title: Tissue-based IL-10 signalling in helminth infection limits IFNγ expression and promotes the intestinal Th2 response

abstract: Type 2 immunity is activated in response to both allergens and helminth infection. It can be detrimental or beneficial, and there is a pressing need to better understand its regulation. The immunosuppressive cytokine IL-10 is known as a T helper 2 (Th2) effector molecule, but it is currently unclear whether IL-10 dampens or promotes Th2 differentiation during infection. Here we show that helminth infection in mice elicits IL-10 expression in both the intestinal lamina propria and the draining mesenteric lymph node, with higher expression in the infected tissue. In vitro, exogenous IL-10 enhanced Th2 differentiation in isolated CD4+ T cells, increasing expression of GATA3 and production of IL-5 and IL-13. The ability of IL-10 to amplify the Th2 response coincided with its suppression of IFN{gamma} expression and, in vivo, we found that, in intestinal helminth infection, IL-10 receptor expression was higher on Th1 cells in the small intestine than on Th2 cells in the same tissue, or on any Th cell in the draining lymph node. In vivo blockade of IL-10 signalling during helminth infection resulted in an expansion of IFN{gamma}+ and Tbet+ Th1 cells in the small intestine and caused a coincident decrease in IL-13, IL-5 and GATA3 expression by intestinal T cells. Together our data indicate that IL-10 signalling promotes Th2 differentiation during helminth infection at least in part by regulating competing Th1 cells in the infected tissue.


title: LACK OF EVIDENCE FOR A VIABLE MICROBIOTA IN MURINE AMNIOTIC FLUID

abstract:
The existence of an amniotic fluid microbiota (i.e., a viable microbial community) in mammals is controversial. Its existence would require a fundamental reconsideration of the role of intra-amniotic microbes in fetal development and pregnancy outcomes. In this study, we determined whether the amniotic fluid of mice harbors a microbiota in late gestation. Bacterial profiles of amniotic fluids located proximally or distally to the cervix were characterized through quantitative real-time PCR, 16S rRNA gene sequencing, and culture (N = 21 mice). These profiles were compared to those of technical controls for background DNA contamination. The load of 16S rDNA in the amniotic fluid exceeded that in controls. Additionally, the 16S rDNA profiles of the amniotic fluid differed from those of controls, with Corynebacterium tuberculostearicum being differentially more abundant in amniotic fluid profiles; however, this bacterium was not cultured. Of the 42 total bacterial cultures of amniotic fluids, only one yielded bacterial growth - Lactobacillus murinus. The 16S rRNA gene of this common murine-associated bacterium was not detected in any amniotic fluid sample, suggesting it did not originate from the amniotic fluid. No differences in 16S rDNA load, 16S rDNA profile, or bacterial culture were observed between amniotic fluids located proximal and distal to the cervix. Collectively, these data show that, although there is a modest DNA signal of bacteria in murine amniotic fluid, there is no evidence that this signal represents a viable microbiota. These findings refute the proposed role of amniotic fluid as a source of microorganisms for in utero colonization.

IMPORTANCEThe prevailing paradigm in obstetrics has been the sterile womb hypothesis, which posits that fetuses are first colonized by microorganisms during labor and/or the vaginal delivery process. However, it has been suggested that fetuses are consistently colonized in utero. One proposed source of colonizers is the amniotic fluid surrounding the fetus. This concept has been derived primarily from investigations that relied on DNA sequencing. Due to the low microbial biomass of amniotic fluid, such studies are susceptible to influences of background DNA contamination. Additionally, even if there is a microbial DNA signature in amniotic fluid, this is not necessarily reflective of a resident microbiota that could colonize the mammalian fetus. In the current study, using multiple microbiologic approaches and incorporating technical controls for DNA contamination, we show that, although there is a low abundance bacterial DNA signal in amniotic fluid, this does not translate to the presence of viable bacteria.

category: microbiology


title: The virulome of Streptomyces scabiei in response to cello-oligosaccharides elicitors

abstract:
2.The development of spots or lesions symptomatic of the common scab disease on root and tuber crops is caused by few pathogenic Streptomyces with Streptomyces scabiei 87-22 as the model species. Thaxtomin phytotoxins are the primary virulence determinants, mainly acting by impairing cellulose synthesis, and their production in S. scabiei is in turn boosted by the cello-oligosaccharides released from host plants. In this work we aimed to determine which molecules and which biosynthetic gene clusters (BGCs) of the specialized metabolism of S. scabiei 87-22 show a production and/or transcriptional response to cello-oligosaccharides. Comparative metabolomic and transcriptomic analyses revealed that molecules of the virulome of S. scabiei induced by cellobiose and cellotriose include i) thaxtomins and concanamycins phytotoxins (and to a lesser extent N-coronafacoyl-L-isoleucine), ii) desferrioxamines, scabichelin and turgichelin siderophores in order to acquire iron essential for housekeeping functions, iii) ectoine for protection against osmotic shock once inside the host, and iv) bottromycins and concanamycins antimicrobials possibly to prevent other microorganisms from colonizing the same niche. Importantly, both cell-oligosaccharides reduced the production of the spore germination inhibitors germicidins and the plant growth regulators rotihibins. The metabolomic study also revealed that cellotriose is in general a more potent elicitor of the virulome compared to cellobiose. This result supports an earlier hypothesis that suggested that the trisaccharide would be the real virulence-triggering factor released from the plant cell wall through the action of thaxtomins. Interestingly, except for thaxtomins, none of these BGCs expression seems to be under direct control of the cellulose utilization repressor CebR suggesting the existence of another master regulator sensing the internalization of cello-oligosaccharides. Finally, we found nine additional cryptic and orphan BGCs that have their expression awakened by cello-oligosaccharides, demonstrating that other and yet to be discovered metabolites are part of the virulome of S. scabiei.

3. Impact statementUnveiling the environmental triggers that signal proper conditions for host colonization and what is the composition of the arsenal of metabolites specialized for this task (the virulome) is key to understand host-pathogen interactions. In this work, focused on the induction of the common scab disease caused by Streptomyces species, we provided further knowledge to both aspects i.e., i) highlighting the capability of cellotriose to trigger the entire virulome and not only the production of thaxtomin phytotoxins, and ii) identifying the set of metabolites that specifically respond to cello-oligosaccharides emanating from the plant under attack. Importantly, we also revealed that the expression of nine cryptic/orphan biosynthetic gene clusters (BGCs) involved in the production of unknown compounds was drastically activated upon cello-oligosaccharides import suggesting that a significant part of the virulome of S. scabiei remains to be discovered. Finally, we unexpectedly found that the expression control of most of the known and cryptic BGCs does not depend on the cello-oligosaccharide utilization repressor CebR which suggests the existence of another and yet unknown master regulator of the virulence in S. scabiei.

4. Significance as a BioResource to the communityNot Applicable

5. OutcomeNot Applicable

6. Data summary[A section describing all supporting external data including the DOI(s) and/or accession numbers(s), and the associated URL.]

The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. RNAseq data were publicly deposited, and our experimental and analytical pipeline were described on the GEO database repository (Accession number: GSE181490)


title: Expression of Remyelination-modulating Genes in Astrocytes is Regulated by MALAT1 and Lnc-DC Long Non-coding RNAs

abstract: This manuscript has been withdrawn by the authors as it was submitted and made public without the full consent of all the authors. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

category: molecular biology


title: AFA: Computationally efficient Ancestral Frequency estimation in Admixed populations: the Hispanic Community Health Study/Study of Latinos

abstract: We developed a computationally efficient method, Ancestral Frequency estimation in Admixed populations (AFA), to estimate the frequencies of bi-allelic variants in admixed populations with an unlimited number of ancestries. AFA uses maximum likelihood estimation by modeling the conditional probability of having an allele given proportions of genetic ancestries. It can be applied using either global or local proportions of genetic ancestries. Simulations mimicking admixture demonstrated the high accuracy of the method. We implemented the method on data from the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), an admixed population with three predominant continental ancestries: Amerindian, European, and African. Comparison of the European and African estimated frequencies to the respective gnomAD frequencies demonstrated high correlations, with Pearson R2=0.97-0.99. We provide a genome-wide dataset of the estimated three ancestral allele frequencies in HCHS/SOL for all available variants with allele frequency between 5%-95% in at least one of the three ancestral populations.

category: genomics


title: Cryo-EM reveals disrupted human p97 allosteric activation by disease mutations and inhibitor binding

abstract: The human AAA ATPase p97, a potential cancer target, plays a vital role in clearing misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders. Here, we present cryo-EM structural analyses of four disease mutants p97R155H, p97R191Q, p97A232E, p97D592N, as well as p97E470D, implicated in resistance to the drug CB-5083. These structures demonstrate that the mutations affect nucleotide-driven p97 allosteric activation by predominantly interfering with either the coupling between the D1 and N-terminal domains (p97R155H and p97R191Q), the inter-protomer interactions (p97A232E), or the coupling between D1 and D2 nucleotide domains (p97D592N, p97E470D). We also show that binding of the competitive inhibitor CB-5083 to the D2 domain prevents conformational changes similar to that seen for mutations that affect coupling between D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations.

category: biochemistry


title: Microphaser - a small-scale phasing approach for improved personalized neopeptidome creation

abstract:
MotivationHaplotype phasing approaches have been shown to improve accuracy of the search space of neoantigen prediction by determining if adjacent variants are located on the same chromosomal copy. However, the aneuploid nature of cancer cells as well as the admixture of different clones in tumor bulk sequencing data are challenging the current diploid based phasing algorithms. We present microphaser, a small-scale phasing approach to improve haplotyping variants in cancer samples. Microphaser aims to create a more accurate neopeptidome for downstream neoantigen prediction.

ResultsMicrophaser achieves large concordance with state-of-the-art phasing-aware neoantigen prediction pipeline neoepiscope, with differences in edge cases and an improved filtering step.

AvailabilityMicrophaser is written in the Rust programming language. It is made available via Github (https://github.com/koesterlab/microphaser)and Bioconda. The corresponding prediction pipeline (https://github.com/snakemake-workflows/dna-seq-neoantigen-prediction) has been written within the Snakemake workflow management system and can be deployed as part of the snakemake-workflows project.

category: bioinformatics


title: Stress-induced collective behavior leads to the formation of multicellular structures and the survival of the unicellular alga Chlamydomonas

abstract: Depending on their nature, living organisms use various strategies to adapt to environmental stress conditions. Multicellular organisms implement a set of reactions involving signaling and cooperation between different types of cells. Unicellular organisms on the other hand must activate defense systems, which involve collective behaviors between individual organisms. In the unicellular model alga Chlamydomonas reinhardtii, the existence and the function of collective behavior mechanisms in response to stress remain largely unknown. Here we report the discovery of a mechanism of abiotic stress response that Chlamydomonas can trigger to form large multicellular structures that can comprise several thousand cells. We show that these aggregates constitute an effective bulwark within which the cells are efficiently protected from the toxic environment. We have generated the first family of mutants that aggregate spontaneously, the socializer mutants (saz), of which we describe here in detail saz1. We took advantage of the saz mutants to implement a large scale multiomics approach that allowed us to show that aggregation is not the result of passive agglutination, but rather genetic reprogramming and substantial modification of the secretome. The reverse genetic analysis we conducted on some of the most promising candidates allowed us to identify the first positive and negative regulators of aggregation and to make hypotheses on how this process is controlled in Chlamydomonas.

category: plant biology


title: Long-term Transverse Imaging of the Hippocampus with Glass Microperiscopes

abstract: The hippocampus consists of a stereotyped neuronal circuit repeated along the septal-temporal axis. This transverse circuit contains distinct subfields with stereotyped connectivity that support crucial cognitive processes, including episodic and spatial memory. However, comprehensive measurements across the transverse hippocampal circuit in vivo are intractable with existing techniques. Here, we developed an approach for two-photon imaging of the transverse hippocampal plane in awake mice via implanted glass microperiscopes, allowing optical access to the major hippocampal subfields and to the dendritic arbor of pyramidal neurons. Using this approach, we tracked dendritic morphological dynamics on CA1 apical dendrites and characterized spine turnover. We then used calcium imaging to quantify the prevalence of place and speed cells across subfields. Finally, we measured the anatomical distribution of spatial information, finding a non-uniform distribution of spatial selectivity along the DG-to-CA1 axis. This approach extends the existing toolbox for structural and functional measurements of hippocampal circuitry.

category: neuroscience


title: Degenerate primed randomly amplified polymorphic DNA (DP-RAPD) fingerprinting of bacteriophages of Vibrio harveyi from shrimp hatcheries in Southern India

abstract: Vibrio harveyi is a significant pathogen of shrimp. Seventy-six bacteriophages infecting luminescent V. harveyi were isolated from a total of 194 water samples drawn from various sources of shrimp hatcheries located in South East coast and Andaman island of India. Degenerate primed randomly amplified polymorphic DNA (DP- RAPD) fingerprinting of these bacteriophages was carried out to determine their genetic relatedness. Similarity matrix based on Dice coefficient followed by construction of dendrogram by unweighted pair group method with arithmetic averages (UPGMA) revealed 12 major clusters. One phage was randomly selected from each of these major clusters for transmission electron microscopic observations. Eleven of them had an icosahedral head (46-115 nm) with a long non-contractile tail (132-329 nm), belonging to the Siphoviridae family and two phages had a short tail (15-27 nm), belonging to the family Podoviridae. The phylogenetic analysis of the phages using DP-RAPD fingerprinting correlated to some extent to the phenotypic nature of the host specifically with regard to sucrose fermentation and source of isolation. However, phages specifically infecting V. harveyi and those belonging to different families did not cluster together in the DP-PCR cluster analysis. Hence, the genetic diversity of phages infecting same host with respect to phenotypic difference was revealed by the DP-RAPD applied in this study.


title: Opportunistic gut sampling indicates differential diet and plastic ingestion risk in Indian dugongs

abstract: Dugongs, exclusively seagrass foragers, are globally threatened marine mammals. Knowledge on their feeding biology has been derived from few direct observations and mostly by analysis of stomach contents. Given limitations in data from Indian populations, dugong strandings serve as an opportunity to understand their dietary composition through gut sampling. In this paper, we utilize the gut contents collected from stranded dugongs to detect differences in the seagrass foraging between two isolated pockets of dugong distribution (Tamil Nadu and Gujarat) and supplement existing knowledge on dugong feeding biology in Indian waters. We extracted, enumerated and identified seagrass species from dugong gut contents. The proportion of seagrass leaf fragments were found higher (>40%) than other fragments in all the gut samples analysed. We recorded two seagrass genera (Halophila spp. and Halodule spp.) from Gujarat and five seagrass genera (Halophila spp., Halodule spp., Cymodocea spp., Enhalus spp., Syringodium spp.) from Tamil Nadu dugong individuals. We also obtained anthropogenic debris such as plastic, fishing net and wood fragments from the gut samples. We suggest enhanced monitoring of seagrass habitats and fine spatial scale threat mapping in entire dugong distribution range in India.

category: ecology


title: Pathological LSD1 mutations cause HDAC-mediated aberrant gene repression during early cell differentiation

abstract: Lysine-specific demethylase 1 (LSD1/KDM1A) demethylates both histone and non-histone substrates, recruits repressive chromatin complexes, and is increased in cancers. De novo LSD1 mutations impairing protein function lead to a rare developmental disorder, but the molecular details of the pathology remains unclear. Using patient-derived fibroblasts, reprogrammed pluripotent stem cells, and differentiated cells, we found over 4000 differentially expressed genes and 68 transcription factors (TFs) whose motif accessibilities changed upon LSD1 mutation. An enhancer-mediated gene regulatory network approach identified transcriptional repressors with impaired activity in fibroblast and stem cells, leading to erroneous activation of their target genes. We also revealed overall decreases in TF target gene expression during early lineage differentiation of LSD1 mutant stem cells, likely caused by increased activity of repressive histone deacetylases (HDACs), co-factors of LSD1. Indeed, an HDACs inhibitor restored changes in gene expression including downregulation phenotype. Our findings characterize the molecular pathogenesis of LSD1 mutations and suggest potential therapeutic strategies for the developmental disorder and cancers caused by LSD1 dysregulations.


title: Pathological LSD1 mutations repress enhancer-mediated gene regulatory networks in early differentiation


title: The epigenetic evolution of gliomas is determined by their IDH1 mutation status and treatment regimen

abstract: Tumor adaptation or selection is thought to underlie therapy resistance of gliomas. To investigate the longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 143 matched initial and recurrent patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) gliomas. IDHwt gliomas showed a longitudinally stable epigenome with relatively low levels of global methylation, whereas the epigenome of IDHmut gliomas showed initial high levels genome-wide of DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. By integrating DNA methylation and gene expression data, adaptive changes of putative master regulators of the cell cycle and of differentiation were seen in IDHmut recurrent tumors. Furthermore, relapses of IDHmut tumors were accompanied by histological progression which in turn influenced survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment differed between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neo-angiogenesis and T-cell infiltration upon treatment for IDHmut gliomas. Our study provides one of the largest cohorts of paired glioma samples profiled with epigenomics, transcriptomics and genomics; and our results demonstrate that the treatment of IDHmut gliomas reshapes the epigenome towards an IDHwt-like phenotype. Accordingly, the prevalent practice of early genotoxic treatment in this patient population may need to be revisited.


title: Fast and efficient digestion of adeno associated virus (AAV) capsid proteins for liquid chromatography mass spectrometry (LC-MS) based peptide mapping and post translational modification analysis (PTMs)

abstract: Adeno-associated virus (AAV) represent a widely used delivery mechanism for gene therapy treatments currently being developed. The size and complexity of these molecules requires the development of sensitive analytical methods for detailed product characterization. Among the quality attributes that need to be monitored, characterization of the AAV capsid protein amino acid sequences and any associated post translational modifications (PTM) present should be performed. As commonly used for recombinant protein analysis, LC-MS based peptide mapping can provide sequence coverage and PTM information to improve product understanding and the development and deployment of the associated manufacturing processes. In the current study, we report a fast and efficient method to digest AAV5 capsid proteins in only 30 minutes prior to peptide mapping analysis. The performance of different proteases in digesting AAV5 was compared and the benefits of using nanoflow liquid chromatography for separation prior to high resolution mass spectrometry to obtain 100% sequence coverage are highlighted. Characterization and quantitation of PTMs on AAV5 capsid proteins when using pepsin as a single protease is reported, thereby demonstrating the potential of this method to aid with complete characterization of AAV serotypes in gene therapy development laboratories.


title: Pout2Prot: an efficient tool to create protein (sub)groups from Percolator output files

abstract: In metaproteomics, the study of the collective proteome of microbial communities, the protein inference problem is more challenging than in single-species proteomics. Indeed, a peptide sequence can not only be present in multiple proteins or protein isoforms of the same species, but also in homologous proteins from closely related species. To assign the taxonomy and functions of the microbial species, specialized tools have been developed, such as Prophane. This tool, however, is not directly compatible with post-processing tools such as Percolator. In this manuscript we therefore present Pout2Prot, which takes Percolator Output (.pout) files from multiple experiments and creates protein group and protein subgroup output files (.tsv) that can be used directly with Prophane. We investigated different grouping strategies, and compared existing protein grouping tools to develop an advanced protein grouping algorithm that offers a variety of different approaches, allows grouping for multiple files, and uses a weighted spectral count for protein (sub)groups to reflect abundance. Pout2Prot is available as a web application at https://pout2prot.ugent.be and is installable via pip as a standalone command line tool and reusable software library. All code is open source under the Apache License 2.0 and is available at https://github.com/compomics/pout2prot.


title: CytokineLink: a cytokine communication map to analyse immune responses in inflammatory and infectious diseases

abstract: Intercellular communication mediated by cytokines is critical to the development of immune responses, particularly in the context of infectious and inflammatory diseases. By releasing these small molecular weight peptides, the source cells can influence numerous intracellular processes in the target cells, including the secretion of other cytokines downstream. However, there are no readily available bioinformatic resources that can model cytokine - cytokine interactions. In this effort, we built a communication map between major tissues and blood cells that reveals how cytokine-mediated intercellular networks form during homeostatic conditions. We collated the most prevalent cytokines from literature, and assigned the proteins and their corresponding receptors to source tissue and blood cell types based on enriched consensus RNA-Seq data from the Human Protein Atlas database. To assign more confidence to the interactions, we integrated literature information on cell - cytokine interactions from two systems immunology databases, immuneXpresso and ImmunoGlobe. From the collated information, we defined two metanetworks: a cell-cell communication network connected by cytokines; and a cytokine-cytokine interaction network depicting the potential ways in which cytokines can affect the activity of each other. Using expression data from disease states, we then applied this resource to reveal perturbations in cytokine-mediated intercellular signalling in inflammatory and infectious diseases (ulcerative colitis and COVID-19, respectively). For ulcerative colitis, with CytokineLink we demonstrated a significant rewiring of cytokine-mediated intercellular communication between non-inflamed and inflamed colonic tissues. For COVID-19, we were able to identify inactive cell types and cytokine interactions that may be important following SARS-CoV-2 infection when comparing the cytokine response with other viruses capable of initiating a cytokine storm. Such findings have potential to inform the development of novel, cytokine-targeted therapeutic strategies. CytokineLink is freely available for the scientific community through the NDEx platform and the project github repository (https://github.com/korcsmarosgroup/CytokineLink).


title: Quantifying the antimicrobial activity of CRISPR-Cas9-accA modified ΔB. subtilis mutants against V. harveyi and E. Coli

abstract: Probiotics are increasingly popular, currently. Probiotics have been described with the ability to treat many disorders of the gastrointestinal tract (GIT) such as irritable bowel syndrome (IBS) and Crohns disease. Types of probiotics include bacterial strains from Lactobacillus and Bifidobacterium. Probiotics can restore balance to gut microbiota by outcompeting pathogenic bacteria for nutrients and secrete antimicrobials to eliminate these bacterial pathogens. However, the viability of most advertised probiotics lose their potency due to being freeze dried into powders during storage or for consuming. Many probiotics become ineffective and produce lower CFUs while traversing through the gastric acids of the digestive system. For these reasons, this study sought to enhance the antimicrobial response of a highly potent probiotic known as Bacillus subtilis. B. subtilis has been used to treat many disorders of the gut and secrete many antimicrobials lethal for pathogenic microbes. B. subtilis was genetically modified to express CRISPR-Cas9 nuclease deletion of the accA gene ({Delta}B.subtilis mutants), which inhibits expression of an essential accA gene a part of the fatty acid synthesis (FAS) metabolic pathway. The CRISPR-Cas9-accA {Delta}B.subtilis mutants were co-cultured with V. harveyi and E. Coli. Bacterial growth, biofilm formation, antimicrobial activity, and antibiotic resistance were quantified. It was found that{Delta} B.subtilis mutants co-cultured with V. harveyi and E. Coli lessened bacterial growth, amplified biofilm with V. harveyi, reduced biofilm formation of E. Coli, the co-cultures with the mutants lacked antimicrobial activity, and increased the antibiotic resistance of V. harveyi and E. Coli. It can be concluded that there is an immense potential for using genetically engineered probiotic strains to enhance the antimicrobial activity of B. subtilis, which can amplify the reduction of pathogenic bacteria. However, the safety and frugality of using B. subtilis as a probiotic requires further consideration.

category: synthetic biology


title: A single shot of a hybrid hAdV5-based anti-COVID-19 vaccine induces a long-lasting immune response and broad coverage against VOC

abstract: Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric hAdV5 vector. The vaccine (named CoroVaxG.3) is based on three pillars: i) high expression of Spike to enhance its immunodominance by using a potent promoter and a mRNA stabilizer; ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; iii) use of Spike stabilized in a prefusion conformation. Transduction with CoroVaxG.3 expressing Spike (D614G) dramatically enhanced Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3 vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha) and P.1 (gamma) Spikes, as well as an authentic WT and P.1 SARS-CoV-2 isolates. Neutralizing antibodies did not wane even after 5 months making this kind of vaccine a likely candidate to enter clinical trials

category: immunology


title: Molecular basis of immune evasion by the delta and kappa SARS-CoV-2 variants

abstract: Worldwide SARS-CoV-2 transmission leads to the recurrent emergence of variants, such as the recently described B.1.617.1 (kappa), B.1.617.2 (delta) and B.1.617.2+ (delta+). The B.1.617.2 (delta) variant of concern is causing a new wave of infections in many countries, mostly affecting unvaccinated individuals, and has become globally dominant. We show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing the impact of individual mutations on immune evasion. Mutations in the B.1.617.1 (kappa) and B.1.617.2 (delta) spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including an unexpected remodeling of the B.1.617.2 (delta) N-terminal domain. The binding affinity of the B.1.617.1 (kappa) and B.1.617.2 (delta) receptor-binding domain for ACE2 is comparable to the ancestral virus whereas B.1.617.2+ (delta+) exhibits markedly reduced affinity. We describe a previously uncharacterized class of N-terminal domain-directed human neutralizing monoclonal antibodies cross-reacting with several variants of concern, revealing a possible target for vaccine development.


title: SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host

abstract: SARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 - the closest bat relative - spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.


title: N-dihydrogalactochitosan reduces mortality in a lethal mouse model of SARS-CoV-2

abstract:
The rapid emergence and global dissemination of SARS-CoV-2 that causes COVID-19 continues to cause an unprecedented global health burden resulting in more than 4 million deaths in the 20 months since the virus was discovered. While multiple vaccine countermeasures have been approved for emergency use, additional treatments are still needed due to sluggish vaccine rollout and vaccine hesitancy. Immunoadjuvant compounds delivered intranasally can guide non-specific innate immune responses during the critical early stages of viral replication, reducing morbidity and mortality. N-dihydrogalactochitosan (GC) is a novel mucoadhesive immunostimulatory polymer of {beta}-0-4-linked N-acetylglucosamine that is solubilized by the conjugation of galactose glycans. We tested GC as a potential countermeasure for COVID-19. GC administered intranasally before and after SARS-CoV-2 exposure diminished morbidity and mortality in humanized ACE2 receptor expressing mice by up to 75% and reduced infectious virus levels in the upper airway and lungs. Our findings demonstrate a new application for soluble immunoadjuvants like GC for preventing severe disease associated with SARS-CoV-2.

Single Sentence SummaryThe immunoadjuvant N-dihydrogalactochitosan diminishes SARS-CoV-2 disease in humanized ACE2 mice representing a new countermeasure against COVID-19.


title: Multiplexed detection of SARS-CoV-2 genomic and subgenomic RNA using in situ hybridization

abstract: The widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N) as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO), but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues as well as extended for other applications including investigating the viral life cycle, viral diagnostics, and drug screening.


title: A global survey of specialized metabolic diversity encoded in bacterial genomes

abstract: Bacterial specialized metabolites are a proven source of antibiotics and cancer therapies, but whether we have sampled all the secondary metabolite chemical diversity of cultivated bacteria is not known. We analysed ~ 170,000 bacterial genomes and ~ 47,000 metagenome assembled genomes (MAGs) using a modified BiG-SLiCE and the new clust-o-matic algorithm. We found that only 3% of the natural products potentially encoded in bacterial genomes have been experimentally characterized. We show that the variation of secondary metabolite biosynthetic diversity drops significantly at the genus level, identifying it as an appropriate taxonomic rank for comparison. Equal comparison of genera based on Relative Evolutionary Distance revealed that Streptomyces bacteria encode the largest biosynthetic diversity by far, with Amycolatopsis, Kutzneria and Micromonospora also encoding substantial diversity. Finally we find that several less-well-studied taxa, such as Weeksellaceae (Bacteroidota), Myxococcaceae (Myxococcota), Pleurocapsa and Nostocaceae (Cyanobacteria), have potential to produce highly diverse sets of secondary metabolites that warrant further investigation.


title: Compendium of secondary metabolite biosynthetic diversity encoded in bacterial genomes


title: Cryptographic-like mechanism allows scatter-hoarders to cache and retrieve their food secretly

abstract: For animals, the ability to hide and retrieve valuable information, such as the location of food, can mean the difference between life and death. Here, we propose that to achieve this, their brain uses spatial cells similarly to how we utilize encryption for data security. Some animals are able to cache hundreds of thousands of food items annually by each individual and later retrieve most of what they themselves stashed. Rather than memorizing their cache locations as previously suggested, we propose that they use a single cryptographic-like mechanism during both caching and retrieval. The model we developed is based on hippocampal spatial cells, which respond to an animals positional attention, such as when the animal enters a specific region (place-cells) or gazes at a particular location (spatial-view-cells). We know that the region that activates each spatial cell remains consistent across subsequent visits to the same area but not between areas. This remapping, combined with the uniqueness of cognitive maps, produces a persistent crypto-hash function for both food caching and retrieval. We also show that the model stores temporal information that helps animals in food caching order preference as previously observed. This behavior, which we refer to as crypto-taxis, might also explain consistent differences in decision-making when animals are faced with a large number of alternatives such as in foraging.

category: animal behavior and cognition


title: The Hippocampus uses cryptographic principles rather than plasticity to enable animals to secretly cache and retrieve their food


title: Defining the pig microglial transcriptome reveals their core signature, regional heterogeneity, and similarity with humans

abstract:
Microglia play key roles in brain homeostasis as well as responses to neurodegeneration and neuroinflammatory processes caused by physical disease and psychosocial stress. The pig is a physiologically-relevant model species for studying human neurological disorders, many of which are associated with microglial dysfunction. Furthermore, pigs are an important agricultural species, and there is a need to understand how microglial function affects their welfare. As a basis for improved understanding to enhance biomedical and agricultural research, we sought to characterise pig microglial identity at genome-wide scale and conduct inter-species comparisons.

We isolated pig hippocampal tissue and microglia from frontal cortex, hippocampus and cerebellum, as well as alveolar macrophages from the lungs and conducted RNA-sequencing (RNAseq). By comparing the transcriptomic profiles between microglia, macrophages, and hippocampal tissue, we derived a set of 365 highly-enriched genes defining the porcine core microglial signature. We found brain regional heterogeneity based on 215 genes showing significant (adjusted p<0.01) regional variations and that cerebellar microglia were most distinct. We compared normalized gene expression for microglia from human, mice and pigs using microglia signature gene lists derived from each species and demonstrated that a core microglial marker gene signature is conserved across species, but that species-specific expression subsets also exist. Importantly, pig and human microglia shared greater similarity than pig and murine microglia.

Our data provide a valuable resource defining the pig microglial transcriptome signature that highlights pigs as a useful large animal species bridging between rodents and humans in which to study the role of microglia during homeostasis and disease.

Main Points- Defined a pig microglial transcriptome signature comprising 365 genes.
- Demonstrated regional variance in the pig microglial transcriptome across the brain.
- Revealed greater similarity between pig and human microglia than mouse.


title: Molecular mimicry between Spike and human thrombopoietin may induce thrombocytopenia in COVID-19

abstract: SARS-CoV-2 causes COVID-19, a disease curiously resulting in varied symptoms and outcomes, ranging from asymptomatic to fatal. Autoimmunity due to cross-reacting antibodies resulting from molecular mimicry between viral antigens and host proteins may provide an explanation. We computationally investigated molecular mimicry between SARS-CoV-2 Spike and known epitopes. We discovered molecular mimicry hotspots in Spike and highlight two examples with tentative autoimmune potential and implications for understanding COVID-19 complications. We show that a TQLPP motif in Spike and thrombopoietin shares similar antibody binding properties. Antibodies cross-reacting with thrombopoietin may induce thrombocytopenia, a condition observed in COVID-19 patients. Another motif, ELDKY, is shared in multiple human proteins such as PRKG1 and tropomyosin. Antibodies cross-reacting with PRKG1 and tropomyosin may cause known COVID-19 complications such as blood-clotting disorders and cardiac disease, respectively. Our findings illuminate COVID-19 pathogenesis and highlight the importance of considering autoimmune potential when developing therapeutic interventions to reduce adverse reactions.


title: Spike mimicry of thrombopoietin may induce thrombocytopenia in COVID-19


title: Potential autoimmunity resulting from molecular mimicry between SARS-CoV-2 Spike and human proteins


title: Investigate Inhibitory Effects of Ginger polyphenols compare to Simvastatin towards HMG-CoA reductase: An Integrated Molecular Docking and Molecular dynamic simulation

abstract: Diabetes is an increasing problem in Ethiopia, affecting up to 6.5% of Ethiopian adults. There are serious complications associated with diabetes including macrovascular and microvascular. Controlling Lipid profiles and blood glucose significantly reduces the risk of complications. Statins are the only current treatment for both type 1 diabetes and Type 2 diabetes dyslipidemia. HMG-CoA reductase plays a central role in the production of cholesterol which, associated with cardiovascular disease (CVD). Statins have been found to reduce cardiovascular disease and mortality in those who are at high risk. Nonetheless, it has adverse effect, such as drug-related hypoglycemia and high cost. These situations lead to develop suitable phytotherapeutic agents with less frequent side effects. Ginger (Zingiber officinale) is widely consumed as a spice, and numerous studies suggest that ginger may have beneficial effects for diabetes and dyslipidemia. But, further studies are needed to investigate effects of binding affinity and binding site residues for major ginger extract polyphenols towards target HMG-CoA reductase. In this study, ADMET web server, Auto-Dock 5.4 and Gromacs 2020 were used. Out of eleven major gingers polyphenols screened three selected based on docking energy compare to Simvastatin for MD simulation. The predicted binding affinity for 6-paradol, 6-shogaol and gingerdione were -8.51, -6.93, -9.24 kcal/mol, respectively. The results of molecular dynamic simulation are consistence with docking. The predicted ligand binding site residues are Arg641, Gly808, Arg641, Met781, Ser794 and Arg595. In conclusion, 6-paradol, 6-shogaol and gingerdione could be possible therapy because, of interactions with target HMG-CoA reductase. Therefore, further wet lab study will be needed, for the better understanding of the mechanism of action of ginger extract by which it modulates liver and kidney vivo condition.


title: Structural Differences In 3C-like protease (Mpro) From SARS-CoV and SARS-CoV-2: Molecular Insights For Drug Repurposing Against COVID-19 Revealed by Molecular Dynamics Simulations.

abstract: A recent fatal outbreak of novel coronavirus SARS-CoV-2, identified preliminary as a causative agent for series of unusual pneumonia cases in Wuhan city, China has infected more than 20 million individuals with more than 4 million mortalities. Since, the infection crossed geographical barriers, the WHO permanently named the causing disease as COVID-2019 by declaring it a pandemic situation. SARS-CoV-2 is an enveloped single-stranded RNA virus causing a wide range of pathological conditions from common cold symptoms to pneumonia and fatal severe respiratory syndrome. Genome sequencing of SARS-CoV-2 has revealed 96% identity to the bat coronavirus and 79.6% sequence identity to the previous SARS-CoV. The main protease (known as 3C-like proteinase/ Mpro) plays a vital role during the infection with the processing of replicase polyprotein thus offering an attractive target for therapeutic interventions. SARS-CoV and SARS-CoV-2 Mpro shares 97% sequence identity, with 12 variable residues but none of them present in the catalytic and substrate binding site. With the high level of sequence and structural similarity and absence of any drug/vaccine against SARS-CoV-2, drug repurposing against Mpro is an effective strategy to combat COVID-19. Here, we report a detailed comparison of SARS-CoV-2 Mpro with SARS-CoV Mpro using molecular dynamics simulations to assess the impact of 12 divergent residues on the molecular microenvironment of Mpro. A structural comparison and analysis is made on how these variable residues affects the intra-molecular interactions between key residues in the monomer and biologically active dimer form of Mpro. The present MD simulations study concluded the change in microenvironment of active-site residues at the entrance (T25, T26, M49 and Q189), near the catalytic region (F140, H163, H164, M165 and H172) and other residues in substrate binding site (V35T, N65S, K88R and N180K) due to 12 mutation incorporated in the SARS-CoV-2 Mpro. It is also evident that SARS-CoV-2 dimer is more stable and less flexible state compared to monomer which may be due to these variable residues, mainly F140, E166 and H172 which are involved in dimerization. This also warrants a need for inhibitor design considering the more stable dimer form. The mutation accumulated in SARS-CoV-2 Mpro indirectly reconfigures the key molecular networks around the active site conferring a potential change in SARS-CoV-2, thus posing a challenge in drug repurposing SARS drugs for COVID-19. The new networks and changes in microenvironment identified by our work might guide attempts needed for repurposing and identification of new Mpro inhibitors.


title: Modeling the SARS-CoV-2 mutation based on geographical regions and time

abstract: The Coronavirus Disease 2019 (COVID-19) epidemic was first detected in late-December 2019. So far, it has caused 203,815,431 confirmed cases and 4,310,623 deaths in the world. We collected sequences from 150,659 COVID-19 patients. Based on the previous phylogenomic analysis, we found three major branches of the virus RNA genomic mutation located in Asia, America, and Europe which is consistent with other studies. We selected sites with high mutation frequencies from Asia, America, and Europe. There are only 13 common mutation sites in these three regions. It infers that the viral mutations are highly dependent on their location and different locations have specific mutations. Most mutations can lead to amino acid substitutions, which occurred in 3/5UTR, S/N/M protein, and ORF1ab/3a/8/10. Thus, the mutations may affect the pathogenesis of the virus. In addition, we applied an ARIMA model to predict the short-term frequency change of these top mutation sites during the spread of the disease. We tested a variety of settings of the ARIMA model to optimize the prediction effect of three patterns. This model can provide good help for predicting short-term mutation frequency changes.


title: The Microtubule Associated Protein Tau Regulates KIF1A Pausing Behavior and Motility

abstract:
Many neurodegenerative diseases result from dysfunction of axonal transport, a highly regulated cellular process responsible for site-specific neuronal cargo delivery. The kinesin-3 family member KIF1A is a key mediator of this process by facilitating long-distance cargo delivery in a spatiotemporally regulated manner. While misregulation of KIF1A cargo delivery is observed in many neurodegenerative diseases, the regulatory mechanisms responsible for KIF1A cargo transport are largely unexplored. Our lab has recently characterized a mechanism for a unique pausing behavior of KIF1A in between processive segments on the microtubule. This behavior, mediated through an interaction between the KIF1A K-loop and the polyglutamylated C-terminal tails of tubulin, helps us further understand how KIF1A conducts long-range cargo transport. However, how this pausing behavior is influenced by other regulatory factors on the microtubule is an unexplored concept. The microtubule associated protein Tau is one potential regulator, as altered Tau function is a pathological marker in many neurodegenerative diseases. However, while the effect of Tau on kinesin-1 and -2 has been extensively characterized, its role in regulating KIF1A transport is greatly unexplored at the behavioral level. Using single-molecule imaging, we have identified Tau-mediated regulation of KIF1A pausing behavior and motility. Specifically, our findings imply a competitive interaction between Tau and KIF1A for the C-terminal tails of tubulin. We introduce a new mechanism of Tau-mediated kinesin regulation by inhibiting the ability of KIF1A to use C-terminal tail reliant pauses to connect multiple processive segments into a longer run length. Moreover, we have correlated this regulatory mechanism to the behavioral dynamics of Tau, further elucidating the function of Tau diffusive and static behavioral state on the microtubule surface. In summary, we introduce a new mechanism of Tau-mediated motility regulation, providing insight on how disruptions in axonal transport can lead to disease state pathology.

SIGNIFICANCEKIF1A mediated cargo transport is essential in many cellular processes such as axonal transport and neuronal development. Defects in KIF1A transport have been implicated in neurodegenerative diseases including Alzheimers disease and frontotemporal dementia. However, the mechanism of KIF1As pathological misregulation remains elusive, highlighting the importance of identifying regulators of KIF1A function. The microtubule associated protein Tau is an attractive potential regulator of KIF1A motility as Tau dysfunction is a hallmark of these neurodegenerative diseases. Here, we demonstrate a direct connection between Tau and KIF1A motility, revealing a unique form of Tau-mediated regulation of axonal transport. Our results provide a molecular foundation for understanding the role of motor protein misregulation in neurodegenerative disease progression.

category: biophysics


title: DNAJB1-PRKACA in HEK293T cells induces LINC00473 overexpression that depends on PKA signaling

abstract: Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in adolescents and young adults in a background of normal liver tissue and has an poor prognosis due to lack of effective chemotherapeutic agents. The DNAJB1-PRKACA gene fusion (DP) has been reported in the majority of FLC tumors, however its oncogenic mechanisms remain unclear. Given the paucity of cellular models, in particular FLC tumor cell lines, we hypothesized that engineering the DP fusion gene in HEK293T cells would provide insight into the cellular effects of the fusion gene. We used CRISPR/Cas9 to engineer HEK293T clones expressing DP fusion gene (HEK-DP) and performed transcriptomic, proteomic, and mitochondrial studies to characterize this cellular model. Proteomic analysis of DP interacting partners identified mitochondrial proteins as well as proteins in other subcellular compartments. HEK-DP cells demonstrated significantly elevated mitochondrial fission, which suggests a role for DP in altering mitochondrial dynamics. Transcriptomic analysis of HEK-DP cells revealed a significant increase in LINC00473 expression, similar to what has been observed in primary FLC samples. LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity.

category: cancer biology


title: FAILURE OF PANCREATIC ALPHA CELLS TO RESPOND TO HYPOGLYCEMIA IS LINKED TO IMPAIRED GLUTAMATE RECEPTOR SIGNALING IN DIABETES

abstract: Glucagon secretion from pancreatic alpha cells is crucial to prevent hypoglycemia. For reasons still unknown, people with type 1 diabetes lose this glucoregulatory mechanism and are susceptible to dangerous hypoglycemia. Here we show that alpha cells in living pancreas slices from donors with type 1 diabetes failed to secrete glucagon in response to decreases in glucose concentration, thus mirroring the in vivo unresponsiveness to hypoglycemia. Glucagon content and responses to KCl depolarization were not affected, suggesting that alpha cells retained their secretory potential. By contrast, alpha cells had severely impaired signaling via glutamate receptors of the AMPA/kainate type. Under healthy conditions, activating these receptors was required to elicit full glucagon responses to decreases in glucose levels. In type 1 diabetes, reactivating residual glutamate receptor function with the positive allosteric modulators cyclothiazide and aniracetam restored glucagon secretion in response to hypoglycemia. These positive allosteric modulators are already approved to treat other conditions and could be repurposed to prevent hypoglycemia and improve management of diabetes.

category: physiology


title: RESTORING GLUTAMATE RECEPTOR SIGNALING IN PANCREATIC ALPHA CELLS RESCUES GLUCAGON RESPONSES IN TYPE 1 DIABETES


title: Reference genome and resequencing of 305 accessions provide insights into spinach evolution, domestication and genetic basis of agronomic traits

abstract: Spinach is a nutritious leafy vegetable belonging to the family Chenopodiaceae. Here we report a chromosome-scale reference genome assembly of spinach, which has a total size of 894.3 Mb and an N50 contig size of 23.8 Mb, with 98.3% anchored and ordered on the six chromosomes. Reconstruction of ancestral Chenopodiaceae karyotype indicates substantial genome rearrangements in spinach after its divergence from ancestral Chenopodiaceae, coinciding with high repeat content in the spinach genome. Resequencing the genomes of 305 cultivated and wild spinach accessions provides insights into spinach genetic diversity and population differentiation. Genome-wide association studies of 20 agronomical traits identify numerous significantly associated regions and candidate genes for these traits. Domestication sweeps in the spinach genome are identified, some of which are associated with important traits (e.g., leaf phenotype, bolting and flowering), demonstrating the role of artificial selection in shaping spinach phenotypic evolution. This study provides not only insights into the spinach evolution and domestication but also valuable resources for facilitating spinach breeding.


title: A CCaMK/Cyclops response element in the promoter of L. japonicus Calcium-Binding Protein 1 (CBP1) mediates transcriptional activation in root symbioses

abstract: Early gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. The Lotus japonicus T-DNA insertion line T90, carrying a promoterless uidA (GUS) gene in the promoter of Calcium Binding Protein1 (CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsible cis- and trans-acting factors, we subjected deletion/modification series of CBP1 promoter:reporter fusions to transactivation and spatio-temporal expression analysis and screened EMS-mutagenized T90 populations for aberrant GUS expression. We identified one cis-regulatory element required for GUS expression in the epidermis and a second element, necessary and sufficient for transactivation by the Calcium and Calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack of GUS expression in T90 white mutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promoters NIN (RNS), RAM1 (AM), and CBP1 (AM and RNS), calling for yet-to-be identified specificity-conferring factors.


title: Human Deep Cortical Neurons Promote Regeneration and Recovery After Cervical Spinal Cord Injury

abstract: Cervical spinal cord injuries (SCI) sever and permanently disrupt sensorimotor neural circuitry. Restoring connectivity within the damaged circuitry is critical to improving function. Herein we report robust regeneration of severed neural circuitry in a rat SCI model following transplantation of human induced pluripotent cells differentiated towards a deep cortical neuron lineage (iPSC-DCNs). In vivo, iPSC-DCNs: (1) integrated within the damaged cord and extended axons to caudal targets, (2) reversed SCI pathophysiology, (3) promoted robust regeneration of severed host supraspinal neural tracts, (4) and improved sensorimotor function. The results herein represent a significant paradigm shift in anatomical and functional outcomes over current preclinical/clinical models and demonstrates the survival and efficacy of human stem cell-derived cortical neurons in a SCI.


title: Altered sleep intensity upon DBS to hypothalamic sleep-wake centers in rats

abstract: Deep brain stimulation (DBS) has been scarcely investigated in the field of sleep research. We hypothesize that DBS onto hypothalamic sleep- and wake-promoting centers will produce significant neuromodulatory effects, and potentially become a therapeutic strategy for patients suffering severe, drug-refractory sleep-wake disturbances. We aimed to investigate whether continuous electrical high-frequency DBS, such as that often implemented in clinical practice, in the ventrolateral preoptic nucleus (VLPO) or the perifornical area of the posterior lateral hypothalamus (PeFLH), significantly modulates sleep-wake characteristics and behavior. We implanted healthy rats with electroencephalographic/electromyographic electrodes and recorded vigilance states in parallel to bilateral bipolar stimulation of VLPO and PeFLH at 125 Hz at 90 A over 24 h to test the modulating effects of DBS on sleep-wake proportions, stability and spectral power in relation to baseline. We unexpectedly found that VLPO DBS at 125 Hz deepens slow-wave sleep as measured by increased delta power, while sleep proportions and fragmentation remain unaffected. Thus, the intensity, but not the amount of sleep or its stability, is modulated. Similarly, the proportion and stability of vigilance states remained altogether unaltered upon PeFLH DBS but, in contrast to VLPO, 125 Hz stimulation unexpectedly weakened SWS, evidenced by reduced delta power. This study provides novel insights into non-acute functional outputs of major sleep-wake centers in the rat brain in response to electrical high-frequency stimulation, a paradigm frequently used in human DBS. In the conditions assayed, while exerting no major effects on sleep-wake architecture, hypothalamic high-frequency stimulation arises as a provocative sleep intensity-modulating approach.


title: Non-mycorrhizal root associated fungi of a tropical montane forest are relatively robust to the long-term addition of moderate rates of nitrogen and phosphorus

abstract:
Andean forests are biodiversity hotspots and globally important carbon (C) repositories. This status might be at risk due to increasing rates of atmospheric nutrient deposition. As fungal communities are key in the recirculation of soil nutrients, assessing their responses to soil eutrophication can help establish a link between microbial biodiversity and the sustainability of the C sink status of this region. Beyond mycorrhizal fungi, which have been studied more frequently, a wide range of other fungi associate with the fine root fraction of trees. Monitoring these communities can offer insights into how communities composed of both facultative and obligate root associated fungi are responding to soil eutrophication.

Here we document the response of non-mycorrhizal root associated fungal (RAF) communities to a long-term nutrient manipulation experiment. The stand level fine root fraction of an old growth tropical montane forest was sampled after seven years of nitrogen (N) and phosphorus (P) additions. RAF communities were characterized by a deep sequencing approach. As per the resource imbalance model, we expected that asymmetries in the availability of C, N and P elicited by fertilization will lead to mean richness reductions and alterations of the community structure.

We recovered moderately diverse fungal assemblages composed by sequence variants classified within a wide set of trophic guilds. While mean richness remained stable, community composition shifted, particularly among Ascomycota and after the addition of P. Fertilization factors, however, only accounted for a minor proportion of the variance in community composition. These findings suggest that, unlike mycorrhizal fungi, RAF communities are less sensitive to shifts in soil nutrient availability. A plausible explanation is that non-mycorrhizal RAF have fundamentally different nutrient acquisition and life history traits, thus allowing them greater stoichiometric plasticity and an array of functional acclimation responses that collectively express as subtle shifts in community level attributes.


title: SATB1 undergoes isoform-specific phase transitions in T cells

abstract: Intracellular space is demarcated into membraneless bodies and compartments via the process of liquid-liquid phase separation (LLPS). Phase separated condensates formed by proteins, RNA and/or other biomolecules are associated with many cellular functions including gene expression regulation. Here we describe how the genome organizer SATB1 utilizes its prion-like domains for LLPS. We have identified two SATB1 isoforms which display distinct biophysical behavior. We further showed how phosphorylation and interaction with nuclear RNA impact their phase transitions, leading to formation of aggregates when dysregulated. Moreover, we show that SATB1 is involved in the regulation of transcription and splicing, both of which displayed deregulation in Satb1 knockout murine thymocytes. Thus, the tight regulation of different SATB1 isoform expression levels, combined with protein post-translational modifications, are imperative for SATB1s physiological roles in T cell development while their deregulation may be linked to disorders such as cancer.


title: SARS-CoV-2 Spike S1 glycoprotein is a TLR4 agonist, upregulates ACE2 expression and induces pro-inflammatory M1 macrophage polarisation.

abstract:
Background and aimsTLR4 is an important innate immune receptor that recognizes bacterial LPS, viral proteins and other pathogen associated molecular patterns (PAMPs). It is expressed on tissue-resident and immune cells. We previously proposed a model whereby SARS-CoV-2 activation of TLR4 via its spike glycoprotein S1 domain increases ACE2 expression, viral loads and hyperinflammation with COVID-19 disease [1]. Here we test this hypothesis in vitro and demonstrate that the SARS-CoV-2 spike S1 domain is a TLR4 agonist in rat and human cells and induces a pro-inflammatory M1 macrophage phenotype in human THP-1 monocyte-derived macrophages.

MethodsAdult rat cardiac tissue resident macrophage-derived fibrocytes (rcTMFs) were treated with either bacterial LPS or recombinant SARS-CoV-2 spike S1 glycoprotein. The expression of ACE2 and other inflammatory and fibrosis markers were assessed by immunoblotting. S1/TLR4 co-localisation/binding was assessed by immunocytochemistry and proximity ligation assays on rcTMFs and human HEK-293 HA-TLR4-expressing cells. THP-1 monocytes were differentiated into M1 or M2 macrophages with LPS/IFN{gamma}, S1/IFN{gamma} or IL-4 and RNA was extracted for RT-qPCR of M1/M2 markers and ACE2.

ResultsTLR4 activation by spike S1 or LPS resulted in the upregulation of ACE2 in rcTMFs as shown by immunoblotting. Likewise, spike S1 caused TLR4-mediated induction of the inflammatory/wound healing marker COX-2 and concomitant downregulation of the fibrosis markers CTGF and Col3a1, similar to LPS. The specific TLR4 TIR domain signalling inhibitor CLI-095 (Resatorvid(R)), blocked the effects of spike S1 and LPS, confirming that spike S1 is a TLR4 agonist and viral PAMP (VAMP). ACE2 expression was also inhibited by the dynamin inhibitor Dynasore(R), suggesting ACE2 expression is mediated by the alternative endosomal/{beta}-interferon pathway. Confocal immunofluorescence microscopy confirmed 1:1 stoichiometric spike S1 co-localisation with TLR4 in rat and human cells. Furthermore, proximity ligation assays confirmed spike S1 and TLR4 binding in human and rat cells. Spike S1/IFN-{gamma} treatment of THP-1-derived macrophages induced pro-inflammatory M1 polarisation as shown by an increase in IL-1{beta} and IL-6 mRNA.

ConclusionsThese results confirm that TLR4 is activated by the SARS-CoV-2 spike protein S1 domain and therefore TLR4 may be a receptor/accessory factor for the virus. By binding to and activating TLR4, spike S1 caused upregulation of ACE2, which may facilitate viral entry into cells. In addition, pro-inflammatory M1 macrophage polarisation via TLR4 activation, links TLR4 activation by spike S1 to inflammation. The clinical trial testing of CLI-095 (Resatorvid(R)) and other TLR4 antagonists in severe COVID-19, to reduce both viral entry into cells and hyperinflammation, is warranted. Our findings likely represent an important development in COVID-19 pathophysiology and treatment, particularly regarding cardiac complications and the role of macrophages.

category: cell biology


title: Dynamic Spatial-Temporal Expression Ratio of X Chromosome to Autosomes but Stable Dosage Compensation in Mammals

abstract: In the evolutionary model of dosage compensation, per-allele expression level of the X chromosome was proposed to have two-fold upregulation, compensating for its dose reduction in males (XY) compared to females (XX). However, the upregulation of X chromosome is still in dispute, and comprehensive evaluations are still lacking. By integrating multi-omics datasets in mammals, we investigated the expression ratios and underlying pattern of X to autosomes (X:AA ratio) and X to orthologs (X:XX ratio) at the transcriptome, translatome, and proteome layers. The results indicated a dynamic spatial-temporal X:AA ratio during development in human and mouse. Meanwhile, by tracing the evolution of orthologous gene expressions in chicken, platypus, and opossum, we found a constant expression ratio between X-linked genes in human and their autosomal orthologs in other species (X:XX ~1) across tissues and developmental stages, demonstrating stable dosage compensation in mammals. We also revealed that different epigenetic regulations could shape the higher tissue- and stage-specificity of X-linked gene expression, and affect X:AA ratios. We conclude that the dynamics of X:AA ratios are attributed to the different gene contents and expression preferences of the X chromosome, instead of the stable dosage compensation.

category: evolutionary biology


title: Cross-sectional genomic perspective of epidemic waves of SARS-CoV-2: a pan India study

abstract: COVID-19 has posed unforeseen circumstances and throttled major economies worldwide. India has witnessed two waves affecting around 31 million people representing 16% of the cases globally. To date, the epidemic waves have not been comprehensively investigated to understand pandemic progress in India. In the present study, we aim for a cross-sectional analysis since its first incidence up to 26th July 2021. We have performed the pan Indian evolutionary study using 20,086 high-quality complete genomes of SARS-CoV-2. Based on the number of cases reported and mutation rates, we could divide the Indian epidemic into seven different phases. First, three phases constituting the pre-first wave had a very less average mutation rate (<11), which increased in the first wave to 17 and then doubled in the second wave (~34). In accordance with the mutation rate, variants of concern (alpha, beta, gamma and delta) and interest (eta and kappa) also started appearing in the first wave (1.5% of the genomes), which dominated the second (~96% of genomes) and post-second wave (100% of genomes) phases. Whole genome-based phylogeny could demarcate the post-first wave isolates from previous ones by the point of diversification leading to incidences of VOCs and VOIs in India. Nation-wide mutational analysis depicted more than 0.5 million events with four major mutations in ~97% of the total 20,086 genomes in the study. These included two mutations in coding (spike (D614G) and NSP 12b (P314L) of RNA dependent RNA polymerase), one silent mutation (NSP3 F106F) and one extragenic mutation (5 UTR 241). Large scale genome-wide mutational analysis is crucial in expanding knowledge on evolution of deadly variants of SARS-CoV-2 and timely management of the pandemic.


title: FragGeneScanRs: better and faster gene prediction for short reads

abstract:
FragGeneScanRs is a better and faster Rust implementation of the FragGeneScan gene prediction model for short and error-prone reads. Its command line interface is backward compatible and adds extra features for more flexible usage. Compared to the original C implementation, shotgun metagenomic reads are processed up to 22 times faster using a single thread, with better scaling for multithreaded execution.

Availability and implementationThe Rust code of FragGeneScanRs is freely available from GitHub under the GPL-3.0 license, with instructions for installation, usage and other documentation.


title: Essential Loop Dynamics Modulates Catalytic Activity in α-Chymotrypsin

abstract: Conformational dynamics of macromolecules including enzymes are essential for their function. The present work reports the role of essential dynamics in alpha-chymotrypsin (CHT) which correlates with its catalytic activity. Detailed optical spectroscopy and classical molecular dynamics (MD) simulation were used to study thermal stability, catalytic activity and dynamical flexibility of the enzyme. The study of the enzyme kinetics reveals an optimum catalytic efficiency at 308K. Polarization gated fluorescence anisotropy with 8-anilino-1-napthelene sulfonate (ANS) have indicated increasing flexibility of the enzyme with an increase in temperature. Examination of the structure of CHT reveal the presence of five loop regions (LRs) around the catalytic S1 pocket. MD simulations have indicated that flexibility increases concurrently with temperature which decreases beyond optimum temperature. Principal component analysis (PCA) of the eigenvectors manifests essential dynamics and gatekeeping role of the five LRs surrounding the catalytic pocket which controls the enzyme activity.


title: Sub-3 Angstrom cryo-EM structure of RNA enabled by engineered homomeric self-assembly

abstract: Many functional RNAs fold into intricate and precise 3D architectures, and high-resolution structures are required to understand their underlying mechanistic principles. However, RNA structural determination is difficult. Herein, we present a nanoarchitectural strategy to enable the efficient single-particle cryogenic electron microscopy (cryo-EM) analysis of RNA-only structures. This strategy, termed RNA oligomerization-enabled cryo-EM via installing kissing-loops (ROCK), involves the engineering of target RNAs by installing kissing-loop sequences onto functionally nonessential stems for the assembly into closed homomeric nanoarchitectures. Assembly with geometric restraints leads to (1) molecular weight multiplication and (2) structural flexibility mitigation, both beneficial for cryo-EM analysis. Together with construct optimization and symmetry-expansion reconstruction, ROCK yields the cryo-EM reconstruction of the Tetrahymena group I intron at an overall resolution of 2.98 [A] (2.85 [A] resolution for the core domains), enabling the de novo model building of the complete intron RNA including previously unknown peripheral domains. When applied to smaller RNAs, ROCK readily produces modest-resolution maps, revealing the conformational rearrangement of the Azoarcus group I intron and the bound ligand in the FMN riboswitch. Our work unleashes the largely unexplored potential of cryo-EM in RNA structural studies.


title: Reconstructing transcriptional histories by CRISPR acquisition of retron-based genetic barcodes

abstract: Biological processes depend on the differential expression of genes over time, but methods to make true physical recordings of these processes are limited. Here we report a strategy for making time-ordered recordings of transcriptional events into living genomes. We do this via engineered RNA barcodes, based on prokaryotic retrons, which are reverse-transcribed into DNA and integrated into the genome using the CRISPR-Cas system. This approach enables the targeted recording of time-ordered transcriptional events in cells. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record via simple, logical rules rather than relying on pre-trained classifiers or post-hoc inferential methods.


title: Durability of SARS-CoV-2-specific T cell responses at 12-months post-infection

abstract:
BackgroundCharacterizing the longevity and quality of cellular immune responses to SARS-CoV-2 is critical to understanding immunologic approaches to protection against COVID-19. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Further analysis of the function, durability, and diversity of the cellular response long after natural infection, over a wider range of ages and disease phenotypes, is needed to further identify preventative and therapeutic interventions.

MethodsWe identified participants in our multi-site longitudinal, prospective cohort study 12-months post SARS-CoV-2 infection representing a range of disease severity. We investigated the function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry. In parallel, the magnitude of SARS-CoV-2-specific antibodies was compared.

ResultsSARS-CoV-2-specific antibodies and T cells were detected at 12-months post-infection. Severity of acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12-months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity.

ConclusionsOur data show that SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12-months post-infection, with higher frequency noted in those who originally experienced severe disease.


title: CsrA-controlled proteins important for Acinetobacter baumannii desiccation tolerance

abstract:
Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that its a good model for studies of the functional basis of this trait. Consistent with results from other A. baumannii strains at 30% RH, we found the global post-transcriptional regulator CsrA to be critically important for desiccation tolerance of AB5075 at 2% RH. To identify CsrA-controlled proteins that may contribute to desiccation tolerance we used proteomics to identify proteins that were differentially present in wild type and csrA mutant cells. Subsequent mutant analysis revealed nine genes that were required for wild type levels of desiccation tolerance, five of which had modest phenotypes. Catalase and a universal stress protein gene were moderately important for desiccation tolerance and two genes of unknown function had very strong desiccation phenotypes. The predicted amino acid sequence of one of these genes predicts an intrinsically disordered protein. This category of proteins is widespread in eukaryotes but less so in prokaryotes. Our results suggest there may be mechanisms responsible for desiccation tolerance that have not previously been explored in bacteria.

IMPORTANCEAcinetobacter baumannii is commonly found in terrestrial environments but can cause nosocomial infections in very sick patients. A factor that contributes to the prevalence of A. baumannii in hospital settings is that it is intrinsically resistant to dry conditions. Here, we established the virulent strain A. baumannii AB5075 as a model for studies of desiccation tolerance at very low relative humidity. Our results show that this trait depends on two proteins of unknown function, one of which is predicted to be an intrinsically disordered protein. This category of protein is critical for the small animals named tardigrades to survive desiccation. Our results suggest that A. baumannii may have novel strategies to survive desiccation that have not previously been seen in bacteria.


title: CsrA-controlled proteins reveal new dimensions of Acinetobacter baumannii desiccation tolerance


title: Identification of the 216kbp gene cluster and structure elucidation of gargantulides B and C, new complex 52-membered macrolides from Amycolatopsis sp.

abstract: Gargantulides B and C, two new and highly complex 52-membered glycosylated macrolactones, were isolated from Amycolatopsis sp. strain CA-230715 during an antibacterial screening campaign. The structures of these giant macrolides were elucidated by 2D NMR spectroscopy and shown to be related to gargantulide A, although containing additional {beta}-glucopyranose and/or -arabinofuranose monosaccharides separately attached to their backbones. Genome sequencing allowed the identification of a strikingly large 216 kbp biosynthetic gene cluster, among the largest type I PKS clusters described so far, and the proposal of a biosynthetic pathway for gargantulides A-C. Additionally, genes putatively responsible for the biosynthesis of the amino sugar {beta}-3,6-deoxy-3-methylamino glucose, reported exclusively in gargantulide macrolides, were also found in the cluster and described in this work. The absolute configurations of gargantulides B and C were assigned based on a combination of NMR and bioinformatics analysis of ketoreductase and enoylreductase domains within the multimodular type I PKS. Furthermore, the absolute stereochemistry of the related macrolide gargantulide A has now been revised and completed. Gargantulides B and C display potent antibacterial activity against a set of drug-resistant Gram-positive bacteria and moderate activity against the clinically relevant Gram-negative pathogen Acinetobacter baumannii.


title: Rampant transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of a human fungal pathogen

abstract: Microorganisms survive and compete by stochastically acquiring mutations that enhance fitness. Although increased mutation rates are often deleterious in multicellular organisms, hypermutation can be beneficial for microbes experiencing strong selective pressures. Infections caused by Cryptococcus neoformans are responsible for [~]15% of AIDS-related deaths and associated with high mortality rates, attributable to a dearth of antifungal drugs and drug resistance. We identified two hypermutator C. neoformans clinical isolates in which Cnl1 transposon insertions were responsible for drug resistance. Whole-genome sequencing revealed both hypermutator genomes harbor a nonsense mutation in the RNAi component ZNF3 and hundreds of Cnl1 elements organized into massive subtelomeric arrays on every chromosome. QTL mapping identified a significant locus associated with hypermutation that included znf3. CRISPR-mediated editing of the znf3 nonsense mutation abolished hypermutation and restored siRNA production. In sum, hypermutation and drug resistance in these isolates results from RNAi loss and a significant burden of Cnl1 elements.


title: Molecular dynamics and theratyping in airway and gut organoids reveal R352Q-CFTR conductance defect

abstract: A significant challenge to making targeted CFTR modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q - with potential role contributing to intrapore chloride ion permeation - in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using ion transport assay and forskolin-induced swelling (FIS) assay respectively. Two CFTR potentiators (VX-770, GLPG1837) and a corrector (VX-809) were tested. Data from R352Q-CFTR were compared to that of participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function which was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics (MD) simulations of R352Q-CFTR were carried out which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico MD simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.


title: Exon-Skipping Antisense Oligonucleotides for Cystic Fibrosis Therapy

abstract: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity, but is expressed at a very low level due to nonsense-mediated mRNA decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable; and read-through drugs are inefficient, partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site-targeting ASOs induced the expression of CFTR mRNA without the PTC-containing exon 23 (CFTR-{Delta}ex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-{Delta}ex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.


title: Functionally distinct roles for eEF2K in the control of ribosome availability and p-body abundance in sensory neurons

abstract: Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies specifically in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. Collectively, the data suggest that eEF2 phosphorylation defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons.


title: A method for the fast and photon-efficient analysis of time-domain fluorescence lifetime image data over large dynamic ranges

abstract: Fluorescence lifetime imaging (FLIM) allows the quantification of subcellular processes in situ, in living cells. A number of approaches have been developed to extract the lifetime from time-domain FLIM data, but they are often limited in terms of dynamic range, speed, photon efficiency or precision. Here, we focus on one of the best performing methods in the field, the center-of-mass (CMM) method, that conveys advantages in terms of speed and photon efficiency over others. In this paper, however, we identify a loss of photon efficiency of CMM for short lifetimes when background noise is present. We sub-sequently present a new development and generalization of the CMM method that provides for the rapid and accurate extraction of fluorescence lifetime over a large lifetime dynamic range. We provide software tools to simulate, validate and analyze FLIM data sets and compare the performance of our approach against the standard CMM and the commonly employed leastsquare minimization (LSM) methods. Our method features a better photon efficiency than standard CMM and LSM and is robust in the presence of background noise. The algorithm is applicable to any time-domain FLIM dataset.


title: Rab11 endosomes coordinate centrosome number and movement following mitotic exit

abstract: The last stage of cell division involves two daughter cells remaining interconnected by a cytokinetic bridge that is cleaved in a process called abscission. During pre-abscission, we identified that the centrosome moves in a Rab11-dependent manner towards the cytokinetic bridge in human cells grown in culture and in an in vivo vertebrate model, Danio rerio (zebrafish). Rab11-endosomes are dynamically organized in a Rab11-GTP dependent manner at the centrosome during pre-abscission and this organization is required for the centrosome protein, pericentrin, to be enriched at the centrosome. Using zebrafish embryos, we found that reduction in pericentrin expression or optogenetically disrupting Rab11-endosome function inhibited centrosome movement towards the cytokinetic bridge and abscission resulting in daughter cells prone to being binucleated and/or having supernumerary centrosomes. These studies suggest that Rab11-endosomes contribute to centrosome function during pre-abscission by regulating pericentrin organization resulting in appropriate centrosome movement towards the cytokinetic bridge and subsequent abscission.


title: Role of YAP in early ectodermal specification anda Huntington's Disease model of human neurulation

abstract: The Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAPs potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and non-neuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntingtons Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAPs transcriptional activity can partially rescue the HD phenotype.

category: developmental biology


title: A temporally controlled sequence of X-chromosomeinactivation and reactivation defines female mouse in vitro germ cells with meiotic potential

abstract:
The early mammalian germ cell lineage is characterized by extensive epigenetic reprogramming, which is required for the maturation into functional eggs and sperm. In particular, the epigenome needs to be reset before parental marks can be established and then transmitted to the next generation. In the female germ line, reactivation of the inactive X-chromosome is one of the most prominent epigenetic reprogramming events, and despite its scale involving an entire chromosome affecting hundreds of genes, very little is known about its kinetics and biological function.

Here we investigate X-chromosome inactivation and reactivation dynamics by employing a tailor-made in vitro system to visualize the X-status during differentiation of primordial germ cell-like cells (PGCLCs) from female mouse embryonic stem cells (ESCs). We find that the degree of X-inactivation in PGCLCs is moderate when compared to somatic cells and characterized by a large number of genes escaping full inactivation. Nevertheless, PGCLCs that fail to undergo X-inactivation show an abnormal gene expression signature and deficiencies in meiotic entry. Subsequent to X-inactivation we observe gradual step-wise X-reactivation, which is mostly completed by the end of meiotic prophase I. Cells deviating from these progressive kinetics and undergoing X-reactivation too rapidly fail to enter a meiotic trajectory. Our data reveals that a fine-tuned X-inactivation and -reactivation cycle is a critical feature of female germ cell developmental competence towards meiosis and oogenesis


title: Whole genome analysis for 163 guide RNAs in Cas9 edited mice reveals minimal off-target activity

abstract: Introductory ParagraphThe Knockout Mouse Phenotyping Program (KOMP2) uses CRISRPR/Cas9 for high-throughput mouse line production to generate null alleles in the inbred C57BL/6N strain for broad-based in vivo phenotyping. In order to assess the risk of spurious S. pyogenes Cas9-induced off-target mutagenesis, we applied whole genome sequencing to compare the genomes of 50 Cas9-derived founder mice representing 163 different gRNAs to 28 untreated inbred control mice. Our analysis pipeline detected 28 off-target sequence variants associated with 21 guides. These potential off-targets were identified in 18/50 (36%) founders with 9/28 (32%) independently validated corresponding to 8 founder animals. In total, only 4.9% (8/163) of all guides exhibited off-target activity resulting in a rate of 0.16 Cas9 off-target mutations per founder analyzed. In comparison, we observed ~1225 unique variants in each mouse regardless of whether or not it was exposed to Cas9. These findings indicate that Cas9-mediated off-target mutagenesis is rare in founder knockout mice generated using guide RNAs designed to minimize off-target risk. Overall, bona fide off-target variants comprise a small fraction of the genetic heterogeneity found in carefully maintained colonies of inbred strains.

category: genetics


title: Structural basis of adenylyl cyclase 9 activation

abstract:
Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPS, (iv) bound to Gs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

One Sentence SummaryCryo-EM reveals activator-induced conformational changes in adenylyl cyclase AC9


title: Early neutrophilia marked by aerobic glycolysis sustains host metabolism and delays cancer cachexia

abstract: An elevated neutrophil-to-lymphocyte ratio negatively predicts the outcome of patients with cancer and is associated with cachexia, the terminal wasting syndrome. Here, we show using murine model systems of colorectal and pancreatic cancer that neutrophilia in the circulation and multiple organs, accompanied by extramedullary hematopoiesis, is an early event during cancer progression. Transcriptomic and metabolic assessment reveals that neutrophils in tumor-bearing animals utilize aerobic glycolysis, alike to cancer cells. Although pharmacological inhibition of aerobic glycolysis slows down tumor growth in C26 tumor-bearing mice, it precipitates cachexia, thereby shortening overall survival. This negative effect may be explained by our observation that acute depletion of neutrophils in pre-cachectic mice impairs systemic glucose homeostasis secondary to altered hepatic lipid processing. Thus, changes in neutrophil number, distribution and metabolism play an adaptive role in host metabolic homeostasis during cancer progression. Our findings provide insight into early events during cancer progression to cachexia, with implications for therapy.


title: NSUN6, an RNA methyltransferase of 5-mC controls glioblastoma response to Temozolomide (TMZ) via NELFB and RPS6KB2 interaction.

abstract: Nop2/Sun RNA methyltransferase (NSUN6) is an RNA 5 - methyl cytosine (5mC) transferase with little information known of its function in cancer and response to cancer therapy. Here, we show that NSUN6 methylates both large and small RNA in glioblastoma and controls glioblastoma response to temozolomide with or without influence of the MGMT promoter status, with high NSUN6 expression conferring survival benefit to glioblastoma patients and in other cancers. Mechanistically, our results show that NSUN6 controls response to TMZ therapy via 5mC mediated regulation of NELFB and RPS6BK2. Taken together, we present evidence that show that NSUN6 mediated 5mC deposition regulates transcriptional pause (by accumulation of NELFB and the general transcription factor complexes (POLR2A, TBP, TFIIA, TFIIE) on the preinitiation complex at TATA binding site to control translation machinery in glioblastoma response to alkylating agents. Our findings open a new frontier into controlling of transcriptional regulation by RNA methyltransferase and 5mC.