathogenicity., Nef is an accessory protein of primate lentiviruses that is essential for efficient replication and pathogenesis of HIV-1. A conserved feature of Nef proteins from different lentiviral lineages is the ability to modulate host protein trafficking and down-regulate a number of cell surface receptors to enhance replication and promote immune evasion. Notably, the inability of Nef to down-regulate CD3 from infected T cells distinguishes HIV-1 Nef and its direct simian precursors from other primate lentiviruses. Why HIV-1 does not employ this potential immune evasion strategy is not fully understood. Using chimeric HIV-1 constructs expressing lentiviral Nef proteins that differ in their ability to down-modulate CD3, we show that retaining CD3 on the surface of infected primary T cells results in increased viral replication and cell-to-cell spread. We identified increased expression of envelope (Env) trimers at the cell surface and increased Env incorporation into virions as the determinants for the Nef- and CD3-dependent enhancement of viral infectivity. Importantly, this was independent of Nef-mediated antagonism of the host restriction factor SERINC5. CD3 retention on the surface of infected primary T cells also correlated with increased T cell signaling, activation, and cell death during cell-to-cell spread. Taken together, our results show that loss of an otherwise conserved function of Nef has a positive effect on HIV-1 replication, allowing for more efficient replication while potentially contributing to HIV-1 pathogenesis by triggering T cell activation and cell death during viral spread.

As part of innate immune defenses, macroautophagy/autophagy targets viruses and viral components for lysosomal degradation and exposes pathogen-associated molecular patterns to facilitate recognition. However, viruses evolved sophisticated strategies to antagonize autophagy and even exploit it to promote their replication. In our recent study, we systematically analyzed the impact of individual SARS-CoV-2 proteins on autophagy. We showed that E, M, ORF3a, and ORF7a cause an accumulation of autophagosomes, whereas Nsp15 prevents the efficient formation of autophagosomes. Consequently, autophagic degradation of SQSTM1/p62 is decreased in the presence of E, ORF3a, ORF7a, and Nsp15. Notably, M does not alter SQSTM1 protein levels and colocalizes with accumulations of LC3B-positive membranes not resembling vesicles. Infection with SARS-CoV-2 prevents SQSTM1 degradation and increases lipidation of LC3B, indicating overall that the infection causes a reduction of autophagic flux. Our mechanistic analyses showed that the accessory proteins ORF3a and ORF7a both block autophagic degradation but use different strategies. While ORF3a prevents the fusion between autophagosomes and lysosomes, ORF7a reduces the acidity of lysosomes. In summary, we found that Nsp15, E, M, ORF3a, and ORF7a of SARS-CoV-2 manipulate cellular autophagy, and we determined the molecular mechanisms of ORF3a and ORF7a., publishedVersion

CARD8 senses pathogen-associated protease activity and assembles a pyroptosis-inducing inflammasome, but detailed regulatory mechanisms have remained elusive. In this issue of Immunity, Sharif et al. use cryo-EM and biochemical assays to unveil how DPP9 sequesters the inflammasome-forming C-terminal fragment of CARD8 to suppress its activation.

Astrocytes from the cerebral cortex (CTX) and cerebellum (CB) share basic molecular programs, but also form distinct spatial and functional subtypes. The regulatory epigenetic layers controlling such regional diversity have not been comprehensively investigated so far. Here, we present an integrated epigenome analysis of methylomes, open chromatin, and transcriptomes of astroglia populations isolated from the cortex or cerebellum of young adult mice. Besides a basic overall similarity in their epigenomic programs, cortical astrocytes and cerebellar astrocytes exhibit substantial differences in their overall open chromatin structure and in gene-specific DNA methylation. Regional epigenetic differences are linked to differences in transcriptional programs encompassing genes of region-specific transcription factor networks centered around Lhx2/Foxg1 in CTX astrocytes and the Zic/Irx families in CB astrocytes. The distinct epigenetic signatures around these transcription factor networks point to a complex interconnected and combinatorial regulation of region-specific transcriptomes. These findings suggest that key transcription factors, previously linked to temporal, regional, and spatial control of neurogenesis, also form combinatorial networks important for astrocytes. Our study provides a valuable resource for the molecular basis of regional astrocyte identity and physiology.

Understanding the contribution of the host’s genetic background to cancer immunity may lead to improved stratification for immunotherapy and to the identification of novel therapeutic targets. We investigated the effect of common and rare germline variants on 139 well-defined immune traits in ∼9000 cancer patients enrolled in TCGA. High heritability was observed for estimates of NK cell and T cell subset infiltration and for interferon signaling. Common variants of IFIH1, TMEM173 (STING1), and TMEM108 were associated with differential interferon signaling and variants mapping to RBL1 correlated with T cell subset abundance. Pathogenic or likely pathogenic variants in BRCA1 and in genes involved in telomere stabilization and Wnt-β-catenin also acted as immune modulators. Our findings provide evidence for the impact of germline genetics on the composition and functional orientation of the tumor immune microenvironment. The curated datasets, variants, and genes identified provide a resource toward further understanding of tumor-immune interactions. We are grateful to the Society for Immunotherapy of Cancer (SITC) for the logistical support of the investigator meeting within the SITC Cancer Immune Responsiveness Workshop (San Francisco CA, US, April 2018; Houston, TX, US, May 2019). We are also grateful to Noah Zaitlen and Andy Dahl for useful discussions on heritability interaction analyses. This work was funded in part by the National Institutes of Health R01CA227466 and K24CA169004 to EZ and T32CA221709 postdoctoral fellowship to RWS, Qatar National Research Fund (QNRF) NPRP11S-0121-180351 grant and the Sidra Precision Medicine Program internal grant (SDR100035 and SDR400023) to DB, Associazione Italiana per la Ricerca sul cancro (AIRC) grant IG2018-21846 to MC, the Cancer Research Institute funding to VT, and the Fundació Bancaria La Caixa, a La Caixa Junior Leader Fellowship (LCF/BQ/PI18/11630003) to EP-P.

The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, the cause of the COVID-19 pandemic. However, this bat virus was most likely unable to directly infect humans since its Spike (S) protein does not interact efficiently with the human ACE2 receptor. Here, we show that a single T403R mutation increases binding of RaTG13 S to human ACE2 and allows VSV pseudoparticle infection of human lung cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S reduces pseudoparticle infection and viral replication. The T403R RaTG13 S is neutralized by sera from individuals vaccinated against COVID-19 indicating that vaccination might protect against future zoonoses. Our data suggest that a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2 by S proteins of bat coronaviruses. This finding could help to better predict the zoonotic potential of animal coronaviruses., The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, but its spike protein doesn’t efficiently bind human ACE2. Here, the authors show that exchange of spike residue 403 between RaTG13 and SARS-CoV-2 spike proteins affects binding to human ACE2 and entry of pseudotyped viruses.

SUMMARYThe circadian clock employs a transcriptional/translational negative feedback loop (TTFL) to anticipate environmental changes due to the Earth’s diurnal cycle, with regulation of organismal physiology believed to stem from temporal transcriptional activation by the positive arm. However, up to 80% of oscillating proteins do not have rhythmic mRNA, establishing circadian post-transcriptional regulation through unknown mechanisms. Given the pervasive conservation of the intrinsically disordered nature of negative-arm clock proteins, we hypothesized that post-transcriptional regulation may stem from conformational shifts in negative-arm proteins that time vacillations in the constituents of negative-arm macromolecular complexes to time cellular physiology. Our investigation of the negative arm clock protein in Neurospora crassa, FREQUENCY (FRQ), demonstrated temporal conformational fluidity correlated with daily changes in physiologically diverse macromolecular complex components. A parallel investigation of the macromolecular complexes centered around Drosophila melanogaster PERIOD (dPER) and human PERIOD (hPER2) found a similar number and physiological diversity of interacting partners in higher eukaryotes. Short linear motifs (SLiMs) associated with the interactors localized to disordered and phosphorylated regions on the PERs and FRQ, with disordered interactors oscillating in the macromolecular complexes over circadian time. This oscillation correlated with oscillations in post-transcriptionally regulated proteins, suggesting the negative arm may tune cellular physiology and proteostasis post-transcriptionally via vacillations in the circadian negative-arm macromolecular protein complexes.

It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human cells. To date, several “Variants of Concern” (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the four SARS-CoV-2 VOCs (Alpha, Beta, Gamma and Delta) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all four VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had little effect, while knock-down of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than four orders of magnitude. In addition, an antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominating Delta variant.IMPORTANCERecent results showed that an early SARS-CoV-2 isolate requires endogenously expressed IFITM proteins for efficient infection. However, whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that out-competed the original strains and currently dominate the pandemic remained to be determined. Here, we demonstrate that depletion of endogenous IFITM2 expression almost entirely prevents the production of infectious Alpha, Beta, Gamma and Delta VOC SARS-CoV-2 virions in a human lung cell line. In comparison, silencing of IFITM1 had little impact, while knock-down of IFITM3 had intermediate effects on viral replication. Finally, an antibody targeting the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. Our results show that SARS-CoV-2 VOCs including the currently dominant Delta variant are dependent on IFITM2 for efficient replication suggesting that IFITM proteins play a key role in viral transmission and pathogenicity.

Interferons are a major part of the anti-viral innate defense system. Successful pathogens, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), need to overcome these defenses to establish an infection. Early induction of interferons (IFNs) protects against severe coronavirus disease 2019 (COVID-19). In line with this, SARS-CoV-2 is inhibited by IFNs in vitro, and IFN-based therapies against COVID-19 are investigated in clinical trials. However, SARS-CoV-2 continues to adapt to the human population resulting in the emergence of variants characterized by increased transmission fitness and/or decreased sensitivity to preventive or therapeutic measures. It has been suggested that the efficient spread of these so-called “Variants of Concern” (VOCs) may also involve reduced sensitivity to IFNs. Here, we examined whether the four current VOCs (Alpha, Beta, Gamma and Delta) differ in replication efficiency or IFN sensitivity from an early isolate of SARS-CoV-2. All viruses replicated in a human lung cell line and in iPSC-derived alveolar type II cells (iAT2). The Delta variant showed accelerated replication kinetics and higher infectious virus production compared to the early 2020 isolate. Replication of all SARS-CoV-2 VOCs was reduced in the presence of exogenous type I, II and III IFNs. On average, the Alpha variant was the least susceptible to IFNs and the Alpha, Beta and Gamma variants show increased resistance against type III IFN. Although the Delta variant has outcompeted all other variants in humans it remained as sensitive to IFNs as an early 2020 SARS-CoV-2 isolate. This suggests that increased replication fitness rather than IFN resistance may be a reason for its dominance. Our results may help to understand changes in innate immune susceptibility of VOCs, and inform clinical trials exploring IFN-based COVID-19 therapies.

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy—termed the "R-clamp”—that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution., Author summary Viral replication depends on interactions with a plethora of host cell proteins. Cellular protein interactions are typically mediated by specialized binding modules, such as the SH3 domain. To gain access to host cell regulation viruses have evolved to contain SH3 domain binding sites in their proteins, a notable example of which is the HIV-1 Nef protein. Here we show that during the primate lentivirus evolution the structural strategy that underlies the avid binding of Nef to cellular SH3 domains, which we have dubbed the R-clamp, has been generated via alternative but functionally interchangeable molecular designs. These patterns of SH3 recognition depend on the amino acid combinations at the positions corresponding to residues 83 and 120 in the consensus HIV-1 Nef sequence, and are distinctly different in Nef proteins from SIVs of Eastern and Central chimpanzees, gorillas, and the four groups of HIV-1 that have independently originated from the latter two. These results highlight the evolutionary plasticity of viral proteins, and have implications on therapeutic development aiming to interfere with SH3 binding of Nef.

Background The incorporation of monoclonal antibodies, such as daratumumab, into multiple myeloma treatment regimens has led to the issue of false-positive interference in both serum protein electrophoresis (SPEP) and immunofixation (IF). The Hydrashift assay removes daratumumab interference from IF, allowing for correct interpretation. Here, we retrospectively examined the use of the Hydrashift assay at a large cancer center and provide guidelines on its most appropriate use. Methods 38 patients with distinct daratumumab peaks on their SPEP were selected and were used to quantify the daratumumab peak on SPEP using the Sebia Phoresis software. A retrospective review of all Hydrashift assays ordered at our institution from July 2018 to March 2020 was performed. Data collected included patient clone type, IF migration patterns, and Hydrashift result. Serial quantification of SPEP results was performed as the corresponding IF transitioned from a true positive to a false positive. Results Daratumumab adds a maximum magnitude of 0.20 g/dL on SPEP. Serial SPEP quantification showed IF transitioned from true positive to false positive when M-spikes ranged from 0.09 g/dL to 0.11 g/dL. Over 20 months, our laboratory performed 280 Hydrashift assays on 96 patients, 43/96 of whom had comigrating daratumumab/IgG-K IF bands. Conclusions The Hydrashift assay is typically unnecessary in patients with large M-spikes, >0.25 g/dL, regardless of clone type. When patient history is available, we recommend the Hydrashift assay be used in patients with comigrating daratumumab/IgG-K bands with M-spikes of

NG2 (nerve/glial antigen 2) glia are uniformly distributed in the gray and white matter of the central nervous system (CNS). They are the major proliferating cells in the brain and can differentiate into oligodendrocytes. NG2 glia do not only receive synaptic input from excitatory and inhibitory neurons, but also secrete growth factors and cytokines, modulating CNS homeostasis. They express several receptors and ion channels that play a role in rapidly responding upon synaptic signals and generating fast feedback, potentially regulating their own properties. Ca2+ influx via voltage-gated Ca2+ channels (VGCCs) induces an intracellular Ca2+ rise initiating a series of cellular activities. We confirmed that NG2 glia express L-type VGCCs in the white and gray matter during CNS development, particularly in the early postnatal stage. However, the function of L-type VGCCs in NG2 glia remains elusive. Therefore, we deleted L-type VGCC subtypes Cav1.2 and Cav1.3 genes conditionally in NG2 glia by crossbreeding NG2-CreERT2 knock-in mice to floxed Cav1.2 and flexed Cav1.3 transgenic mice. Our results showed that ablation of Cav1.2 and Cav1.3 strongly inhibited the proliferation of cortical NG2 glia, while differentiation in white and gray matter was not affected. As a consequence, no difference on myelination could be detected in various brain regions. In addition, we observed morphological alterations of the nodes of Ranvier induced by VGCC-deficient NG2 glia, i.e., shortened paired paranodes in the corpus callosum. Furthermore, deletion of Cav1.2 and Cav1.3 largely eliminated N-methyl-D-aspartate (NMDA)-dependent long-term depression (LTD) and potentiation in the hippocampus while the synaptic input to NG2 glia from axons remained unaltered. We conclude that L-type VGCCs of NG2 glia are essential for cell proliferation and proper structural organization of nodes of Ranvier, but not for differentiation and myelin compaction. In addition, L-type VGCCs of NG2 glia contribute to the regulation of long-term neuronal plasticity.

Astrocytes constitute the main glial component of the mammalian blood brain barrier (BBB). However, in the olfactory bulb (OB), the olfactory nerve layer (ONL) is almost devoid of astrocytes, raising the question which glial cells are part of the BBB. We used mice expressing EGFP in astrocytes and tdTomato in olfactory ensheathing cells (OECs), a specialized type of glial cells in the ONL, to unequivocally identify both glial cell types and investigate their contribution to the BBB in the olfactory bulb. OECs were located exclusively in the ONL, while somata of astrocytes were located in deeper layers and extended processes in the inner sublamina of the ONL. These processes surrounded blood vessels and contained aquaporin-4, an astrocytic protein enriched at the BBB. In the outer sublamina of the ONL, in contrast, blood vessels were surrounded by aquaporin-4-negative processes of OECs. Transcardial perfusion of blood vessels with lanthanum and subsequent visualization by electron microscopy showed that blood vessels enwrapped by OECs possessed intact tight junctions. In acute olfactory bulb preparations, injection of fluorescent glucose 6-NBDG into blood vessels resulted in labeling of OECs, indicating glucose transport from the perivascular space into OECs. In addition, Ca2+ transients in OECs in the outer sublamina evoked vasoconstriction, whereas Ca2+ signaling in OECs of the inner sublamina had no effect on adjacent blood vessels. Our results demonstrate that the BBB in the inner sublamina of the ONL contains astrocytes, while in the outer ONL OECs are part of the BBB.

Semen is the vehicle for virion dissemination in the female reproductive tract (FRT) in male-to-female HIV transmission. Recent data suggests that higher frequency semen exposure is associated with activation of anti-HIV mechanisms in HIV negative sex workers. Here, we use a non-human primate (NHP) model to show that repeated vaginal exposure to semen significantly reduces subsequent infection by repeated low-dose vaginal SIVmac251 challenge. Repeated semen exposures result in lower CCR5 expression in circulating CD4+ T-cells, as well as higher expression of Mx1 (in correlation with IFNε expression) and FoxP3 in the cervicovaginal mucosa, and increased infiltration of CD4+ T-cells. Establishing in vivo evidence of competing effects of semen on transmission impacts our basic understanding of what factors may determine HIV infectivity in humans. Our results clearly indicate that repeated semen exposure can profoundly modulate the FRT microenvironment, paradoxically promoting host resistance against HIV acquisition., High frequency semen exposure has been associated with activation of anti-HIV mechanisms in HIV negative sex workers. Here, Abdulhaqq et al. show that repeated vaginal exposure to semen reduces vaginal infection by SIV in non-human primates, and is associated with lower CCR5 expression in CD4 T-cells and a local type-I interferon response.

Since several years, the enteric nervous system (ENS) is getting more and more in the focus of gastrointestinal research. While the main interest was credited for years to the enteric neurons and their functional properties, less attention has been paid on the enteric glial cells (EGCs). Although the similarity of EGCs to central nervous system (CNS) astrocytes has been demonstrated a long time ago, EGCs were investigated in more detail only recently. Similar to the CNS, there is not "the" EGC, but also a broad range of diversity. Based on morphology and protein expression, such as glial fibrillary acidic protein (GFAP), S100, or Proteolipid-protein-1 (PLP1), several distinct glial types can be differentiated. Their heterogeneity in morphology, localization, and transcription as well as interaction with surrounding cells indicate versatile functional properties of these cells for gut function in health and disease. Although NG2 is found in a subset of CNS glial cells, it did not colocalize with the glial marker S100 or GFAP in the ENS. Instead, it in part colocalize with PDGFRα, as it does in the CNS, which do stain fibroblast-like cells in the gastrointestinal tract. Moreover, there seem to be species dependent differences. While GFAP is always found in the rodent ENS, this is completely different for the human gut. Only the compromised human ENS shows a significant amount of GFAP-positive glial cells. So, in general we can conclude that the EGC population is species specific and as complex as CNS glia. Anat Rec, 302:1333-1344, 2019. © 2019 Wiley Periodicals, Inc.

Latent viral reservoirs in long-living cell populations are the main obstacle to a cure of HIV/AIDS. HIV-1 latency is controlled by the activation status of infected cells and their ability to return to a resting phenotype associated with silencing of viral gene expression. These cellular features are not just determined by the host since HIV-1 has evolved sophisticated mechanisms to alter cellular activation and survival to its advantage. Especially the HIV-1 accessory proteins Nef and Vpu exert numerous activities to promote viral replication and immune evasion affecting the size and preservation of the viral reservoir. Here, we review how antagonistic and synergistic functions of Nef and Vpu might affect HIV-1 latency. We also discuss whether these two accessory factors represent suitable targets to improve the ‘shock and kill’ cure strategy.

The chloroplast organelle in mesophyll cells of higher plants represents a sunlight-driven metabolic factory that eventually fuels life on our planet. Knowledge of the ultrastructure and the dynamics of this unique organelle is essential to understanding its function in an ever-changing and challenging environment. Recent technological developments promise unprecedented insights into chloroplast architecture and its functionality. The review highlights these new methodical approaches and provides structural models based on recent findings about the plasticity of the thylakoid membrane system in response to different light regimes. Furthermore, the potential role of the lipid droplets plastoglobuli is discussed. It is emphasized that detailed structural insights are necessary on different levels ranging from molecules to entire membrane systems for a holistic understanding of chloroplast function.

NG2 glia represent a distinct type of macroglial cells in the CNS and are unique among glia because they receive synaptic input from neurons. They are abundantly present in white and grey matter. While the majority of white matter NG2 glia differentiates into oligodendrocytes, the physiological impact of grey matter NG2 glia and their synaptic input are ill defined yet. Here we asked whether dysfunctional NG2 glia affect neuronal signaling and behavior. We generated mice with inducible deletion of the K+ channel Kir4.1 in NG2 glia and performed comparative electrophysiological, immunohistochemical, molecular and behavioral analyses. Focussing on the hippocampus, we found that loss of the Kir4.1 potentiated synaptic depolarizations of NG2 glia and enhanced the expression of myelin basic protein. Notably, while mice with targeted deletion of the K+ channel in NG2 glia showed impaired long term potentiation at CA3-CA1 synapses, they demonstrated improved spatial memory as revealed by testing new object location recognition. Our data demonstrate that proper NG2 glia function is critical for normal brain function and behavior.

Simian immunodeficiency virus infecting sooty mangabeys (SIVsmm) has been transmitted to humans on at least nine occasions, giving rise to human immunodeficiency virus type 2 (HIV-2) groups A to I. SIVsmm isolates replicate in human T cells and seem capable of overcoming major human restriction factors without adaptation. However, only groups A and B are responsible for the HIV-2 epidemic in sub-Saharan Africa, and it is largely unclear whether adaptive changes were associated with spread in humans. To address this, we examined the sensitivity of infectious molecular clones (IMCs) of five HIV-2 strains and representatives of five different SIVsmm lineages to various APOBEC3 proteins. We confirmed that SIVsmm strains replicate in human T cells, albeit with more variable replication fitness and frequently lower efficiency than HIV-2 IMCs. Efficient viral propagation was generally dependent on intact vif genes, highlighting the need for counteraction of APOBEC3 proteins. On average, SIVsmm was more susceptible to inhibition by human APOBEC3D, -F, -G, and -H than HIV-2. For example, human APOBEC3F reduced infectious virus yield of SIVsmm by ∼80% but achieved only ∼40% reduction in the case of HIV-2. Functional and mutational analyses of human- and monkey-derived alleles revealed that an R128T polymorphism in APOBEC3F contributes to species-specific counteraction by HIV-2 and SIVsmm Vifs. In addition, a T84S substitution in SIVsmm Vif increased its ability to counteract human APOBEC3F. Altogether, our results confirm that SIVsmm Vif proteins show intrinsic activity against human APOBEC3 proteins but also demonstrate that epidemic HIV-2 strains evolved an increased ability to counteract this class of restriction factors during human adaptation. IMPORTANCE Viral zoonoses pose a significant threat to human health, and it is important to understand determining factors. SIVs infecting great apes gave rise to HIV-1. In contrast, SIVs infecting African monkey species have not been detected in humans, with one notable exception. SIVsmm from sooty mangabeys has crossed the species barrier to humans on at least nine independent occasions and seems capable of overcoming many innate defense mechanisms without adaptation. Here, we confirmed that SIVsmm Vif proteins show significant activity against human APOBEC3 proteins. Our analyses also revealed, however, that different lineages of SIVsmm are significantly more susceptible to inhibition by various human APOBEC3 proteins than HIV-2 strains. Mutational analyses suggest that an R128T substitution in APOBEC3F and a T84S change in Vif contribute to species-specific counteraction by HIV-2 and SIVsmm. Altogether, our results support that epidemic HIV-2 strains acquired increased activity against human APOBEC3 proteins to clear this restrictive barrier.

A group of vascular plants called homoiochlorophyllous resurrection plants evolved unique capabilities to protect their photosynthetic machinery against desiccation-induced damage. This study examined whether the ontogenetic status of the resurrection plant Craterostigma pumilum has an impact on how the plant responds to dehydration at the thylakoid membrane level to prepare cells for the desiccated state. Thus, younger plants (6 months) counterparts. Ultrastructural analysis provided evidence that younger plants suppressed senescence-like programs that are realized in older plants. During dehydration, older plants degrade specific subunits of the photosynthetic apparatus such as the D1 subunit of PSII and subunits of the cytochrome b6f complex. The latter leads to a controlled down-regulation of linear electron transport. In contrast, younger plants increased photoprotective high-energy quenching mechanisms and maintained a high capability to replace damaged D1 subunits. It follows that depending on the ontogenetic state, either more degradation-based or more photoprotective mechanisms are employed during dehydration of Craterostigma pumilum.

Arthropod venoms offer a promising resource for the discovery of novel bioactive peptides and proteins, but the limited size of most species translates into minuscule venom yields. Bioactivity studies based on traditional fractionation are therefore challenging, so alternative strategies are needed. Cell-free synthesis based on synthetic gene fragments is one of the most promising emerging technologies, theoretically allowing the rapid, laboratory-scale production of specific venom components, but this approach has yet to be applied in venom biodiscovery. Here, we tested the ability of three commercially available cell-free protein expression systems to produce venom components from small arthropods, using U2-sicaritoxin-Sdo1a from the six-eyed sand spider Hexophtalma dolichocephala as a case study. We found that only one of the systems was able to produce an active product in low amounts, as demonstrated by SDS-PAGE, mass spectrometry, and bioactivity screening on murine neuroblasts. We discuss our findings in relation to the promises and limitations of cell-free synthesis for venom biodiscovery programs in smaller invertebrates.

We present a protocol for analyzing the impact of SARS-CoV-2 proteins in interferon (IFN) signaling using luciferase reporter assays. Here, the induction of defined promoters can be quantitatively assessed with high sensitivity and broad linear range. The results are similar to those obtained using qPCR to measure endogenous mRNA induction. The assay requires stringent normalization and confirmation of the results in more physiological settings. The protocol is adaptable for other viruses and other innate immune stimuli., Graphical Abstract

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy - termed the “R-clamp” - that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.AUTHOR SUMMARYViral replication depends on interactions with a plethora of host cell proteins. Cellular protein interactions are typically mediated by specialized binding modules, such as the SH3 domain. To gain access to host cell regulation viruses have evolved to contain SH3 domain binding sites in their proteins, a notable example of which is the HIV-1 Nef protein. Here we show that during the primate lentivirus evolution the structural strategy that underlies the avid binding of Nef to cellular SH3 domains, which we have dubbed the R-clamp, has been generated via alternative but functionally interchangeable molecular designs. These patterns of SH3 recognition depend on the amino acid combinations at the positions corresponding to residues 83 and 120 in the consensus HIV-1 Nef sequence, and are distinctly different in Nef proteins from SIVs of Eastern and Central chimpanzees, gorillas, and the four groups of HIV-1 that have independently originated from the latter two. These results highlight the evolutionary plasticity of viral proteins, and have implications on therapeutic development aiming to interfere with SH3 binding of Nef.

After acute brain injuries various response cascades are evoked that direct the formation of the glial scar. Here, we report that acute lesions associated with a disruption of the blood-brain barrier trigger a re-programming within the oligodendrocyte lineage. In PLP-DsRed1/GFAP-EGFP and PLP-EGFPmem/GFAP-mRFP1 transgenic mice with cortical injuries, we transiently found PLP transgene-labelled cells with activated GFAP promoter activity adjacent to the lesion site. We termed them AO cells, based on their concomitant activity of astro- and oligodendroglial genes. By fate mapping using PLP- and GFAP-split Cre complementation and NG2-CreERT2 mice we observed that major portions of AO cells surprisingly differentiated into astrocytes. Using repeated long-term in vivo two-photon laser-scanning microscopy (2P-LSM) we followed oligodendrocytes after injury. We observed their conversion into astrocytes via the AO cell stage with silencing of the PLP promoter and simultaneous activation of the GFAP promoter. In addition, we provide evidence that this oligodendrocyte-to-astrocyte conversion depends on local cues. At the lesion site higher expression levels of various glial differentiation factors were detected. And indeed, local injection of IL-6 promoted the formation of AO cells. In summary, our findings highlight the plastic potential of oligodendrocytes in acute brain trauma. An altered environmental milieu affects gene expression programs of mature oligodendrocytes and induces a plastic differentiation stage with astrogliogenic potential via transitional AO cells.

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Heterologous COVID-19 vaccination regimens combining vector- and mRNA-based vaccines are already administered, but data on solicited adverse reactions, immunological responses and elicited protection are limited. We aimed to evaluate the reactogenicity, humoral and cellular immune responses towards different SARS-CoV-2 variants after a heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination and analyzed a cohort of 26 individuals aged 25-46 (median 30.5) years that received a ChAdOx1 nCoV-19 prime followed by a BNT162b2 boost after an 8- week interval. Self-reported solicited symptoms after ChAdOx1 nCoV-19 prime were in line with previous reports and less severe after the BNT162b2 boost. Antibody titers increased significantly over time resulting in strong neutralization titers two weeks after the BNT162b2 boost. Neutralizing activity against the prevalent strain B.1.1.7 (Alpha) and immune-evading VOC B.1.351 (Beta) was ∼4-fold higher than in individuals receiving homologous BNT162b2 vaccination. No difference was seen in neutralization of VOI B.1.617 (Kappa). In addition, the heterologous vaccination induced CD4+ and CD8+ T cells reactive to SARS-CoV-2 spike peptides of all analyzed variants; Wuhan-Hu-1, B.1.1.7, B.1.351, and P.1 (Gamma). In conclusion, heterologous ChAdOx1 nCoV-19 / BNT162b2 prime-boost vaccination regimen is not associated with serious adverse events and results in a potent humoral immune response and elicits T cell reactivity. Variants B.1.1.7, B.1.351 and B.1.617.1 are potently neutralized by sera of all participants and reactive T cells recognize spike peptides of all tested variants. These results suggest that this heterologous vaccination regimen is at least as immunogenic and protective as homologous vaccinations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 pandemic, most likely emerged from bats1. A prerequisite for this devastating zoonosis was the ability of the SARS-CoV-2 Spike (S) glycoprotein to use human angiotensin-converting enzyme 2 (ACE2) for viral entry. Although the S protein of the closest related bat virus, RaTG13, shows high similarity to the SARS-CoV-2 S protein it does not efficiently interact with the human ACE2 receptor2. Here, we show that a single T403R mutation allows the RaTG13 S to utilize the human ACE2 receptor for infection of human cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S significantly reduced ACE2-mediated virus infection. The S protein of SARS-CoV-1 that also uses human ACE2 also contains a positive residue (K) at this position, while the S proteins of CoVs utilizing other receptors vary at this location. Our results indicate that the presence of a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2. This finding could help to predict the zoonotic potential of animal coronaviruses.

Cortical neural circuits are complex but very precise networks of balanced excitation and inhibition (E/I). Yet, the molecular and cellular mechanisms that form the E/I balance are just beginning to emerge. Here, using conditional GABAB receptor-deficient mice we identified a GABA/TNF-related cytokine (TNFSF12)-mediated bidirectional communication pathway between Parvalbumin-positive (PV+) fast spiking interneurons and oligodendrocyte precursor cells (OPCs) that determines the density and function of interneurons in the developing medial prefrontal cortex (mPFC). Interruption of the GABAergic signaling to OPCs resulted in reduced myelination and hypoactivity of interneurons, strong changes of cortical network activities and impaired cognitive behavior. In conclusion, glial transmitter receptors are pivotal elements in finetuning distinct brain functions.

Cofilin-1 (CFL1) overexpression in pancreatic cancer correlates with high invasiveness and shorter survival. Besides a well-documented role in actin remodeling, additional cellular functions of CFL1 remain poorly understood. Here, we unraveled molecular tumor-promoting functions of CFL1 in pancreatic cancer. For this purpose, we first show that a knockdown of CFL1 results in reduced growth and proliferation rates in vitro and in vivo, while apoptosis is not induced. By mechanistic modeling we were able to predict the underlying regulation. Model simulations indicate that an imbalance in actin remodeling induces overexpression and activation of CFL1 by acting on transcription factor 7-like 2 (TCF7L2) and aurora kinase A (AURKA). Moreover, we could predict that CFL1 impacts proliferation and apoptosis via the signal transducer and activator of transcription 3 (STAT3). These initial model-based regulations could be substantiated by studying protein levels in pancreatic cancer cell lines and human datasets. Finally, we identified the surface protein CD44 as a promising therapeutic target for pancreatic cancer patients with high CFL1 expression., publishedVersion

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are thought to restrict numerous viral pathogens including severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudovirus infection studies of cells that overexpress IFITMs. Here, we verified that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. Strikingly, however, endogenous IFITM expression was essential for efficient infection of genuine SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral entry. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. Intriguingly, IFITM-derived peptides and targeting antibodies inhibited SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are important cofactors for SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and suitable targets for therapeutic approaches.