title: Stress resilience is established during development and is regulated by complement factors

abstract:
Individuals in a population respond differently to stressful situations. While resilient individuals recover efficiently, others are susceptible to the same stressors. However, it remains challenging to identify resilience in mammalian embryos to determine if stress resilience is established as a trait during development or acquired later in life. Using a new behavioural paradigm in zebrafish larvae, we show that resilience is a trait that is determined and exhibited early in life. Resilient and susceptible individuals retained these traits throughout life and passed them on to the next generation. Resilient larvae showed higher expression of resilience-associated genes and larvae lacking neuropeptide Y and miR218 were significantly under-represented in the resilient population. Unbiased transcriptome analysis revealed that multiple factors of the innate immune complement cascade were downregulated in resilient larvae in response to stressors. Pharmacological inhibition and genetic knockouts of critical complement factors led to an increase in resilience. We conclude that resilience is established early during development as a stable trait, and that neuropeptides and the complement pathway play positive and negative roles in determining resilience respectively.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/478444v1_ufig1.gif" ALT="Figure 1">
View larger version (59K):
org.highwire.dtl.DTLVardef@359261org.highwire.dtl.DTLVardef@11d1e62org.highwire.dtl.DTLVardef@b5d346org.highwire.dtl.DTLVardef@141bae1_HPS_FORMAT_FIGEXP  M_FIG C_FIG Why some individual individuals recover better than others from stressful situations is unclear. We show that resilience to stress is established during zebrafish development as a stable and heritable trait. Resilience is augmented by brain-derived neuropeptides and attenuated by innate immune complement factors specifically expressed in the liver.

HighlightsO_LIWildtype zebrafish larvae show differences in their dynamics of recovering from stress, with some being more resilient than others.
C_LIO_LIResilience is a stable and heritable trait.
C_LIO_LIResilient fish show specific stress-responsive transcriptional changes.
C_LIO_LINeuropeptide Y and miRNA218 positively affect resilience, while innate immune complement factors attenuate resilience.
C_LI


title: Proximity of astrocyte leaflets to the synapse determines memory strength

abstract: SummaryAstrocyte distal processes, known as leaflets or perisynaptic astrocyte processes (PAPs), fine-tune synaptic activity by clearing neurotransmitters and limiting extrasynaptic glutamate diffusion. While learning and memory depends on orchestrated synaptic activity of neuronal ensembles within the hippocampus, it is becoming increasingly evident that astrocytes residing in the environment of these synapses play a central role in shaping memories. However, how astroglial synaptic coverage contributes to mnemonic processing remains largely unknown. Here, we targeted astrocyte leaflet structure in vivo by depleting Ezrin, an integral leaflet-structural protein, in astrocytes of the adult hippocampal CA1 using a CRISPR-Cas9 genetic approach. This resulted in significantly smaller astrocyte territories and reduced astroglial synaptic coverage. In addition, using genetically encoded glutamate sensors and whole-cell patch-clamp recordings from pyramidal neurons, we found that Ezrin deletion and the resultant manipulation of leaflet structure boosted extrasynaptic glutamate diffusion and NMDA-receptor activation. Importantly, these cellular phenotypes translated to enhanced fear memory expression that was accompanied by increased activation of CA1 pyramidal neurons in the days after learning occurred. We show that Ezrin is critical for astrocyte morphology as well as for adult hippocampal synapse integrity and function. Our data show that astrocyte leaflet structure gates memory strength by regulating glutamate spillover in the vicinity of memory-related synaptic activity.

category: neuroscience


title: Elevated photic response is followed by a rapid decay and depressed state in ictogenic networks

abstract:
The switch between non-seizure and seizure states involves profound alterations in network excitability and synchrony. Both increased and decreased excitability may underlie the state transitions, as shown in epilepsy patients and animal models. Inspired by video-electroencephalography recordings in patients, we developed a framework to study spontaneous and photic-evoked neural and locomotor activity in zebrafish larvae. We combined high-throughput behavioral tracking and whole-brain in vivo two-photon calcium imaging to perform side-by-side comparison of multiple zebrafish seizure and epilepsy models. Our setup allowed us to dissect behavioral and physiological features that are divergent or convergent across multiple models. We observed that locomotor and neural activity during interictal and spontaneous ictal periods exhibit great diversity across models. Yet, during photic stimulation, hyperexcitability and rapid response dynamics was well conserved across multiple models, highlighting the reliability of photic-evoked seizure activity for high-throughput assays. Intriguingly, in several models, we observed that the initial elevated photic response is often followed by fast decay of neural activity and a prominent depressed state. We argue that such depressed states are likely due to homeostatic mechanisms triggered by excessive neural activity. An improved understanding of the interplay between elevated and depressed excitability states might suggest tailored epilepsy therapies.

KEY POINTSO_LIFeatures of spontaneous locomotor and neural activity varies across zebrafish epilepsy and seizure models.
C_LIO_LIWe propose photic stimulation as a reliable tool to investigate behavioral and physiological phenotypes in zebrafish epilepsy and seizure models.
C_LIO_LIWe observed elevated activity with faster dynamics in response to photic stimulation in all tested zebrafish models.
C_LIO_LIPhotic-evoked neural responses were often followed by depressed state in seizure-prone networks
C_LI


title: Distinct developmental phenotypes result from mutation of Set8/KMT5A and histone H4 lysine 20 in Drosophila melanogaster

abstract: Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8 or mammalian KMT5A prevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. Here, we show that human KMT5A can functionally substitute for Set8 during Drosophila development and that the catalytic SET domains of the two enzymes are fully interchangeable. We also uncovered a role in eye development for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set8null mutants are inviable, we found that an R634G mutation in the SET domain predicted to ablate catalytic activity resulted in viable adults, suggesting important non-catalytic functions of Set8. Similarly, flies that were engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but they are phenotypically distinct from H4K20R, Set8null, and Set8R634G animals. Taken together, our results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophila development.

category: genetics


title: Real-time neurofeedback to alter interpretations of a naturalistic narrative

abstract: We explored the potential of using real-time fMRI (rt-fMRI) neurofeedback training to bias interpretations of naturalistic narrative stimuli. Participants were randomly assigned to one of two possible conditions, each corresponding to a different interpretation of an ambiguous spoken story. While participants listened to the story in the scanner, neurofeedback was used to reward neural activity corresponding to the assigned interpretation. After scanning, final interpretations were assessed. While neurofeedback did not change story interpretations on average, participants with higher levels of decoding accuracy during the neurofeedback procedure were more likely to adopt the assigned interpretation. Thus, we believe that individualized neurofeedback shaped interpretations successfully when the signal was accurate, although more work is needed to improve this method and validate the result. While naturalistic stimuli introduce a unique set of challenges in providing effective and individualized neurofeedback, we believe that this technique holds promise for individualized cognitive therapy.


title: SARS-CoV-2 leverages airway epithelial protective mechanism for viral infection

abstract: Despite much concerted effort to better understand SARS-CoV-2 viral infection, relatively little is known about the dynamics of early viral entry and infection in the airway. Here we analyzed a single-cell RNA sequencing dataset of early SARS-CoV-2 infection in a humanized in vitro model, to elucidate key mechanisms by which the virus triggers a cell-systems-level response in the bronchial epithelium. We find that SARS-CoV-2 virus preferentially enters the tissue via ciliated cell precursors, giving rise to a population of infected mature ciliated cells, which signal to basal cells, inducing further rapid differentiation. This feed-forward loop of infection is mitigated by further cell-cell communication, before interferon signaling begins at three days post-infection. These findings suggest hijacking by the virus of potentially beneficial tissue repair mechanisms, possibly exacerbating the outcome. This work both elucidates the interplay between barrier tissues and viral infections, and may suggest alternative therapeutic approaches targeting non-immune response mechanisms.

category: systems biology


title: An intranasal lentiviral booster broadens immune recognition of SARS-CoV-2 variants and reinforces the waning mRNA vaccine-induced immunity that it targets to lung mucosa

abstract: As the COVID-19 pandemic continues and new SARS-CoV-2 variants of concern emerge, the adaptive immunity initially induced by the first-generation COVID-19 vaccines wains and needs to be strengthened and broadened in specificity. Vaccination by the nasal route induces mucosal humoral and cellular immunity at the entry point of SARS-CoV-2 into the host organism and has been shown to be the most effective for reducing viral transmission. The lentiviral vaccination vector (LV) is particularly suitable for this route of immunization because it is non-cytopathic, non-replicative and scarcely inflammatory. Here, to set up an optimized cross-protective intranasal booster against COVID-19, we generated an LV encoding stabilized Spike of SARS-CoV-2 Beta variant (LV::SBeta-2P). mRNA vaccine-primed and -boosted mice, with waning primary humoral immunity at 4 months post-vaccination, were boosted intranasally with LV::SBeta-2P. Strong boost effect was detected on cross-sero-neutralizing activity and systemic T-cell immunity. In addition, mucosal anti-Spike IgG and IgA, lung resident B cells, and effector memory and resident T cells were efficiently induced, correlating with complete pulmonary protection against the SARS-CoV-2 Delta variant, demonstrating the suitability of the LV::SBeta-2P vaccine candidate as an intranasal booster against COVID-19.

category: immunology


title: Combating the SARS-CoV-2 Omicron variant with non-Omicron neutralizing antibodies

abstract: The highly mutated and transmissible Omicron variant has provoked serious concerns over its decreased sensitivity to the current coronavirus disease 2019 (COVID-19) vaccines and evasion from most anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (NAbs). In this study, we explored the possibility of combatting the Omicron variant by constructing bispecific antibodies based on non-Omicron NAbs. We engineered ten IgG-like bispecific antibodies with non-Omicron NAbs named GW01, 16L9, 4L12, and REGN10987 by fusing the single-chain variable fragments (scFvs) of two antibodies through a linker and then connecting them to the Fc region of IgG1. Surprisingly, eight out of ten bispecific antibodies showed high binding affinity to the Omicron receptor-binding domain (RBD) and exhibited extreme breadth and potency against pseudotyped SARS-CoV-2 variants of concern (VOCs) including Omicron, as well as authentic Omicron(+R346K) variants. Six bispecific antibodies containing the cross-NAb GW01 neutralized Omicron variant and retained their abilities to neutralize other sarbecoviruses. Bispecific antibodies inhibited Omicron infection by binding to the ACE2 binding site. A cryo-electron microscopy (cryo-EM) structure study of the representative bispecific antibody FD01 in complex with the Omicron spike (S) revealed 5 distinct trimers and one unique bi-trimer conformation. The structure and mapping analyses of 34 Omicron S variant single mutants elucidated that two scFvs of the bispecific antibody synergistically induced the RBD-down conformation into 3-RBD-up conformation, enlarged the interface area, accommodated the S371L mutation, improved the affinity between a single IgG and the Omicron RBD, and hindered ACE2 binding by forming bi-trimer conformation. Our study offers an important foundation for anti-Omicron NAb design. Engineering bispecific antibodies based on non-Omicron NAbs may provide an efficient solution to combat the Omicron variant.

category: microbiology


title: Susceptibility of wild canids to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

abstract:
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proven to be a promiscuous virus, capable of infecting a variety of different animal species, but much work remains in determining the susceptibility of common wildlife species to the virus. Here, we demonstrate that following experimental inoculation with SARS-CoV-2, red fox (Vulpes vulpes) become infected and can shed virus in oral and respiratory secretions. Conversely, experimentally challenged coyotes (Canis latrans) did not become infected or shed virus. Our results add red fox to the animal species known to be susceptible to SARS-CoV-2 and suggest that they may contribute to continued maintenance and transmission of the virus.

Article Summary LineExperimental infection of red fox (Vulpes vulpes) and coyotes (Canis latrans) with SARS-CoV-2 revealed that red fox are susceptible to infection and can shed virus, while coyotes do not become infected.


title: Omicron-specific mRNA vaccine induced potent neutralizing antibody against Omicron but not other SARS-CoV-2 variants

abstract: The emerging SARS-CoV-2 variants of concern (VOC) harbor mutations associated with increasing transmission and immune escape, hence undermine the effectiveness of current COVID-19 vaccines. In late November of 2021, the Omicron (B.1.1.529) variant was identified in South Africa and rapidly spread across the globe. It was shown to exhibit significant resistance to neutralization by serum not only from convalescent patients, but also from individuals receiving currently used COVID-19 vaccines with multiple booster shots. Therefore, there is an urgent need to develop next generation vaccines against VOCs like Omicron. In this study, we develop a panel of mRNA-LNP-based vaccines using the receptor binding domain (RBD) of Omicron and Delta variants, which are dominant in the current wave of COVID-19. In addition to the Omicron- and Delta-specific vaccines, the panel also includes a "Hybrid" vaccine that uses the RBD containing all 16 point-mutations shown in Omicron and Delta RBD, as well as a bivalent vaccine composed of both Omicron and Delta RBD-LNP in half dose. Interestingly, both Omicron-specific and Hybrid RBD-LNP elicited extremely high titer of neutralizing antibody against Omicron itself, but few to none neutralizing antibody against other SARS-CoV-2 variants. The bivalent RBD-LNP, on the other hand, generated antibody with broadly neutralizing activity against the wild-type virus and all variants. Surprisingly, similar cross-protection was also shown by the Delta-specific RBD-LNP. Taken together, our data demonstrated that Omicron-specific mRNA vaccine can induce potent neutralizing antibody response against Omicron, but the inclusion of epitopes from other variants may be required for eliciting cross-protection. This study would lay a foundation for rational development of the next generation vaccines against SARS-CoV-2 VOCs.


title: Visible blue light inactivates SARS-CoV-2 variants and inhibits Delta replication in differentiated human airway epithelia

abstract: The emergence of SARS-CoV-2 variants that evade host immune responses has prolonged the COVID-19 pandemic. Thus, the development of an efficacious, variant-agnostic therapeutic for the treatment of early SARS-CoV-2 infection would help reduce global health and economic burdens. Visible light therapy has the potential to fill these gaps. In this study, visible blue light centered around 425 nm efficiently inactivated SARS-CoV-2 variants in cell-free suspensions and in a translationally relevant well-differentiated tissue model of the human large airway. Specifically, 425 nm light inactivated cell-free SARS-CoV-2 variants Alpha, Beta, Delta, Gamma, Lambda, and Omicron by up to 99.99% in a dose-dependent manner, while the monoclonal antibody bamlanivimab did not neutralize the Beta, Delta, and Gamma variants. Further, we observed that 425 nm light reduced virus binding to host ACE-2 receptor and limited viral entry to host cells in vitro. Further, the twice daily administration of 32 J/cm2 of 425 nm light for three days reduced infectious SARS-CoV-2 Beta and Delta variants by >99.99% in human airway models when dosing began during the early stages of infection. In more established infections, logarithmic reductions of infectious Beta and Delta titers were observed using the same dosing regimen. Finally, we demonstrated that the 425 nm dosing regimen was well-tolerated by the large airway tissue model. Our results indicate that blue light therapy has the potential to lead to a well-tolerated and variant-agnostic countermeasure against COVID-19.


title: HOW 1 CONCERNING IS A SARS-COV-2 VARIANT OF2 CONCERN? COMPUTATIONAL PREDICTIONS AND3 THE VARIANTS LABELING SYSTEM.

abstract: We herein report a study to evaluate the use of computational prediction of SARS-CoV-2 genetic variations in improving the current variants labeling system. First, we reviewed the basis of the system developed by the World Health Organization (WHO) for the labeling of SARS-CoV-2 genetic variants and the adaptations made to it by the United States Center of Diseases Control (CDC). We observed that the labeling system is based upon the virus major attributes. However, we found that the labeling criteria of the SARS-CoV-2 variants derived from these attributes are not accurately defined and are used differently by the two health management agencies. Consequently, discrepancies exist between the labels given by WHO and CDC to same variants. Our observations suggest that giving the VOC label to a new variant is premature and might not be appropriate. Therefore, we carried out a comparative computational study to predict the effects of the mutations on the virus structure and functions of five VOCs. By linking these data to the criteria used by WHO and the CDC for variant labeling, we ascertained that comparative computational predictions of the impact of genetic variations are a better ground for rapid and more accurate labelling of SARS-CoV-2 variants. We propose to label all emergent variants VUM or VBM and to carry out computational predictive studies and thorough variants comparison, upon which more appropriate and informative labels can be attributed. Furthermore, harmonization of the variants labeling system would be globally beneficial to communicate about and fight COVID-19 pandemic.

category: molecular biology


title: HOW CONCERNING IS A SARS-COV-2 VARIANT OF CONCERN? COMPUTATIONAL PREDICTIONS AND THE VARIANTS LABELING SYSTEM


title: The predictive value of double-stranded RNA for A-to-I editing detection

abstract:
Structured AbstractO_ST_ABSMotivationC_ST_ABSAdenosine-to-inosine (A-to-I) RNA editing, a crucial reaction for many processes that contribute to transcriptome plasticity, is both widely common across the transcriptome and difficult to predict due to a lack of distinctive genomic characteristics that can be obtained and analyzed computationally. An exception to this is the secondary structure of RNA molecules, which has been shown to have a major impact on the selectivity and specificity of the enzymes responsible for A-to-I editing. Yet, this information is rarely used for the task of editing site prediction.

ResultsHere, we demonstrated the value of using base-pairing probabilities of RNA nucleotides to classify genomic sites as A-to-I RNA editing sites, using large-scale truth data which we compiled and make available for use in training future models. Our analysis suggests that the span of four bases from -2 (upstream) to +1 (downstream) of a putative editing site is most informative in this regard. A classifier trained on base-pairing probabilities alone performed with a positive predictive value (PPV) of 0.68, a negative predictive value (NPV) of 0.64, and an area under the receiver operating characteristic curve (AUC) of 0.71. By identifying structure-related features that are informative for detecting A-to-I RNA editing sites and quantifying their predictive value, this work advances our understanding of A-to-I editing determinants.

AvailabilityAll source codes and data are available at https://github.com/Ally-s-Lab/P-BEP

category: bioinformatics


title: Investigating the performance of deep learning methods for Hi-C resolution improvement

abstract:
MotivationHiC is a widely used technique to study the 3D organization of the genome. Due to its high sequencing cost, most of the generated datasets are of coarse quality, consequently limiting the quality of the downstream analyses. Recently, multiple deep learning-based methods have been proposed to improve the quality of these data sets by increasing their resolution through upscaling. However, the existing works do not thoroughly evaluate these methods using HiC reproducibility metrics across different HiC experiments to establish their applicability in real-world scenarios. This study extensively compares deep learning-based HiC upscaling methods on real-world, low-quality HiC datasets. We evaluate these models using HiC reproducibility metrics on data from three cell lines - GM12878 (lymphoblastoid), K562 (human erythroleukemic), and IMR90 (lung fibroblast) - obtained from different HiC experiments.

ResultsWe show that the deep-learning techniques evaluated in this study, trained on downsampled data, cannot upscale real-world, low-quality HiC matrices effectively. More importantly, we also show that retraining these methods on examples of real-world data improves their performance and similarity on target experimental data sets. However, even with retraining, our downstream analyses on the output of these methods suggest that these methods fail to capture the biological signals in the real-world inputs with high sparsity. Therefore, our study highlights the need for rigorous evaluation and identifies specific areas that need improvement concerning current deep learning-based HiC upscaling methods.

AvailabilityImplementation of our evaluation pipeline is available at https://github.com/rsinghlab/Investigation-of-HiC-Resolution-Improvement-Methods


title: In silico analysis of predicted differential MHC binding and CD8+ T-cell immune escape of SARS-CoV-2 B.1.1.529 variant mutant epitopes

abstract:
IntroductionThe B.1.1.529 (Omicron) SARS-CoV-2 variant has raised global concerns due to its high number of mutations and its rapid spread. It is of major importance to understand the impact of this variant on the acquired and induced immunity. Several preliminary studies have reported the impact of antibody binding and to this date, there are few studies on Omicrons CD8+ T-cell immune escape.

MethodsWe first assessed the impact of Omicron and B.1.617.2 (Delta) variant mutations on the SARS-CoV-2 spike epitopes submitted to the Immune Epitope Database (IEDB) with positive out-come on MHC ligand or T-cell assays (n=411). From those epitopes modified by a mutation, we found the corresponding homologous epitopes in Omicron and Delta. We then ran the netMHCpan computational MHC binding prediction on the pairs of IEDB epitopes and matching homologous epitopes over top 5 MHC I alleles on some selected populations. Lastly, we applied a Fisher test to find mutations enriched for homologous epitopes with decreased predicted binding affinity.

ResultsWe found 31 and 78 IEDB epitopes modified by Delta and Omicron mutations, respectively. The IEDB spike protein epitopes redundantly cover the protein sequence. The WT pMHC with a strong predicted binding tend to have homologous mutated pMHC with decreased binding. A similar trend is observed in Delta over all HLA genes, while in Omicron only for HLA-B and HLA-C. Finally, we obtained one and seven mutations enriched for homologous mutated pMHC with decreased MHC binding affinity in Delta and Omicron, respectively. Three of the Omicron mutations, VYY143-145del, K417N and Y505H, are replacing an aromatic or large amino acid, which are reported to be enriched in immunogenic epitopes. K417N is common with Beta variants, while Y505H and VYY143-145del are novel Omicron mutations.

ConclusionIn summary, pMHC with Delta and Omicron mutations show decreased MHC binding affinity, which results in a trend specific to SARS-CoV-2 variants. Such epitopes may decrease overall presentation on different HLA alleles suggesting evasion from CD8+ T-cell responses in specific HLA alleles. However, our results show B.1.1.529 (Omicron) will not totally evade the immune system through a CD8+ immune escape mechanism. Yet, we identified mutations in B.1.1.529 (Omicron) introducing amino acids associated with increased immunogenicity.

AvailabilityAll the code and results from this study are available at https://github.com/TRON-bioinformatics/omicron-analysis.


title: Analysis of receptors responsible for the dysfunction of the human immune system by different viral infections

abstract: There are difficulties in creating direct anti-viral drugs for all viruses, including new, suddenly arising infections, such as COVID-19. Therefore, pathogenetic therapy is often used to treat severe viral infections. Despite significant distinctions in the etiopathogenesis of viral diseases, they are often associated with the substantial dysfunction of the immune system. To identify shared mechanisms of immune dysfunction during infection by nine different viruses (cytomegalovirus, Ebstein-Barr virus, human T-cell leukemia virus type 1, Hepatitis B and C viruses, human immunodeficiency virus, Dengue virus, SARS-CoV, and SARS-CoV-2), we applied analysis of corresponding transcription profiles from peripheral blood mononuclear cells (PBMC). As a result, we revealed common pathways, cellular processes, and master regulators for studied viral infections. We found that all nine viral infections cause immune activation, exhaustion, cell proliferation disruption, and increased susceptibility to apoptosis. An application of network analysis allowed us to identify receptors of PBMC that are the proteins at the top of signaling pathways, which may be responsible for the observed transcription changes. The identified relationships between some of them and virus-induced immune disfunction are new, with little or no information in the literature, e.g., receptors for autocrine motility factor, insulin, prolactin, angiotensin II, and immunoglobulin epsilon.


title: Differences between Omicron SARS-CoV-2 RBD and other variants in their ability to interact with cell receptors and monoclonal antibodies

abstract: SARS-CoV-2 has caused immeasurable damage worldwide and available treatments with high efficacy are still scarce. With the continuous emergence of new variants of the virus, such as Omicron, Alpha, Beta, Gamma, and Delta - the so-called variants of concern, the available therapeutic and prevention strategies had to return to the experimental trial to verify their effectiveness against them. This work aims to expand the knowledge about the SARS-CoV-2 receptor-binding domain (RBD) interactions with cell receptors and monoclonal antibodies (mAbs). Special attention is given to the Omicron variant and its comparison with the others, including its sublineage BA.2 and two new ones (B.1.640.1 and B.1.640.2/IHU) recently found in France. By using constant-pH Monte Carlo simulations, the free energy of interactions between the SARS-CoV-2 receptor-binding domain (RBD) from different variants and several partners (Angiotensin-Converting Enzyme-2 (ACE2) polymorphisms and several mAbs) were calculated. It was evaluated both the impact of mutations for the RBD-ACE2 and how strongly each of mAb can bind to the virus RBD, which can indicate their therapeutic potential for neutralization. RBD-ACE2-binding affinities were higher for two ACE2 polymorphisms typically found in Europeans (rs142984500 and rs4646116), indicating that these types of polymorphisms may be related to genetic susceptibility to COVID-19. The antibody landscape was computationally investigated with the largest set of mAbs so far in the literature. From the 33 studied binders, groups of mAbs were identified with weak (e.g. S110 and Ab3b4), medium (e.g. CR3022), and strong binding affinities (e.g. P01, S2K146 and S230). All the mAbs with strong binding capacity could also bind to the RBD from SARS-CoV-1, SARS-CoV-2 wt, and all studied variants. These mAbs and especially their combination are amenable to experimentation and clinical trials because of their high binding affinities and neutralization potential for current known virus mutations and a universal coronavirus.

category: biophysics


title: Enrichment of specific GABAergic neuronal types in the mouse perirhinal cortex

abstract: GABAergic neurons represent 10-15% of the neuronal population of the cortex but exert a powerful control over information flow in cortical circuits. GABAergic neurons show an extraordinary diversity in their morphology, physiology, molecular markers and connectivity. This diversity allows GABAergic neurons to participate in a wide variety of microcircuit motifs. The diversity of GABAergic neurons has been shown to be conserved across cortical regions. The GABAergic population can be broadly divided in three major classes parvalbumin, somatostatin and 5HT3aR groups. The largest GABAergic class in the cortex is represented by the parvalbumin-expressing fast-spiking neurons, which provide powerful somatic inhibition to their postsynaptic targets. Recently, the density of parvalbumin-expressing neurons has been shown to be lower in associative areas of the mouse cortex, including the perirhinal cortex, as compared to sensory and motor areas. In the present study we investigated whether this reduction in parvalbumin-expressing neurons leads to a decreased GABAergic population, or to an enrichment of other GABAergic cell-types. We found that the GABAergic population of the perirhinal cortex is comparable to that of a primary sensory area, and it is enriched of neurons belonging to the 5HT3aR group. We also demonstrate that, despite the low density of parvalbumin-expressing neurons, the perirhinal cortex contains a comparable population of fast-spiking neurons, most of which do not express parvalbumin. Our results demonstrate a yet uncharacterized diversity within the fast-spiking population across cortical regions.


title: Reduction of layer 5 mediated long-range cortical communication by antipsychotic drugs

abstract: Psychosis is characterized by a diminished ability of the brain to distinguish externally driven activity patterns from self-generated activity patterns. Antipsychotic drugs are a class of small molecules with relatively broad binding affinity for a variety of neuromodulator receptors that, in humans, can prevent or ameliorate psychosis. How these drugs influence the function of cortical circuits and in particular their ability to identify self-generated activity patterns is still largely unclear. Here we used widefield calcium imaging to determine the cell type specific functional effects of antipsychotic drugs in mouse dorsal cortex during visuomotor integration. By comparing cell type specific activation patterns between locomotion onsets that were experimentally coupled to self-generated visual feedback and locomotion onsets that were not coupled, we show that deep cortical layers were differentially activated in these two conditions. We then show that the antipsychotic drug clozapine disrupted visuomotor integration at locomotion onsets also primarily in deep cortical layers. Given that one of the key components of visuomotor integration in cortex are long-range cortico-cortical connections, we tested whether the effect of clozapine was detectable in the correlation structure of activity patterns across dorsal cortex. We found that clozapine, as well as two other antipsychotic drugs, aripiprazole and haloperidol, resulted in a strong reduction in correlations of layer 5 activity between cortical areas and impaired the spread of visuomotor prediction errors generated in visual cortex. Our results are consistent with the interpretation that a major functional effect of antipsychotic drugs is an alteration of the activation of internal representations as a consequence of reduced long-range layer 5 mediated communication.


title: A critical test of deep convolutional neural networks ability to capture recurrent processing in the brain using visual masking

abstract:
Recurrent processing is a crucial feature in human visual processing supporting perceptual grouping, figure-ground segmentation, and recognition under challenging conditions. There is a clear need to incorporate recurrent processing in deep convolutional neural networks (DCNNs) but the computations underlying recurrent processing remain unclear. In this paper, we tested a form of recurrence in deep residual networks (ResNets) to capture recurrent processing signals in the human brain. Though ResNets are feedforward networks, they approximate an excitatory additive form of recurrence. Essentially, this form of recurrence consists of repeating excitatory activations in response to a static stimulus. Here, we used ResNets of varying depths (reflecting varying levels of recurrent processing) to explain electroencephalography (EEG) activity within a visual masking paradigm. Sixty-two humans and fifty artificial agents (10 ResNet models of depths - 4, 6, 10, 18 and 34) completed an object categorization task. We show that deeper networks (ResNet-10, 18 and 34) explained more variance in brain activity compared to shallower networks (ResNet-4 and 6). Furthermore, all ResNets captured differences in brain activity between unmasked and masked trials, with differences starting at [~]98ms (from stimulus onset). These early differences indicated that EEG activity reflected  pure feedforward signals only briefly (up to [~]98ms). After [~]98ms, deeper networks showed a significant increase in explained variance which peaks at [~]200ms, but only within unmasked trials, not masked trials. In summary, we provided clear evidence that excitatory additive recurrent processing in ResNets captures some of the recurrent processing in humans.

Significance statementThe challenge of modeling recurrent processes is not trivial and the operationalization of recurrent processing is highly contested. In this paper, we tested the ability of deep residual networks (ResNets) to explain recurrent processes in the human brain. Though ResNets are feedforward networks, they have been shown to equate operations in recurrent neural networks. In this study, we show that deeper networks explained more variance in brain activity than shallower networks. However, all networks still performed far from the noise ceiling. Thus, we conclude that recurrent processing in ResNets captures a form of recurrent processing in humans though other types of recurrent processing (inhibition, multiplicative) that are not present in current regular deep neural networks (alexnet, cornet, resnet) are necessary for building better visual models.


title: Vamos en bici: Study protocol of an investigation of cognitive and neural changes following language training, physical exercise training, or a combination of both

abstract: As the relative number of elderly people as well as the average life expectancy increases, identifying potential means to alter the ongoing trajectory of aging and specifically the trajectory of cognitive performance is of great importance. Some modifiable lifestyle factors, such as physical and cognitive activity, have shown positive effects on cognition and brain structure, and the combination of the two might even show a boosted, interactive effect. In this study protocol, we describe in detail our data which was acquired to explore how cognitive stimulation in the form of acquiring a new language, physical exercise on stationary bikes or the combination of the two interventions affect brain structure, cognitive performance, and psychosocial functioning. One-hundred and forty-two older healthy adults (63-78 years) were randomly assigned to one of four six-month intervention programs, comprising (a) foreign language learning, (b) physical exercise training on a bicycle ergometer, (c) a combination of language learning and physical exercise, or (d) a book club (serving as an active control condition). We collected a rich neuroimaging data set, comprising T1-weighted structural, resting state functional, high resolution hippocampal, myelin water fraction, diffusion-weighted, arterial spin labeling, and multi-parameter images. Using a cognitive battery, we collected data from the domains of episodic memory, working memory, perceptual speed, and fluid intelligence. We performed comprehensive physical assessments including cardiopulmonary exercise testing, and additionally collected data on psychosocial functioning (e.g., well-being, perceived stress, control beliefs). We assume that physical activity boosts brain plasticity per se by inducing structural and neurochemical changes in brain regions that are important for learning and memory and therefore may facilitate the effects of cognitive training. (269 words)


title: An actionable map of arm and hand muscle responses to electrical stimulation of the dorsal cervical spinal cord in humans

abstract:
While epidural stimulation of the lumbar spinal cord has emerged as a powerful modality for recovery of movement, how it should be targeted to the cervical spinal cord to activate arm and hand muscles is not well-understood, particularly in humans. We sought to map muscle responses to posterior epidural cervical spinal cord stimulation in humans. We hypothesized that lateral stimulation over the dorsal root entry zone would be most effective, and responses would be strongest in the muscles innervated by the stimulated segment. Eighteen people undergoing clinically indicated cervical spine surgery were enrolled. During surgery, stimulation was performed in midline and lateral positions at multiple exposed segments; eight arm and three leg muscles were recorded on each side of the body. Across all segments and muscles tested, lateral stimulation produced stronger muscle responses than midline. These stronger responses were due to a combination of a decreased response threshold and steeper recruitment slope. Muscles innervated at a cervical segment had the largest responses from stimulation at that segment, but responses were also observed in muscles innervated at other cervical segments and in leg muscles. The cervical responses were clustered in rostral (C4-C6) and caudal (C7-T1) cervical segments. Strong responses to lateral stimulation are likely due to the proximity of stimulation to afferent axons. Small changes in responses to stimulation of adjacent cervical segments argues for local circuit integration, and distant muscle responses suggest activation of long propriospinal connections. This map can help guide cervical stimulation to improve arm and hand function.

Significance StatementEpidural stimulation of the human lumbar spinal cord for movement recovery after injury has been guided by a map of muscle responses to stimulation, but no such map exists for the human cervical spinal cord. A map of muscle responses to cervical epidural stimulation during clinically indicated surgery revealed a much stronger effect of changes in midline versus lateral stimulation compared with stimulation at different cervical segments. In addition, muscle responses were highly correlated between each segment within the upper cervical enlargement (C4-C6) and within the lower cervical enlargement (C7-T1), but these regions were not as similar to each other. Thus, a map of cervical spinal cord stimulation reveals its internal organization and may help guide therapy.


title: Neural correlates of perceived emotions in human insula and amygdala

abstract: The emotional status of a speaker is an important non-linguistic cue carried by human voice and can be perceived by a listener in vocal communication. Understanding the neural circuits involved in processing emotions carried by human voice is crucial for understanding the neural basis of social interaction. Previous studies have shown that human insula and amygdala responded more selectively to emotional sounds than non-emotional sounds. However, it is not clear whether the neural selectivity to emotional sounds in these brain structures is determined by the emotion presented by a speaker or by the emotion perceived by a listener. In this study, we recorded intracranial electroencephalography (iEEG) responses to emotional human voices while subjects performed emotion recognition tasks. We found that the iEEG responses of Heschls gyrus (HG) and posterior insula were determined by the presented emotion, whereas the iEEG responses of anterior insula and amygdala were driven by the perceived emotion. These results suggest that the anterior insula and amygdala play a crucial role in representing subjectively perceived emotion.


title: High Frequencies of PD-1+TIM3+TIGIT+CTLA4+ Functionally Exhausted SARS-CoV-2-Specific CD4+ and CD8+ T Cells Associated with Severe Disease in Critically ill COVID-19 Patients

abstract:
SARS-CoV-2-specific memory T cells that cross-react with common cold coronaviruses (CCCs) are present in both healthy donors and COVID-19 patients. However, whether these cross-reactive T cells play a role in COVID-19 pathogenesis versus protection remain to be fully elucidated. In this study, we characterized cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells, targeting genome-wide conserved epitopes in a cohort of 147 non-vaccinated COVID-19 patients, divided into six groups based on the degrees of disease severity. We compared the frequency, phenotype, and function of these SARS-CoV-2-specific CD4+ and CD8+ T cells between severely ill and asymptomatic COVID-19 patients and correlated this with -CCCs and {beta}-CCCs co-infection status. Compared with asymptomatic COVID-19 patients, the severely ill COVID-19 patients and patients with fatal outcomes: (i) Presented a broad leukocytosis and a broad CD4+ and CD8+ T cell lymphopenia; (ii) Developed low frequencies of functional IFN-{gamma}-producing CD134+CD138+CD4+ and CD134+CD138+CD8+ T cells directed toward conserved epitopes from structural, non-structural and regulatory SARS-CoV-2 proteins; (iii) Displayed high frequencies of SARS-CoV-2-specific functionally exhausted PD-1+TIM3+TIGIT+CTLA4+CD4+ and PD-1+TIM3+TIGIT+CTLA4+CD8+ T cells; and (iv) Displayed similar frequencies of co-infections with {beta}-CCCs strains but significantly fewer co-infections with -CCCs strains. Interestingly, the cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcome seen in asymptomatic COVID-19 patients. Our results demonstrate that, the critically ill COVID-19 patients displayed fewer co-infection with -CCCs strain, presented broad T cell lymphopenia and higher frequencies of cross-reactive exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells. In contrast, the asymptomatic COVID-19 patients, appeared to present more co-infections with -CCCs strains, associated with higher frequencies of functional cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells. These findings support the development of broadly protective, T-cell-based, multi-antigen universal pan-Coronavirus vaccines.

KEY POINTSO_LIA broad lymphopenia and lower frequencies of SARS-CoV-2-specific CD4+ and CD8+ T-cells were associated with severe disease onset in COVID-19 patients.
C_LIO_LIHigh frequencies of phenotypically and functionally exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells, co-expressing multiple exhaustion markers, and targeting multiple structural, non-structural, and regulatory SARS-CoV-2 protein antigens, were detected in severely ill COVID-19 patients.
C_LIO_LICompared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented more functional cross-reactive CD4+ and CD8+ T cells targeting conserved epitopes from structural, non-structural, and regulatory SARS-CoV-2 protein antigens.
C_LIO_LIThe cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcomes seen in asymptomatic COVID-19 patients.
C_LIO_LICompared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented higher rates of co-infection with the -CCCs strains.
C_LIO_LICompared to patients with mild or asymptomatic COVID-19, severely ill symptomatic patients and patients with fatal outcomes had more exhausted SARS-CoV-2-speccific CD4+ and CD8+ T cells that preferentially target cross-reactive epitopes that share high identity and similarity with the {beta}-CCCs strains.
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title: Heightened NLRP3 inflammasome activation is associated with aging and CMML diseases severity

abstract: Aging causes chronic low-grade inflammation known as inflamm-aging. It is a risk factor for chronic myelomonocytic leukemia (CMML), a hematological malignancy that is most prevalent in older people. Recent studies suggest a critical role for the NLRP3 inflammasome in inflamm-aging. However, the mechanisms involved in NLRP3 activation in aging and its involvement in CMML progression are not fully understood. Here, we report that aging increases interleukin-1{beta} production upon NLRP3 activation in human CD14+ monocytes. Interestingly, we found that Toll-like receptor (TLR) 1/2 agonist Pam3Cysk4 directly activates NLRP3 inflammasome without the requirement of second activation signal in monocytes from older but not from younger healthy donors. Further, we observed a dichotomous response to NLRP3 inflammasome activation in monocytes from CMML patients. Intriguingly, CMML patients with heightened NLRP3 activation showed increased treatment dependency and disease severity. Collectively, our results suggest that aging causes increased sensitivity to NLRP3 inflammasome activation at cellular level, which may explain increased inflammation and immune dysregulation in older individuals. Furthermore, NLRP3 inflammasome activation was dysregulated in CMML and positively correlated with disease severity.


title: Differences in the gut permeability marker levels, and abundance and nuclear antigen reactivity of fecal immunoglobulin A (IgA) subclasses in systemic lupus erythematosus patients and healthy controls

abstract: Systemic lupus erythematosus (SLE) is characterized by the production of anti-nuclear autoantibodies. Here, for the first time, we show that the abundances of gut permeability marker Zonulin and IgA1- and IgA2-subclasses are significantly higher in the fecal samples of SLE patients compared to HCs. Importantly, IgA-total, IgA1- and IgA2-subclasses from SLE patients showed higher nAg reactivity titers. Notably, we found that not only the nuclear antigen (nAg) reactive fecal IgA1:IgA2 ratio is higher in SLE patients, but also the abundance and nAg reactivity of fecal IgA and subclasses, IgA1 particularly, correlate with the fecal levels of Zonulin, which is produced primarily in the small intestine. These observations suggest that higher amounts of nAg-reactive IgA are produced, particularly, in the proximal gut as indicated by higher IgA1:IgA2 abundance and nAg reactivity ratios and correlation of these features with the Zonulin levels.


title: Fecal immunoglobulin A (IgA) and its subclasses in systemic lupus erythematosus patients are nuclear antigen reactive and this feature correlates with gut permeability marker levels


title: Virucidal activity and mechanism of action of cetylpyridinium chloride against SARS-CoV-2

abstract:
ObjectiveSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. Recent studies have shown the importance of the throat and salivary glands as sites of virus replication and transmission. The viral host receptor, angiotensin-converting enzyme 2 (ACE2), is broadly enriched in epithelial cells of the salivary glands and oral mucosae. Oral care products containing cetylpyridinium chloride (CPC) as a bactericidal ingredient are known to exhibit antiviral activity against SARS-CoV-2 in vitro. However, the exact mechanism of action remains unknown.

MethodsThis study examined the antiviral activity of CPC against SARS-CoV-2 and its inhibitory effect on the interaction between the viral spike (S) protein and ACE2 using an enzyme-linked immunosorbent assay.

ResultsCPC (0.05%, 0.1% and 0.3%) effectively inactivated SARS-CoV-2 within the contact times (20 and 60 s) in directions for use of oral care products in vitro. The binding ability of both the S protein and ACE2 were reduced by CPC.

ConclusionsOur results suggest that CPC inhibits the interaction between S protein and ACE2, and thus, reduces infectivity of SARS-CoV-2 and suppresses viral adsorption.


title: Role of mucin gene for growth in Anas platyrynchos - a novel report

abstract: Mucin gene is expressed at the mucous membrane of the inner layer of the internal organs. Intestinal mucin 2 (MUC2), a major gel-forming mucin, represents a primary barrier component of mucus layers. This is the first report of role of mucin gene in growth traits in animals. In the current study, we had randomly studied Bengal ducks (Anas platyrynchos) reared from day old to 10 weeks of age under organized farm and studied the growth parameters as well as body weight and average daily body weight gain. We had characterized mucin gene for Bengal duck and observed glycosylation and EGF1 (EGF like domain signature) as important domain for growth traits in duck. We observed better expression profile for mucin gene in high growing ducks in comparison to that of lower growing ducks with real time PCR. Hence mucin gene may be employed as a marker for growth traits.

category: genomics


title: Indigenous peoples in eastern Brazil: insights from 19th century genomes and metagenomes

abstract:
Although Brazil was inhabited by more than 3,000 Indigenous populations prior to European colonization, todays Indigenous peoples represent less than 1% of Brazils census population. Some of the decimated communities belonged to the so-called "Botocudos" from central-eastern Brazil. These peoples are thought to represent a case of long-standing genetic continuity bearing a strong craniometric resemblance to that of the oldest Indigenous Americans ("Paleoamericans"). Yet, little is known about their origins and genetic relationship to other Native Americans, as only two "Botocudo" genomes have been sequenced so far and those were surprisingly of Polynesian ancestry. To deepen our knowledge on the genomic history of pre-contact Indigenous Americans and the pathogens they were exposed to, we carbon-dated and sequenced 24 ancient Brazilians (including 22 "Botocudos") whose remains were hosted at the National Museum of Rio de Janeiro and recovered prior to the tragic 2018 fire. The resulting genomes depth of coverage ranged from 0.001x to 24x. Their genetic ancestry was found to be Indigenous American without gene flow from external populations such as Europeans, Africans or Polynesians. Unlike Mesoamericans, the "Botocudos" and Amazonians do not seem to have experienced a population expansion once in the Americas. Moreover, remarkably, their genomes exhibit amongst the lowest levels of heterozygosity worldwide and long runs of homozygosity, which could be explained by unique social practices or a very small effective size. Finally, whole genomes of likely ancient pathogens were recovered, including lineages of Human parvovirus B19 that were possibly introduced after the European contact.

Significance statementTo better understand the genetic relationship among Indigenous populations in Brazil, we sequenced the genomes of 24 ancient individuals (22 of which labelled as "Botocudos", a term used to describe hunter-gatherer tribes) whose remains were hosted at the Museu Nacional of Rio de Janeiro prior to the tragic fire that consumed it in 2018. Unlike two previously published "Botocudo" genomes, the 22 "Botocudos" from this study have Indigenous American-related ancestry without any Polynesian-related ancestry, and they are similarly related to several Native Brazilian populations. Finally, unlike Eurasian hunter-gatherers, the "Botocudos" exhibit among the lowest heterozygosity and longest runs of homozygosity worldwide - compatible with a very small effective size and suggesting a unique social structure among hunter-gatherers in the Americas.


title: Spatial Venomics - Cobra Venom System Reveals Spatial Differentiation of Snake Toxins by Mass Spectrometry Imaging

abstract: Among venomous animals, toxic secretions have evolved as biochemical weapons associated with various highly specialized delivery systems on many occasions. Despite extensive research, there is still limited knowledge of the functional biology of most animal toxins, including their venom production and storage, as well as the morphological structures within sophisticated venom producing tissues that might underpin venom modulation. Here we report on the spatial exploration of a snake venom gland system by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), in combination with standard proteotranscriptomic approaches, to enable in situ toxin mapping in spatial intensity maps across a venom gland sourced from the Egyptian cobra (Naja haje). MALDI-MSI toxin visualization on the elapid venom gland reveals high spatial heterogeneity of different toxin classes at the proteoform level, which may be the result of physiological constraints on venom production and/or storage that reflects the potential for venom modulation under diverse stimuli.

category: biochemistry


title: Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

abstract: Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including E. coli RseP. Our observations are consistent with a rearrangement of the RseP domains surrounding the active center to expose the substrate-binding site where a conserved electrostatic linkage between the transmembrane and membrane-associated domains mediates the conformational changes, suggesting that RseP has a gating mechanism to regulate substrate entry. Mutational analysis also supports that the substrate transmembrane helix is unwound by strand addition to the intramembrane {beta} sheet and is clamped at the active center for efficient cleavage. Furthermore, this substrate accommodation mechanism appears to be common across distinct intramembrane proteases.


title: A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA

abstract: Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader Replication Factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFCs central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFCs external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.


title: Neural network for the prediction of treatment response in triple negative breast cancer

abstract:
AO_SCPLOWBSTRACTC_SCPLOWThe automatic analysis of stained histological sections is becoming increasingly popular. Deep Learning is today the method of choice for the computational analysis of such data, and has shown spectacular results for large datasets for a large variety of cancer types and prediction tasks. On the other hand, many scientific questions relate to small, highly specific cohorts. Such cohorts pose serious challenges for Deep Learning, typically trained on large datasets.

In this article, we propose a modification of the standard nested cross-validation procedure for hyper-parameter tuning and model selection, dedicated to the analysis of small cohorts. We also propose a new architecture for the particularly challenging question of treatment prediction, and apply this workflow to the prediction of response to neoadjuvant chemotherapy for Triple Negative Breast Cancer.


title: Making sense of disorder: Investigating intrinsically disordered proteins in the tardigrade proteome via a computational approach

abstract: Tardigrades, also known as water bears, are a phylum of microscopic metazoans with the extraordinary ability to endure environmental extremes. When threatened by suboptimal habitat conditions, these creatures enter a suspended animation-like state called cryptobiosis, in which metabolism is diminished, similar to hibernation. In this state, tardigrades benefit from enhanced extremotolerance, withstanding dehydration efficiently for years at a time in a type of cryptobiosis called anhydrobiosis. Recent studies have demonstrated that the tardigrade proteome is at the heart of cryptobiosis. Principally, intrinsically disordered proteins (IDPs) and tardigrade-specific intrinsically disordered proteins (TDPs) are known to help protect cell function in the absence of water. Importantly, TDPs have been successfully expressed in cells of other species experimentally, even protecting human tissue against stress in vitro. However, previous work has failed to address how to strategically identify TDPs in the tardigrade proteome holistically. The overarching purpose of this current study, consequently, was to generate a list of IDPs/TDPs associated with tardigrade cryptobiosis that are high-priority for further investigation. Firstly, a novel database containing 44,836 tardigrade proteins from 338 different species was constructed to consolidate and standardize publicly available data. Secondly, a support vector machine (SVM) was created to sort the newly constructed database entries on the binary basis of disorder (i.e., IDP versus non-IDP). Features of this model draw from disorder metrics and literature curation, correctly classifying 160 of the 171 training set proteins (~93.6%). Of the 5,415 putative IDPs/TDPs our SVM identified, we present 82 (30 having confident subclass prediction and 52 having experimental detection in previous studies). Subsequently, the role each protein might play in tardigrade resilience is discussed. By and large, this supervised machine learning classifier represents a promising new approach for identifying IDPs/TDPs, opening doors to harness the tardigrades remarkable faculties for biomaterial preservation, genetic engineering, astrobiological research, and ultimately, the benefit of humankind.


title: Tension at intercellular junctions is necessary for accurate orientation of cell division in the epithelium plane

abstract:
The direction in which a cell divides is set by the orientation of its mitotic spindle and is important for determining cell fate, controlling tissue shape and maintaining tissue architecture. Division perpendicular to the plane of the substrate can promote tissue stratification during development or wound healing, but also metastasis when orientation is aberrant. Much is known about the molecular mechanisms involved in setting the spindle orientation. However, less is known about the contribution of mechanical factors, such as tissue tension, in ensuring spindle orientation in the plane of the epithelium, despite epithelia being continuously subjected to mechanical stresses. Here, we used suspended epithelial monolayers devoid of extracellular matrix and subjected to varying levels of tissue tension to study the orientation of division relative to the tissue plane. We found that decreasing tissue tension by compressing the monolayers or by inhibiting myosin contractility leads to a higher frequency of out-of-plane divisions. Reciprocally, accurate in-plane division can be restored by increasing tissue tension by increasing cell contractility or by tissue stretching. By considering the full three-dimensional geometry of the epithelium, we show that spindles are sensitive to tissue tension, independently of cell shape, through its impact on the tension at subcellular surfaces. Overall, our data suggest that accurate spindle orientation in the plane of the epithelium necessitates the presence of a sufficiently large tension at intercellular junctions.

Significance statementIn growing epithelia, divisions are typically oriented in the plane of the tissue to drive expansion. In some organs, divisions are then re-oriented so that they occur perpendicular to the epithelium plane to drive tissue stratification and cell differentiation. When uncontrolled, this switch in orientation can lead to defects in tissue organisation and, in cancer, likely contribute to metastasis. While much is known about the molecular mechanisms controlling mitotic spindle orientation, less is known about the role of mechanical factors. Here we use mechanical and chemical perturbations to show that mechanics plays a role in controlling the plane of division. Overall, our data suggest that the orientation of spindles in the epithelium plane requires sufficient tension across intercellular junctions.


title: Active site center redesign increases protein stability preserving catalysis

abstract:
The stabilization of natural proteins is a long-standing desired goal in protein engineering. Optimizing the hydrophobicity of the protein core often results in extensive stability enhancements. However, the presence of totally or partially buried catalytic charged residues, essential for protein function, has limited the applicability of this strategy. Here, focusing on the thioredoxin, we aimed to augment protein stability by removing buried charged residues in the active site without loss of catalytic activity. To this end, we performed a charged-to-hydrophobic substitution of a buried and functional group, resulting in a significant stability increase yet abolishing catalytic activity. Then, to simulate the catalytic role of the buried ionizable group, we designed a combinatorial library of variants targeting a set of seven surface residues adjacent to the active site. Notably, more than 50% of the library variants restored, to some extent, the catalytic activity. The combination of experimental study of 2% of the library with the prediction of the whole mutational space by Partial Least-squares regression revealed that a single point mutation at the protein surface is sufficient to fully restore the catalytic activity without thermostability cost. As a result, we engineered one of the highest thermal stability reported for a protein with a natural occurring fold (138 {degrees}C). Further, our hyperstable variant preserves the catalytic activity both in vitro and in vivo.

SIGNIFICANCEThe major driving force of protein folding is the hydrophobic effect, and increasing the protein core hydrophobicity essentially increases protein stability. Active sites often contain buried ionizable groups, which can be essential for function but dramatically reduce protein stability. Thus, increasing the protein core hydrophobicity cannot be applied to enzyme active sites without a functional cost. We propose a method to enhance protein stability by overcoming this obstacle. We show that catalytic properties of buried charges can be mimicked with surface mutations, thus paving the way to unlock the optimization of the hydrophobic core to stabilize enzymes.


title: Highly efficient, all-organic bioluminescence-photosensitizer conjugate eradicates early-stagetumors and prevents metastasis in mice

abstract: Photodynamic therapy (PDT) is an established treatment modality using light-activatable drugs. Despite its unique cytotoxic mechanism, the shallow penetration of light has been a serious drawback limiting the applications of PDT. Here, we report bioluminescence-activated PDT (BL-PDT) using efficient bioluminescence resonance energy transfer (BRET) conjugates of clinically approved photosensitizers, Ce6, and luciferase proteins. A high photon-to-Ce6 conversion efficiency (80%), along with intracellular delivery by membrane-fusion liposomes, enabled effective cancer cell killing in vitro. In a syngeneic mouse model of aggressive 4T1 triple-negative breast cancer, as well as a xenograft model of MDA-MB-231 tumors, BL-PDT resulted in complete tumor remission and prevention of metastasis, as well as neo-adjuvant effects. Our result shows the promise of molecularly activable, clinically viable, depth-unlimited phototherapy.

category: cancer biology


title: A novel role of 3,5-cAMP in the regulation of actin cytoskeleton in Arabidopsis.

abstract: The role of cyclic adenosine monophosphate (3,5-cAMP) in plants is not well understood, and here, we report a novel role of 3,5-cAMP in regulating the actin cytoskeleton. The 3,5-cAMP treatment affects the thermal stability of 51 proteins, including a vegetative actin isoform, ACTIN2. Consistent with the above results, the increase in 3,5-cAMP levels, obtained either by feeding or by chemical modulation of 3,5-cAMP metabolism, is sufficient to partially rescue the short hypocotyl phenotype of the actin2 actin7 mutant, severely compromised in actin level. No such complementation was measured for a positional isomer of 3,5-cAMP, 2,3-cAMP, attesting to the specificity of 3,5-cAMP treatment. Moreover, supplementation of 3,5-cAMP partly counters the activity of an actin-depolymerizing drug latrunculin B. In vitro characterization of the 3,5-cAMP - actin interaction argues against the direct binding. Instead, based on the proteomics characterization of the act2act7 mutant supplemented with 3,5-cAMP, we hypothesize that 3,5-cAMP affects cytoskeleton dynamic by modulation of calcium signaling, and actin binding proteins.

category: plant biology


title: Cerebellar transcranial direct current stimulation disrupts neuroplasticity of intracortical motor circuits.

abstract: While previous research using transcranial magnetic stimulation (TMS) suggest that cerebellum (CB) influences the neuroplastic response of primary motor cortex (M1), the role of different indirect (I) wave inputs in M1 mediating this interaction remains unclear. The aim of this study was therefore to assess how CB influences neuroplasticity of early and late I-wave circuits. 22 young adults (22 {+/-} 2.7 years) participated in 3 sessions in which I-wave periodicity repetitive transcranial magnetic stimulation (iTMS) was applied over M1 during concurrent application of cathodal transcranial direct current stimulation over CB (tDCSCB). In each session, iTMS either targeted early I-waves (1.5 ms interval; iTMS1.5), late I-waves (4.5 ms interval; iTMS4.5), or had no effect (variable interval; iTMSSham). Changes due to the intervention were examined with motor evoked potential (MEP) amplitude using TMS protocols measuring corticospinal excitability (MEP1mV) and the strength of CB-M1 connections (CBI). In addition, we indexed I-wave activity using short-interval intracortical facilitation (SICF) and low-intensity single-pulse TMS applied with posterior-anterior (MEPPA) and anterior-posterior (MEPAP) current directions. Following both active iTMS sessions, there was no change in MEP1mV, CBI or SICF (all P > 0.05), suggesting that tDCSCB broadly disrupted the excitatory response that is normally seen following iTMS. However, although MEPAP also failed to facilitate after the intervention (P > 0.05), MEPPA potentiated following both active iTMS sessions (both P < 0.05). This differential response between current directions suggests that the disruptive effects of CB modulation on M1 plasticity may be selectively mediated by AP-sensitive circuits (also likely recruited with MEP1mV, CBI, and SICF).


title: Origin of hexagonal metric of grid cells in navigating spaces

abstract: To achieve the computational goal of navigating both natural and mental spaces, the brain adopts a hexagonal metric of grid cells in the entorhinal cortex (ERC) to chart the spaces. However, little is known about how and why the hexagonal metric emerges. Here we designed an object matching task where participants adjusted two parts of object variants to match their prototype in fMRI scanner. Unbeknownst to the participants, the object variants were arranged on a ring centering on the prototype in an object space constructed by the object parts. When the participants navigated from a location in the ring to the center in the space, we observed hexagonal signals in the ERC, and more importantly, a spatial rhythm of 3Hz in the hippocampus (HPC). Accordingly, we built a computational model depicting the spatial rhythm of 3Hz as scaffolds to transform discrete spatial locations into structured states of cognitive map that allows continuous updates of location estimates with each movement made. The formal proof shows that to support the cognitive map in the HPC, the spatial input from the ERC must be in a hexagonal pattern. To further examine the biological plausibility of the model, we built various forms of neural networks in their simplest architecture to transform locations in space into structured states of cognitive map, and units with hexagonal patterns emerge ubiquitously. In short, with empirical experiments, formal proof, and neural network simulation, our study reveals the origin of the hexagonal metric of grid cells mechanistically.


title: Glia of C. elegans coordinate the heat shock response independent of the neuronal thermosensory circuit and serotonin

abstract: As organisms age, they lose the ability to induce appropriate stress responses, becoming vulnerable to protein toxicity and tissue damage. Neurons can signal to peripheral tissues to induce protective organelle-specific stress responses. Recent work has demonstrated a novel and independent role of glia in inducing such responses. Here, we show that overexpression of heat shock factor 1 (hsf-1) in the four astrocyte-like cephalic sheath cells of C. elegans is sufficient to induce a non-cell autonomous cytosolic unfolded protein response (UPR), also known as the heat shock response (HSR), in distal cells. These animals upregulate the HSR in peripheral cells and have increased lifespan and resistance to heat stress. This glial HSR regulation is independent of the canonical neuronal thermosensory circuit and of known neurotransmitters but is dependent on the small clear vesicle release protein UNC-13. Additionally, HSF-1 and the FOXO transcription factor DAF-16 are partially required in peripheral tissues for increase of non-autonomous HSR, lifespan, and thermotolerance. We find that cephalic sheath glial hsf-1 over-expression leads to increased pathogen resistance, which suggests a role for this signaling pathway in immune function.


title: Impact of APOE e3 and e4 genotypes on plasma proteome signatures in Alzheimer's disease

abstract: The {varepsilon}4 allele of the apolipoprotein E (APOE) gene is a high-risk factor for Alzheimers disease (AD). However, approximately 25%-40% of patients with AD do not carry the APOE{varepsilon}4 allele, and the pathophysiological mechanisms underlying AD are less evident in these individuals. The main objective of this study was to understand better the changes in plasma that may contribute to disease pathogenesis in AD and how APOE{varepsilon}3 and APOE{varepsilon}4 contribute to biomarker profiles in AD. We conducted an in-depth plasma proteomics analysis using intensive depletion of high-abundant plasma proteins using the Agilent multiple affinity removal liquid chromatography (LC) column-Human 14 (Hu14) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) technique. In this study, we identified a high number of protein expression alterations in plasma which were found uniquely in APOE{varepsilon}3 and APOE{varepsilon}4 carriers. These differentially expressed proteins (DEPs) were associated with several molecular functions, including complement cascade, glycolysis, metabolism, plasma lipoprotein assembly, remodelling, and clearance. In addition to unique changes in both APOE genotypes, many proteins were also dysregulated in the presence of both APOE{varepsilon}3 and APOE{varepsilon}4 genotypes depicting the involvement of these proteins in the pathogenesis of AD regardless of the APOE genotypes. We also compared the plasma proteomes of{varepsilon} 4 and{varepsilon} 3 carriers in normal controls, which provided insight into factors that may provide protection from progression to AD despite the presence of the{varepsilon} 4 allele. Furthermore, our findings also identified some proteins previously discovered in AD CSF and brain proteomics signatures that could provide clinically meaningful information.


title: Do place cells dream of deceptive moves in a signaling game?

abstract: We consider the possibility of applying game theory to analysis and modeling of neurobiological systems. Specifically, the basic properties and features of information asymmetric signal games are considered and discussed as having potential to explain diverse neurobiological phenomena at levels of biological function that include gene regulation, molecular and biochemical signaling, cellular and metabolic function, as well as neuronal action potential discharge to represent cognitive variables that may underlie memory and behavior. We begin by arguing that there is a pressing need for conceptual frameworks that can permit analysis and integration of information and explanations across the many scales of diverse levels of biological function if we are to understand cognitive functions like learning, memory, and perception. The present work focuses on systems level neuroscience organized around the connected brain regions of the entorhinal cortex and hippocampus. These areas are intensely studied in rodent subjects as model neuronal systems that undergo activity-dependent synaptic plasticity to form and represent memories and spatial knowledge used for purposeful navigation. Examples of cognition-related spatial information in the observed neuronal discharge of hippocampal place cell populations and medial entorhinal head-direction cell populations are used to illustrate possible challenges to information maximization concepts that may be natural to explain using the ideas and features of information asymmetric signaling games.


title: Acyl ghrelin attenuates neurochemical and motor deficits in the 6OHDA model of Parkinsons disease

abstract:
The feeding-related hormone, acyl-ghrelin, protects dopamine neurons in murine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-based models of experimental Parkinsons disease (PD). However, the potential protective effect of acyl-ghrelin on substantia nigra pars compacta (SNpc) dopaminergic neurones and consequent behavioural correlates in the more widely used 6-hydroxydopamine (6-OHDA) rat medial forebrain bundle (MFB) lesion model of PD are unknown. To address this question, acyl-ghrelin levels were raised directly by mini-pump infusion for 7-days prior to unilateral injection of 6-OHDA into the MFB with assessment of amphetamine-induced rotations on days 27 and 35, and immunohistochemical analysis of dopaminergic neurone survival. While acyl-ghrelin treatment was insufficient to elevate food intake or body weight, it attenuated amphetamine-induced circling behaviour and SNpc dopamine neurone loss induced by 6-OHDA. These data support the notion that elevating circulating acyl-ghrelin may be a valuable approach to slow or impair progression of neurone loss in PD.

HighlightsO_LIAcyl-ghrelin attenuates SNpc dopamine cell loss in rat 6-OHDA-lesion model of PD
C_LIO_LIAcyl-ghrelin attenuates motor deficits in rat 6-OHDA-lesion model of PD
C_LI


title: Recipient LAG3 deficiency results in antibody-mediated rejection of mouse renal allografts

abstract: Lymphocyte activation gene-3 (LAG3) is a coinhibitory receptor expressed by a wide range of immune cells. While LAG3 is predominantly studied in T cells, its functions in humoral immune responses remain poorly characterized. The goal of this study was to test the role of recipient LAG3 in a mouse model of renal allograft rejection. Compared to WT animals, naive B6.LAG3-/- mice have increased splenic cellularity and higher frequencies of CD44hi memory T cells, CXCR5hi follicular T cells, and B220+CD138+ plasma cells yet do not develop spontaneous autoimmunity. Furthermore, naive B6.LAG3-/- (H-2Db) mice have increased frequencies of memory T cells recognizing H-2Dd, H-2Ds, H-2Dq and H-2Dk alloantigens. In addition, naive non-transplanted B6.LAG3-/- mice have elevated levels of serum IgG reactive to allogenic class I and class II MHC molecules. Whereas 4/4 B6.WT recipients spontaneously accept fully MHC-mismatched C3H (H-2k) renal allografts for longer than 60 d, recipient LAG3 deficiency led to rapid allograft rejection and elevated serum creatinine levels. Graft histology at rejection revealed minimal T cell infiltration, diffuse C4d staining, atrophic peritubular capillaries, endothelial swelling and edema characteristic of antibody mediated rejection (AMR). Compared to WT, LAG3-/- recipients had elevated frequencies of anti-donor IFN{gamma} producing T cells and increased levels of anti-donor MHC antibodies. Recipient CD8+ T cell depletion did not alter the rejection kinetics in LAG3-/- recipients, while B cell depletion significantly extended C3H kidney allograft survival. Conditional knockout mice were used to determine the importance of LAG3 expression by T and B lymphocytes. The absence of LAG3 on either T or B lymphocytes had no impact on graft rejection with both groups accepting kidney allografts for longer than 30 days, suggesting that LAG3 expression on either T or B cells is sufficient to regulate immune responses to kidney allografts. These results suggest that LAG3 is a regulator of both T and B cell responses to kidney allografts and is an attractive potential therapeutic target for AMR prevention and treatment.


title: High-resolution structural analysis of enterovirus-reactive polyclonal antibodies in complex with whole virions

abstract: Non-polio enteroviruses (NPEVs) cause serious illnesses in young children and neonates including aseptic meningitis, encephalitis, neonatal sepsis and inflammatory muscle disease, among others. Over 100 serotypes have been described to date but except for the EV-A71, there are no available vaccines or therapeutics against NPEVs. Efforts towards rationally designed pan-NPEV vaccines would greatly benefit from structural biology methods for rapid and comprehensive evaluation of vaccine candidates and elicited antibody responses. Towards this goal, we tested if electron-microscopy-based polyclonal epitope mapping (EMPEM), a method where structural analysis is performed using serum-derived polyclonal antibodies (pAbs), can be applied to an NPEV. EMPEM was performed on immune complexes featuring CV-A21 viral particles and CV-A21-specific pAbs isolated from mice. Notably, this is the first example of structural analysis of polyclonal immune complexes comprising whole virions. We introduce a data processing workflow that allows reconstruction of different pAbs at near-atomic resolution. The analysis resulted in identification of several antibodies targeting two immunodominant epitopes, near the 3-fold and 5-fold axis of symmetry; the latter overlapping with the receptor binding site. These results demonstrate that EMPEM can be applied to map broad-spectrum polyclonal immune responses against intact virions and define potentially cross-reactive epitopes.


title: Ornithine Lipid Activates Both TLR4 and the non-canonical NLRP3 Inflammasome.

abstract:
Toll-like receptors (TLRs) are proteins that act as the sentinels of mammalian cells by detecting bacteria and viruses motifs such as the phospholipid lipopolysaccharides (LPS) from Gram-negative bacterial membrane, among others. Bacteria like Vibrio cholerae, when grow in phosphate-depleted medium are unable to produce LPS and other phospholipids and therefore increase the synthesis of ornithine lipids (OLs) to keep membrane integrity and survival. We found that, although the huge structural differences between OL and LPS, our immune system is still able to detect OL and trigger immune response through TLR4 and the non-canonical Nucleotide-binding domain and leucine-rich repeat-containing pyrin protein 3 (NLRP3) inflammasome. Similar to LPS, OL induced TLR4-dependent tumor necrosis factor (TNF)- secretion and Nuclear Factor (NF)-{kappa}B activation and therefore also elicited the priming of the NLRP3 inflammasome. Moreover, incubation of macrophages with OL causes caspase-dependent cleavage of gasdermin D (GSDMD) and consequent K+ efflux-dependent NLRP3 activation and interleukin (IL)-1{beta} secretion.

Results abstract (if needed)In bone-marrow derived murine macrophages (BMDM) from wild type mice, OLs induced hallmarks of NF-{kappa}B activation such as TNF- secretion and the expression of pro-IL-1{beta} and NLRP3. OL induced NF-{kappa}B activation dependent on TLR4 as demonstrated by the NF-{kappa}B activation measured in HEK293 reporting cells expressing uniquely TLR4 and by the decrease of TNF- secretion in macrophages caused by antibodies blocking TLR4. OLs also induced IL-1{beta} secretion dependent on cellular K+ efflux and NLRP3/caspase-1 inflammasome, as it was blocked by specific inhibitors and absent on Nlrp3-/-, Pycard-/- or Casp1/11-/- macrophages.


title: Myxoma virus lacking the host range determinant M062 stimulates cGAS-dependent type 1 interferon response and unique transcriptomic changes in human macrophages

abstract:
The evolutionarily successful poxviruses possess effective and diverse strategies to circumvent or overcome host defense mechanisms. Poxviruses encode many immunoregulatory proteins to evade host immunity to establish a productive infection and have unique means of inhibiting DNA sensing-dependent type 1 interferon (IFN-I) responses, a necessity given their dsDNA genome and exclusively cytoplasmic life cycle. We found that the key DNA sensing inhibition by poxvirus infection was dominant during the early stage of poxvirus infection before DNA replication. In an effort to identify the poxvirus gene products which subdue the antiviral proinflammatory responses (e.g., IFN-I response), we investigated the function of one early gene that is the known host range determinant from the highly conserved poxvirus host range C7L superfamily, myxoma virus (MYXV) M062. Host range factors are unique features of poxviruses that determine the species and cell type tropism. Almost all sequenced mammalian poxviruses retain at least one homologue of the poxvirus host range C7L superfamily. In MYXV, a rabbit-specific poxvirus, the dominant and broad-spectrum host range determinant of the C7L superfamily is the M062R gene. The M062R gene product is essential for MYXV infection in almost all cells tested from different mammalian species and specifically inhibits the function of host Sterile  Motif Domain-containing 9 (SAMD9), as M062R-null ({Delta}M062R) MYXV causes abortive infection in a SAMD9-dependent manner. In this study we investigated the immunostimulatory property of the {Delta}M062R. We found that the replication-defective {Delta}M062R activated host DNA sensing pathway during infection in a cGAS-dependent fashion and that knocking down SAMD9 expression attenuated proinflammatory responses. Moreover, transcriptomic analyses showed a unique feature of the host gene expression landscape that is different from the dsDNA-stimulated inflammatory state. This study establishes a link between the anti-neoplastic function of SAMD9 and the regulation of innate immune responses.

Author SummaryPoxviruses encode a group of genes called host range determinants to maintain or expand their host tropism. The mechanism by which many viral host range factors function remains elusive. Some host range factors possess immunoregulatory functions responsible for evading or subduing host immune defense mechanisms. Most known immunoregulatory proteins encoded by poxviruses are dispensable for viral replication in vitro. The uniqueness of MYXV M062R is that it is essential for viral infection in vitro and belongs to one of the most conserved poxvirus host range families, the C7L superfamily. There is one known host target of the MYXV M062 protein, SAMD9. SAMD9 is constitutively expressed in mammalian cells and exclusively present in the cytoplasm and has an anti-neoplastic function. Humans with deleterious mutations in SAMD9 present disease that ranges from lethality at a young age to a predisposition to myelodysplastic syndromes (MDS) that often require bone marrow transplantation. More importantly, SAMD9 serves as an important antiviral intrinsic molecule to many viruses. The cellular function of SAMD9 remains unclear mostly due to the difficulty of studying this protein, i.e., its large size, long half-life, and its constitutive expression in most cells. In this study we used M062R-null MYXV as a tool to study SAMD9 function and report a functional link between SAMD9 and the regulation of the proinflammatory responses triggered by cGAS-dependent DNA sensing.


title: Diurnal differences in intracellular replication within splenic macrophages correlates with the outcome of pneumococcal infection.

abstract:
Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within a specific subset of splenic macrophages, we sought to investigate events within the spleen that correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of moderate septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. These findings correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs), known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size in active phase-infected mice compared to those in rest phase-infected mice which decreased in size over time and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF- concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. In summary, these data link the difference in susceptibility to invasive pneumococcal infection to variation in the ability of MMMs to successfully control and digest phagocytosed bacteria.

Author summaryCircadian rhythms are present within the majority of multicellular organisms and influence almost all aspects of our physiology. As such, circadian rhythm disorders have been shown to result in an increased susceptibility to certain diseases. The effects of host circadian rhythm have been also mirrored in rodent studies, with the outcome of Streptococcus pneumoniae infection being dependent on the time of challenge. Whilst studies into the functional effects of circadian rhythm on the host immune system are present, knowledge of how these contribute to the control of invasive S. pneumoniae infection are lacking, especially considering the recent breakthrough in understanding the stages of pneumococcal pathogenesis. We show here that mice infected with S. pneumoniae during their active phase developed septicaemia quicker than those infected during their rest phase. We demonstrate that this is likely due to increased replication of pneumococci specifically within a subset of splenic macrophages, which subsequently results in increased numbers of pneumococci in the blood and higher levels of pro-inflammatory cytokines which result in septicaemia. These data provide novel insights into how circadian rhythm influences the immune functionality of the spleen, and how the regulation of function of one macrophage subtype can significantly alter the course of infection.


title: Whole-Genome Sequence of African Swine Fever Virus isolate from India provides insights into diversity and evolution

abstract: African swine fever (ASF) was first reported in 1921, and since then has posed a major threat to the world pig industry and still remains a major challenge as there is no vaccine or therapy available. In May 2020, the first incidence of ASF was recorded in India, followed by a number of outbreaks in the north eastern part of India. In this study, we report the first whole genome of an Indian isolate of ASF virus (ASF/IND/20/CAD/543) using next generation sequencing and compared with the other ASFV complete genome. On phylogenetic analysis, the virus was assigned to genotype II on the basis of p72 genotyping. However, the whole genome based phylogeny distinguished it from other genotype II isolates of clade 1.1.1. This study adds to our understanding of ASFVs genetic diversity and molecular evolution.


title: Significant Broad Spectrum Antiviral activity of Bi121 Against Different Variants of SARS-CoV-2

abstract: The SARS-CoV-2 pandemic infected 343 million people with over 5.59 million deaths. New mutated lineages of SARS-CoV-2 such as Omicron are evolving faster. Broad-spectrum viral inhibitors that block the initial stage of infection by reducing virus proliferation and disease severity is an unmet global medical need. We studied Bi121, a standardized polyphenolic-rich compound isolated from Pelargonium sidoides, against recombinant Vesicular Stomatitis Virus (rVSV)-pseudotyped SARS-CoV-2S (spike) that represent mutations in the spike protein of six different variants of SARS-CoV-2. Bi121 was effective in neutralizing all six rVSV-{Delta}G-SARS-CoV-2S variants expressing different mutations. The antiviral activity of Bi121 was then assessed against three variants of SARS-CoV-2 (USA WA1/2020, Hongkong/VM20001061/2020, B.1.167.2 (Delta)) using RT-qPCR and plaque assays in two different cell lines (Vero cells and HEK-ACE2). Bi121 showed significant activity toward all the three variants tested, suggesting a broad-spectrum activity.


title: Repair of Mismatched Templates during Rad51-dependent Break-Induced Replication

abstract:
Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3 end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. When every 10th base is mismatched between donor and recipient, BIR is 44% efficient compared to fully homologous sequences; but BIR still occurs about 10% when every 6th base is mismatched. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, but where they are clustered with spacings of every 6th bp. These different arrangements of uneven mismatch distribution were in general less efficient in recombination as templates with evenly distributed mismatches. A donor with all 10 mismatches clustered every 6th base at the 3 invading end of the DSB was not impaired compared to the case where mismatches were clustered at the 5 end. These data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but sequence-specific factors are also important. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3 end. Mismatch incorporation depends on the 3 [->] 5 proofreading exonuclease activity of DNA polymerase {delta}, with little contribution from Msh2/Mlh1 mismatch repair proteins. Surprisingly, the probability of a mismatch 27 nt from the 3 end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase {delta} "chews back" the 3 end of the invading strand without any mismatch-dependent cues from the strand invasion structure.

Author SummaryDNA double-strand breaks (DSBs) are the most lethal forms of DNA damage and inaccurate repair of these breaks presents a serious threat to genomic integrity and cell viability. Break-induced replication (BIR) is a homologous recombination pathway that results in a nonreciprocal translocation of chromosome ends. We used budding yeast Saccharomyces cerevisiae to investigate Rad51-mediated BIR, where the invading 3 end of the DSB and a donor template share 108 bp of homology. We examined the tolerance of differently distributed mismatches on a homologous donor template and found that BIR efficiency was the same whether the mismatches were clustered at the 3 invading end or at the 5 end. We confirmed that mismatches are incorporated into the BIR product in a strongly polar fashion as far as about 40 nucleotides from the 3 end. We conclude that the proofreading activity of DNA polymerase {delta} "chews back" the 3 end of the invading strand even when the sequences removed have no mismatches for the first 26 nucleotides. These observations enrich our understanding of the details of Rad51-mediated strand invasion and provide insight into the mechanism of the 3 to 5 proofreading activity of DNA polymerase during homologous recombination.


title: Chromatin-associated microprocessor assembly is regulated by PRP40, the U1 snRNP auxiliary protein

abstract: Cotranscriptional processing of RNA polymerase II-generated primary transcripts is a well-documented phenomenon. We recently showed that in plants, miRNA biogenesis is also a cotranscriptional event. Here, we report that Arabidopsis PRP40, the U1 snRNP auxiliary protein, positively regulates the recruitment of SE, the core component of the plant microprocessor, to miRNA genes. The association of DCL1, the microprocessor endoribonuclease, with chromatin was altered in prp40ab mutant plants. Impaired cotranscriptional microprocessor assembly was accompanied by RNA polymerase II accumulation at miRNA genes and retention of miRNA precursors at their transcription sites in the prp40ab mutant plants. We show that cotranscriptional microprocessor assembly, regulated by AtPRP40, positively affects RNAPII transcription of miRNA genes and is important to reach the correct levels of produced miRNAs.