Absence of cardiolipin from the outer leaflet of a mitochondrial inner membrane mimic restricts Opa1-mediated fusion Cardiolipin is a tetra-acylated di-phosphatidylglycerol lipid enriched in the matrix-facing (inner) leaflet of the mitochondrial inner membrane. Cardiolipin plays an important role in regulating mitochondria function and dynamics. Yet, the mechanisms connecting cardiolipin distribution and mitochondrial protein function remain indirect. In our previous work, we established an in vitro system reconstituting mitochondrial inner membrane fusion mediated by Opa1. We found that the long form of Opa1 (l-Opa1) works together with the proteolytically processed short form (s-Opa1) to mediate fast and efficient membrane fusion. Here, we extend our reconstitution system to generate supported lipid bilayers with asymmetric cardiolipin distribution. Using this system, we find the presence of cardiolipin on the inter-membrane space-facing (outer) leaflet is important for membrane tethering and fusion. We discuss how the presence of cardiolipin in this leaflet may influence protein and membrane properties, and future applications for this approach.
ATP dependent polymerization dynamics of bacterial actin proteins involved in Spiroplasma swimming MreB is a bacterial protein belonging to the actin superfamily. It polymerises into an antiparallel double-stranded filament that generally functions in cell shape determination by maintaining cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five classes of MreB homologs (SpeMreB1-5) that are likely to be involved in swimming motility. Here, we investigated the structure, ATPase activity, and polymerisation dynamics of SpeMreB3 and SpeMreB5. SpeMreB3 polymerised into an antiparallel double-stranded filament, and SpeMreB5 formed sheets, including the antiparallel filament, upon the binding of a nucleotide. SpeMreB3 showed slow Pi release owing to the lack of an amino acid motif conserved in the catalytic centre of MreB family proteins. Our crystal structures of SpeMreB3 and analyses of the mutant variants showed that the amino acid motif most likely plays a role in eliminating the proton of the nucleophilic water for ATP hydrolysis. Our sedimentation assay suggests that SpeMreB3 has a lower polymerisation activity than SpeMreB5, while their polymerisation dynamics are qualitatively similar to those of other actin superfamily proteins, in which ATP hydrolysis stabilises the filament, and Pi release leads to depolymerisation.
Effect of Adolescent Stress on Adult Morphine-Induced Behavioral Sensitization is Dependent Upon Genetic Background 1Deaths related to opioid use have skyrocketed in the United States, leading to a public health epidemic. Research has shown that both biological (genes) and environmental (stress) precursors are linked to opioid use. In particular, stress during adolescence - a critical period of frontal lobe development - influences the likelihood of abusing drugs. However, little is known about the biological mechanisms through which adolescent stress leads to long-term risk of opioid use, or whether genetic background moderates this response. Male and female C57BL/6J and BALB/cJ mice were exposed to chronic variable social stress (CVSS) or control conditions throughout adolescence and then tested for morphine locomotor sensitization or morphine consumption in adulthood. To examine possible mechanisms that underlie stress-induced changes in morphine behaviors, we assessed physiological changes in response to acute stress exposure and prefrontal cortex miRNA gene expression. Adolescent stress did not influence morphine sensitization or consumption in BALB/cJ animals, and there was limited evidence of stress effects in female C57BL/6J mice. In contrast, male C57BL/6J mice exposed to adolescent CVSS had blunted morphine sensitization compared to control animals; no differences were observed in the acute locomotor response to morphine administration or morphine consumption. Physiologically, C57BL/6J mice exposed to CVSS had an attenuated corticosterone recovery following an acute stressor and downregulation of twelve miRNA in the prefrontal cortex compared to control mice. The specificity of the effects for C57BL/6J versus BALB/cJ mice provides evidence of a gene by environmental interaction influencing opioid behaviors. Long-term differences in stress reactivity or miRNA expression suggests two possible biological mechanisms to evaluate in future research. A novel Fiji/ImageJ plugin for the rapid analysis of blebbing cells When confined, cells have recently been shown to undergo a phenotypic switch to what has been termed, fast amoeboid (leader bleb-based) migration. However, as this is a nascent area of research, few tools are available for the rapid analysis of cell behavior. Here, we demonstrate that a novel Fiji/ImageJ-based plugin, Analyze_Blebs, can be used to quickly obtain cell migration parameters and morphometrics from time lapse images. As validation, we show that Analyze_Blebs can detect significant differences in cell migration and morphometrics, such as the largest bleb size, upon introducing different live markers of F-actin, including F-tractin and LifeAct tagged with green and red fluorescent proteins. We also demonstrate, using flow cytometry, that live markers increase total levels of F-actin. Furthermore, that F-tractin increases cell stiffness, which was found to correlate with a decrease in migration, thus reaffirming the importance of cell mechanics as a determinant of Leader Bleb-Based Migration (LBBM).

Highlight summaryA new plugin, Analyze_Blebs, enables the rapid analysis of cell migration and morphometrics of fast amoeboid cells. Morphometrics combined with cell stiffness are found to be predictive of confined, fast amoeboid (leader bleb-based), migration. DeGlyPHER: an ultrasensitive method for analysis of viral spike N-glycoforms Viruses can evade the host immune system by displaying numerous glycans on their surface "spike-proteins" that cover immune epitopes. We have developed an ultrasensitive "single pot" method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for analysis of site-specific glycosylation of any glycoprotein. kmtricks: Efficient and flexible construction of Bloom filters for large sequencing data collections When indexing large collections of short-read sequencing data, a common operation that has now been implemented in several tools (Sequence Bloom Trees and variants, BIGSI, ..) is to construct a collection of Bloom filters, one per sample. Each Bloom filter is used to represent a set of k-mers which approximates the desired set of all the non-erroneous k-mers present in the sample. However, this approximation is imperfect, especially in the case of metagenomics data. Erroneous but abundant k-mers are wrongly included, and non-erroneous but low-abundant ones are wrongly discarded. We propose kmtricks, a novel approach for generating Bloom filters from terabase-sized collections of sequencing data. Our main contributions are 1/ an efficient method for jointly counting k-mers across multiple samples, including a streamlined Bloom filter construction by directly counting, partitioning and sorting hashes instead of k-mers, which is approximately four times faster than state-of-the-art tools; 2/ a novel technique that takes advantage of joint counting to preserve low-abundant k-mers present in several samples, improving the recovery of non-erroneous k-mers. Our experiments highlight that this technique preserves around 8x more k-mers than the usual yet crude filtering of low-abundance k-mers in a large metagenomics dataset.

Availabilityhttps://github.com/tlemane/kmtricks

FundingThe work was funded by IPL Inria Neuromarkers, ANR Inception (ANR-16-CONV-0005), ANR Prairie (ANR-19-P3IA-0001), ANR SeqDigger (ANR-19-CE45-0008). Maf/ham1-like pyrophosphatases of non-canonical nucleotides are host-specific partners of viral RNA-dependent RNA polymerases Cassava brown streak disease (CBSD), dubbed the "Ebola of plants", is a serious threat to food security in Africa caused by two viruses of the family Potyviridae: cassava brown streak virus (CBSV) and Ugandan (U)CBSV. Intriguingly, U/CBSV, along with another member of this family and one secoviridae, are the only known RNA viruses encoding a protein of the Maf/ham1-like family, a group of widespread pyrophosphatase of non-canonical nucleotides (ITPase) expressed by all living organisms. Despite the socio-economic impact of CDSD, the relevance and role of this atypical viral factor has not been yet established. Here, using an infectious cDNA clone and reverse genetics, we demonstrate that UCBSV requires the ITPase activity for infectivity in cassava, but not in the model plant Nicotiana benthamiana. HPLC MS/MS experiments showed that, quite likely, this host-specific constraint is due to an unexpected high concentration of non-canonical nucleotides in cassava. Finally, protein analyses and experimental evolution of mutant viruses indicated that keeping a fraction of the yielded UCBSV ITPase covalently bound to the viral RNA-dependent RNA polymerase (RdRP) optimizes viral fitness, and this seems to be a feature shared by the other members of the Potyviridae family expressing Maf/ham1-like proteins. All in all, our work (i) reveals that the over-accumulation of non-canonical nucleotides in the host might have a key role in antiviral defense, and (ii) provides the first example of an RdRP-ITPase partnership, reinforcing the idea that RNA viruses are incredibly versatile at adaptation to different host setups. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants. An individualized causal framework for learning intercellular communicationnetworks that define microenvironments of individual tumors Cells within a tumor microenvironment (TME) dynamically communicate and influence each others cellular states through an intercellular communication network (ICN). In cancers, intercellular communications underlie immune evasion mechanisms of individual tumors. We developed an individualized causal analysis framework for discovering tumor-specific ICNs. Using head and neck squamous cell carcinoma (HNSCC) tumors as a testbed, we first mined single-cell RNA-sequencing data to discover gene expression modules (GEMs) that reflect the states of transcriptomic processes within tumor and stromal single cells. By deconvoluting bulk transcriptomes of HNSCC tumors profiled by The Cancer Genome Atlas (TCGA), we estimated the activation states of these transcriptomic processes in individual tumors. Finally, we applied individualized causal network learning to discover an ICN within each tumor. Our results show that cellular states of cells in TMEs are coordinated through ICNs that enable multi-way communications among epithelial, fibroblast, endothelial, and immune cells. Further analyses of individual ICNs revealed structural patterns that were shared across subsets of tumors, leading to the discovery of 4 different subtypes of networks that underlie disparate TMEs of HNSCC. Patients with distinct TMEs exhibited significantly different clinical outcomes. Our results show that the capability of estimating individual ICNs reveals heterogeneity of ICNs and sheds light on the importance of intercellular communication in impacting disease development and progression. The multi-level phenotypic impact of synonymous substitutions: heterologous gene expression in human cells. Redundancy in the genetic code allows for differences in transcription and/or translation efficiency between sequences carrying synonymous polymorphisms, potentially leading to phenotypic changes. It is commonly admitted that the evolution codon usage bias ("CUB", the over-representation of certain codons in a genome, a gene or in positions along a gene) are driven by a combination of neutral and selective processes, but their relative contribution is a matter of debate, especially in mammals. Particularly, integrative studies quantifying the phenotypic impact of CUB at different molecular and cellular levels are lacking. Here we report a multiscale analysis of the effects of synonymous codon recoding during heterologous gene expression in human cells.

Six synonymous versions of the shble antibiotic resistance gene were generated, fused to a fluorescent reporter, and independently expressed in HEK293 cells. Multiscale phenotype was assessed by means of: i) mRNA-to-DNA and protein-to-mRNA ratios for each shble version; ii) cellular fluorescence, using flow cytometry, as a proxy for single-cell level expression; and iii) real-time cell proliferation in absence or presence of antibiotic, as a proxy for the cell fitness.

We show that differences in CUB strongly impact the molecular and the cellular phenotype: i) they result in large differences in mRNA and in protein levels, as well in mRNA-to-protein ratio; ii) they introduce splicing events not predicted by current algorithms; iii) they lead to reproducible phenotypic heterogeneity; iv) they lead to a trade-off between the benefit of antibiotic resistance and the burden of heterologous expression.

We interpret that CUB modulate mRNA availability and suitability for translation in human cells, leading to differences in protein levels and eventually eliciting phenotypic differences.

AUTHOR SUMMARYThe genetic code is redundant, with several codons encoding for the same amino acid. These synonymous codons are not used with equal frequencies. Instead, codon usage bias (CUB) varies between species, genes, and even positions along a gene. At each of these levels, CUB are shaped by the overall balance between mutational biases and selection forces. To shed light on the molecular mechanisms underlying molecular and organism phenotypes, integrative studies quantifying the phenotypic impact of CUB at different levels of biological integration are necessary. Here, we monitored the multiscale changes induced by six synonymous versions of an antibiotic resistance gene independently expressed in a human cell line. We show that: 1. both mRNA levels, protein levels, and their ratios are affected by CUB; 2. potential effects on mRNA (splicing and availability for translation) seem to constitute the main level of action; 3. cell fitness is severely impacted by a trade-off between the burden of heterologous expression and the benefits of antibiotic resistance. This integrative study provides new insights on translation regulation and the associated phenotypic impact in human cells, associated to CU. Mutagenic distinction between the receptor-binding and fusion subunits of the SARS-CoV-2 spike glycoprotein and its upshot We observe that a residue R of the spike glycoprotein of SARS-CoV-2 which has mutated in one or more of the current Variants of Concern or Interest or under Monitoring rarely participates in a backbone hydrogen bond if R lies in the S1 subunit and usually participates in one if R lies in the S2 subunit. A partial explanation for this based upon free energy is explored as a potentially general principle in the mutagenesis of viral glycoproteins. This observation could help target future vaccine cargos for the evolving coronavirus as well as more generally. A study of the Delta and Omicron variants suggests that Delta was an energetically necessary intermediary in the evolution from Wuhan-Hu-1 to Omicron. An adaptive teosinte mexicana introgression modulates phosphatidylcholine levels and is associated with maize flowering time Native Americans domesticated maize (Zea mays ssp. mays) from lowland teosinte parviglumis (Zea mays ssp.parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability, and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 (HPC1), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern US, Canada and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time. Genomic insights into longan evolution from a chromosome-level genome assembly and population genomics of longan accessions Longan (Dimocarpus longan) is a subtropical fruit best known for its nutritious fruit and regarded as a precious tonic and traditional medicine since ancient times. High-quality chromosome-scale genome assembly is valuable for functional genomic study and genetic improvement of longan. Here, we report a chromosome-level reference genome sequence for longan cultivar JDB with an assembled genome of 455.5 Mb in size anchored to fifteen chromosomes, representing a significant improvement of contiguity (contig N50=12.1 Mb, scaffold N50= 29.5 Mb) over a previous draft assembly. A total of 40,420 protein-coding genes were predicted in D. longan genome. Synteny analysis suggests longan shares the widespread gamma event with core eudicots, but has no other whole genome duplications. Comparative genomics showed that D. longan genome experienced significant expansions of gene families related to phenylpropanoid biosynthesis and UDP-glucosyltransferase. Deep genome sequencing analysis of longan cultivars identified longan biogeography as a major contributing factor for genetic diversity, and revealed a clear population admixture and introgression among cultivars of different geographic origins, postulating a likely migration trajectory of longan overall confirmed by existing historical records. Finally, genome-wide association studies (GWAS) of longan cultivars identified quantitative trait loci (QTL) for six different fruit quality traits and revealed a shared QTL containing three genes for total soluble solid and seed weight. The chromosome-level reference genome assembly, annotation and population genetic resource for D. longan will facilitate the molecular studies and breeding of desirable longan cultivars in the future. Metabarcoding faecal samples to investigate spatiotemporal variation in the diet of the endangered Westland petrel (Procellaria westlandica) As top predators, seabirds can be indirectly impacted by climate variability and commercial fishing activities through changes in marine communities. However, high mobility and foraging behaviour enables seabirds to exploit prey distributed patchily in time and space. This capacity to adapt to environmental change can be described through the study of their diet. Traditionally, the diet of seabirds is assessed through the morphological identification of prey remains in regurgitates. This sampling method is invasive for the bird and limited in terms of taxonomic resolution. However, the recent progress in DNA-based approaches is now providing a non-invasive means to more comprehensively and accurately characterize animal diets. Here, we used a non-invasive metabarcoding approach to characterize the diet of the Westland petrel (Procellaria westlandica), which is an endangered burrowing species, endemic to the South Island of New Zealand. We collected 99 fresh faecal samples at two different seasons and in two different sub-colonies. Our aims were to describe the diet of the Westland petrel, investigate seasonal and spatial variation in the petrels diet, and assess potential impacts of the New Zealand fishery industry. We found that amphipods were the most common prey, followed by cephalopods and fish. Our results could be the result of natural foraging behaviour, but also suggest a close link between the composition of prey items and New Zealands commercial fishing activities. In particular, the high abundance of amphipods could be the result of Westland petrels feeding on discarded fisheries waste (fish guts). Our results also showed significant differences in diet between seasons (before hatching vs chick-rearing season) and between sampling sites (two sub-colonies 1.5 km apart), indicating plasticity in the foraging strategy of the Westland petrel. Due to its non-invasive nature, metabarcoding of faecal samples can be applied to large numbers of samples to help describe dietary variation in seabirds and indicate their ecological requirements. In our example, dietary DNA (dDNA) provided valuable information regarding the dietary preferences of an iconic species in New Zealands unique biodiversity. dDNA can thus inform the conservation of endangered or at-risk species that have elusive foraging behaviours. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Multi-omics investigation of tacrolimus nephrotoxicity BackgroundTacrolimus, an immunosuppressive drug prescribed to a majority of transplanted patients is nephrotoxic, through still unclear mechanisms. This study aims to evaluate the impact of tacrolimus on a lineage of proximal tubular cells using a multi-omics approach.

MethodsLLC-PK1 cells were exposed to 5 M of tacrolimus for 24h. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, FBP1 and FBP2 was measured using RT-qPCR.

ResultsIn our cell model, tacrolimus impacted different metabolic pathways including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p<0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis. Apart from glucose synthesis, gluconeogenesis is an important process in kidney mediated acid-base balance control.

ConclusionThe variations found using this multi-omics approach clearly point towards a dysregulation of energy production in epithelial cells of the renal tubule, and potentially of their functions, that may be implicated in tacrolimus nephrotoxicity in the clinics. LPS-TLR4 pathway exaggerates alcohol-induced acute-on-chronic liver failure via provoking NETs formation Backgrounds: Intrahepatic infiltration of neutrophils is a character of alcoholic acute-on-chronic liver failure (AACLF) and neutrophil extracellular traps (NETs) are an important strategy for neutrophils to fix and kill invading microorganisms. Intestinal bacteria and the gut-liver axis have been thought to play a key role in many liver diseases also including AACLF. However, whether NETs appear in AACLF and play a role in AACLF is still unsure. Methods: WT, NE KO, and TLR4 KO mice were used to build the AACLF model, and the intestinal bacteria were eliminated at the same time and LPS was given. Then the formation of NETs and AACLF related markers were detected. Results: The serum MPO-DNA and LPS concentration was increased in AACLF patients and a correlation was revealed between these two indexes. More intrahepatic NETs formed in AACLF mice by testing MPO-DNA, Cit H3, and NE. These markers decreased with gut detergent and restored markers with gut detergent plus LPS supplement. While NETs formation failed to change with gut microbiome or combine LPS supplement in TLR4 KO mice. As we tested AACLF related characters, liver injury, intrahepatic fat deposition, inflammation, and fibrosis alleviated with depletion of NE. These related marks were also attenuated with gut sterilization by antibiotics and recovered with combined treatment with antibiotics plus LPS. But the liver injury, intrahepatic fat, fibro deposition, and liver inflammation-related markers did show a significant difference in TLR4 KO mice when they received the same treatment. Conclusion: Intestinal-derived LPS promotes NETs formation in AACLF through the TLR4 pathway and further accelerates the AACLF process by NETs. TeamTree analysis: a new approach to evaluate scientific production Advances in science and technology depend on the work of research teams and the publication of results through peer-reviewed articles representing a growing socio-economic resource. Current methods to mine the scientific literature regarding a field of interest focus on content, but the workforce credited by authorship remains largely unexplored, and appropriate measures of scientific production are debated. Here, a new bibliometric approach named TeamTree analysis is introduced that visualizes the development and composition of the workforce driving a field. A new citation-independent measure that scales with the H index estimates impact based on publication record, genealogical ties and collaborative connections. This author-centered approach complements existing tools to mine the scientific literature and to evaluate research across disciplines. Using total RNA quality metrics for time since deposition estimates in degrading bloodstains Determining the time since deposition (TSD) of bloodstains would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. The physicochemical changes occurring to biomolecules as a bloodstain degrades can be used to approximate the TSD of bloodstains. Our study aims to quantify the timewise degradation trends and temperature dependence found in total RNA from bloodstains without the use of amplification, expanding the scope of the RNA TSD research which has previously targeted mRNA molecules. Whole bovine blood was stored in plastic microcentrifuge tubes at 21{degrees}C or 4{degrees}C and tested over different timepoints spanning one week. Nine RNA metrics were visually assessed and quantified using linear and mixed models; the RNA Integrity Number equivalent (RINe) and the DV200 demonstrated strong negative trends over time and statistical independence. The RINe model fit was high (R2 = 0.60), and while including the biological replicate as a random effect increased the fit for all RNA metrics, no significant differences were found between biological replicates stored at the same temperature for the RINe and DV200 metrics. Importantly, this suggests that these standardized metrics can likely be directly compared between scenarios and individuals, with DV200 having an inflection point at [~]28 hrs. This study provides a novel approach for blood TSD estimates, producing metrics that are not affected by inter-individual variation and improving our understanding of the rapid degradation occurring in bloodstains.

HIGHLIGHTSO_LIAmplification-free analysis of total RNA in degrading bloodstains.
C_LIO_LIShort-term RNA degradation assessment using high-resolution size measurements.
C_LIO_LITotal RNA quality and quantity metrics were assessed across a one-week.
C_LIO_LITotal RNA quality metrics demonstrated the strongest timewise trends.
C_LIO_LIBiological replicates produced similar results for RNA quality metrics.
C_LI Perceiving and remembering speech depend on multifractal nonlinearity in movements producing and exploring speech Speech perception and memory for speech require active engagement. Gestural theories have emphasized mainly the effect of speakers movements on speech perception. They fail to address the effects of listener movement, focusing on communication as a boundary condition constraining movement among interlocutors. The present work attempts to break new ground by using multifractal geometry of physical movement as a common currency for supporting both sides of the speaker-listener dyads. Participants self-paced their listening to a narrative, after which they completed a test of memory querying their narrative comprehension and their ability to recognize words from the story. The multifractal evidence of nonlinear interactions across timescales predicted the fluency of speech perception. Self-pacing movements that enabled listeners to control the presentation of speech sounds constituted a rich exploratory process. The multifractal nonlinearity of this exploration supported several aspects of memory for the perceived spoken language. These findings extend the role of multifractal geometry in the speakers movements to the narrative case of speech perception. In addition to posing novel basic research questions, these findings make a compelling case for calibrating multifractal structure in text-to-speech synthesizers for better perception and memory of speech. Modelling homing suppression gene drive in haplodiploid organisms Gene drives have shown great promise for suppression of pest populations. These engineered alleles can function by a variety of mechanisms, but the most common is the CRISPR homing drive, which converts wild-type alleles to drive alleles in the germline of heterozygotes. Some potential target species are haplodiploid, in which males develop from unfertilized eggs and thus have only one copy of each chromosome. This prevents drive conversion, a substantial disadvantage compared to diploids where drive conversion can take place in both sexes. Here, we study homing suppression gene drives in haplodiploids and find that a drive targeting a female fertility gene could still be successful. However, such drives are less powerful than in diploids and suffer more from functional resistance alleles. They are substantially more vulnerable to high resistance allele formation in the embryo due to maternally deposited Cas9 and gRNA and also to somatic cleavage activity. Examining spatial models where organisms move over a continuous landscape, we find that haplodiploid suppression drives surprisingly perform nearly as well as in diploids, possibly due to their ability to spread further before inducing strong suppression. Together, these results indicate that gene drive can potentially be used to effectively suppress haplodiploid populations. Differential fate of acellular vascular scaffolds in vivo involves macrophages and T-cell subsets Biological scaffold or implant is a popular choice for the preparation of tissue-engineered organs and has the potential to address donor shortage in the clinics. However, biological scaffolds prepared by physical or chemical agents cause damage to the extracellular matrix by potentially inducing immune responses after implantation. The current study explores an alternative route for the preparation of acellular scaffolds and explores the fate of the prepared scaffolds in a milieu of immune cells following implantation without using immunosuppressant. Using the syngeneic (Lewis male-Lewis female) and allogeneic (Brown Norway male-Lewis female) models and different tissue routes (subcutaneous vs omentum) for transplantation, normal blood vascular scaffolds were implanted which was converted to acellular vascular scaffolds by in vivo natural decellularization at the end of 2 months of observation. We also prepared chemically decellularized acellular scaffolds from normal untreated blood vascular scaffolds using a cocktail of chemicals which was also similarly placed in subcutaneous and omentum sites. Here, we applied in-depth quantitative proteomics along with histology and image analysis to comprehensively describe and compare the proteome of the natural and chemically decellularized scaffold. Our data confirm that site-specific advantages exist in modulating the ECM and regulating the immune responses (macrophage and T cells) following implantation, which possibly led to the production of an acellular scaffold (natural decellularization) under in vivo conditions. The current approach opens up the possibility to create tailor-made acellular scaffolds to build functional blood vessels. In addition, the identification of different tissue sites facilitates differential immune response against the scaffolds. This study provides a rich resource aimed toward an enhanced mechanistic understanding to study immune responses under similar settings in the field of transplantation and regenerative medicine.

Impact statementThe development of a scaffold helps in the preparation of a functional organ in the clinics. In the current study, we prepared an acellular vascular scaffold by utilizing site specific tissue changes and vis-a-vis compared with a conventionally chemically prepared biological scaffold at genomic and protein level, which helped us to identify immunological trigger following implantation. The current study which was carried out without any immunosuppressive agents could help to establish (a) alternative strategies for preparing biological scaffolds as well as (b) implantable sites as potential bioreactors to circumvent any adverse immune reactions for acceptance of the scaffold/implant post implantation. STING mediates immune responses in the closest living relatives of animals Animals have evolved unique repertoires of innate immune genes and pathways that provide their first line of defense against pathogens. To reconstruct the ancestry of animal innate immunity, we have developed the choanoflagellate Monosiga brevicollis, one of the closest living relatives of animals, as a model for studying mechanisms underlying pathogen recognition and immune response. We found that M. brevicollis is killed by exposure to Pseudomonas aeruginosa bacteria. Moreover, M. brevicollis expresses STING, which, in animals, activates innate immune pathways in response to cyclic dinucleotides during pathogen sensing. M. brevicollis STING increases the susceptibility of M. brevicollis to P. aeruginosa-induced cell death and is required for responding to the cyclic dinucleotide 23 cGAMP. Furthermore, similar to animals, autophagic signaling in M. brevicollis is induced by 23 cGAMP in a STING-dependent manner. This study provides evidence for a pre-animal role for STING in antibacterial immunity and establishes M. brevicollis as a model system for the study of immune responses. First detection of herpesvirus and frequency of mycoplasma infection in free-ranging Hermann s tortoises (Testudo hermanni), and in potential pet vectors AO_SCPLOWBSTRACTC_SCPLOWTwo types of pathogens cause highly contagious upper respiratory tract diseases (URTD) in Chelonians: testudinid herpesviruses (TeHV) and a mycoplasma (Mycoplasma agassizii). In captivity, these infections are frequent and can provoke outbreaks. Pet trade generates international flow of tortoises, often without sanitary checking; individuals intentionally or accidentally released in the wild may spread pathogens. A better understanding of the transmission of infectious agents from captivity to wild tortoises is needed. Many exotic individuals have been introduced in populations of the endangered western Hermanns tortoise (Testudo hermanni hermanni), notably spur-thighed tortoises (Testudo graeca). We assessed the presence of TeHV and mycoplasma in native western Hermanns tortoises and in potential pet vectors in south-eastern France. Using a large sample (N=572 tortoises), this study revealed, by PCR, the worrying presence of herpesvirus in 7 free-ranging individuals (3 sub-populations). Additionally, Mycoplasma agassizii was detected, by PCR, in 15 of the 18 populations sampled with a frequency ranging from 3.4% (1 of 29 tortoises) to 25% (3 of 12 tortoises). Exotic spur-thighed tortoises showed high frequency of mycoplasma infection in captivity (18.2%) and in individuals (50%) found in native Hermanns tortoise sub-populations, suggesting that this species could be a significant vector. The paucity of information of TeHV on European tortoise URTD in natural settings, especially in combination with mycoplasma, prompts for further studies. Indeed, sick tortoises remain concealed and may not be easily detected in the field. Our results indicate a good health for most infected tortoise but it should be screened in the field as well as in captivity. HSP90 modulates T2R bitter taste receptor nitric oxide production and innate immune responses in human airway epithelial cells and macrophages Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two airway innate immune cell types are activated by bitter products, including some secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells and increases phagocytosis by macrophages. Using biochemistry and live cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). We used immunofluorescence to show that H441 cells express eNOS and certain T2Rs and that the bitterant denatonium benzoate activates NO production in an HSP90-dependent manner in cells grown either as submerged cultures and at air liquid interface. In primary sinonasal epithelial cells, we determined that HSP-90 inhibition reduces T2R-stimulated NO production and ciliary beating which are crucial for pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves an innate immune role by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream calcium signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases like chronic rhinosinusitis, asthma, or cystic fibrosis. Admp regulates tail bending by controlling ventral epidermal cell polarity via phosphorylated myosin translocation. The tails transient but pronounced ventral bending is found in many chordate embryos and provides an interesting model of how tissue interactions contribute to the overall shape (Lu et al., 2020). Here, we identify one of the upstream regulators of tail bending. Admp knock-down embryo (Admp MO) completely inhibited ventral tail bending. Admp MO showed specific inhibition of the cell-cell intercalation ventral epidermis. In the early stages of the intercalation, each ventral epidermal cell exhibited a boat-shaped morphology (boat cell) with a narrow apical surface in which phosphorylated myosin (pMLC) accumulated. In the Admp MO, the number of the boat cells reduced and pMLC was observed at the basal side in the Admp MO embryo. Finally, laser cutter experiments showed a pMLC-dependent relaxation of the ventral tail bending, which was observed when each ventral midline epidermal cell was cut along the apico-basal axis. Based on these results, we propose a novel function for Admp, in which Admp controls the pMLC translocation to the apical side of the ventral epidermal cells and leads the tail bending by ventral resistance to the notochord extension.

Summary StatementAdmp is an upstream regulator of the tail bending in the protochordate Ciona tailbud embryo, regulating tissue polarity of the ventral midline epidermis by the translocation of the phosphorylated myosin.