Training-induced bioenergetic improvement in human skeletal muscle is associated with non-stoichiometric changes in the mitochondrial proteome without reorganization of respiratory chain content Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible and inexpensive therapeutic intervention improving mitochondrial bioenergetics and quality of life. By combining a multi-omics approach with biochemical and in silico normalization, we removed the bias arising from the training-induced increase in human skeletal muscle mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritized mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the increase in mitochondrial content. We demonstrate that enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and that training-induced supercomplex formation does not confer enhancements in mitochondrial bioenergetics. Our study provides a new analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding and calling for careful reinterpretation of previous findings.
Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies RNA binding motif protein 20 (RBM20) is a key regulator of alternative splicing in the heart, and its mutation leads to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous DCM-associated missense mutations in RBM20 (R636S) as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulated contractile dysfunction of RBM20-associated DCM and revealed greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP revealed a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing revealed that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO, impacting distinct cardiac signaling pathways. Splicing defects specific to KO or R636S mutations were supported by data from R636S gene-edited pig hearts and eCLIP. Super-resolution microscopy verified that mutant RBM20 maintains limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a novel pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways that are likely to mediate differential contractile phenotypes and stress-associated heart pathology.
Consumer risk perception towards pesticide stained tomatoes in Uganda. BackgroundTomatoes are consumeddaily. Unfortunately, abuse of pesticides application by vegetable growers in Uganda increases risks of exposing consumers through the pesticide residues, as it may be above European Union Maximum Residue Limits (used as a standard in Uganda). This study is aimed to determine consumer attitudes and risk perception towards pesticide stained tomatoes available on the Ugandan local markets.

MethodsA cross-sectional study sampled 468 household consumers in four districts one from each region of Uganda. In each district, 60 household members from three randomly selected Sub Counties were interviewed. In addition, in each district 9 tomato handlers (3 tomato farmers, 3 tomato retailers and 3 tomato wholesalers) participated in Focus Group Discussion (FGDs) perdistrict. Collected data was entered into MS-Excel 13 and exported into STATA SE 14.0 for cleaning and analysis at below a 5% level of significance and 95% Confidence Intervals (CI). Proportion of risk perceptions and attitudes were computed and presented as percentages while factors associated with risk perception were determined using Fisher exact test. Qualitative data was analyzed using deductive and inductive approaches under thematic content analysis.

ResultsMore than half, 54.2% (253/468), of the respondents were females, mean age was 37 years (SD=13.13, ranging from 18 to 88 years). Half of the respondents, 50.9% (238/467), were farmers by occupation and 40.3% (188/468) had completed upper primary education. Only 5.0% (20/396) of consumers reported a high risk perception towards tomatoes stained with pesticide residues, the rest, 95.0% (376/396), were buying pesticide stained tomatoes despite their awareness about the possible health effects. The main reason for buying the pesticide stained tomatoes was that a majority, 59.0% (230/390), lacked an alternative to stained tomatoes. However, consumers generally had a negative attitude towards pesticide stained tomatoes, with 67.0% (313/468) of the consumers disagreeing to a statement that tomatoes sold on the market are safe. Consumer risk perception was significantly associated with consumer awareness about residues in the tomatoes; where the proportino of consumers who were aware of the risk of pesticide stained tomatoes were 42.8 times more likely not to buy stained tomatoes compared to the proportion of those who were not aware. OR, 42.8 (95% CI: 10.76-170.28). However, level of education P(0.975), gender P(0.581) and age-group P(0.680) were not associated with consumer risk perception after Fisher-Exact tests analysis. (95% CI and 5% level of significance).

ConclusionConsumer risk perception on pesticide stained tomatoes among Ugandan consumers ranked low with a majority of consumers buying tomatoes stained with pesticide residuesdue to lack of an alternative, except for a few who had a high risk perception about the pesticide health effects. Loss of the DYRK1A protein kinase results in reduction of ribosomal protein genes expression, ribosome mass and translation defects Ribosomal proteins (RPs) are evolutionary conserved proteins that are essential for protein translation. RP expression must be tightly regulated to ensure the appropriate assembly of ribosomes and to respond to the growth demands of cells. The elements regulating the transcription of RP genes (RPGs) have been characterized in yeast and Drosophila, yet how cells regulate the production of RPs in mammals is less well understood. Here, we show that a subset of RPG promoters is characterized by the presence of the palindromic TCTCGCGAGA motif and marked by the recruitment of the protein kinase DYRK1A. The presence of DYRK1A at these promoters is associated with enhanced binding of the TATA-binding protein, TBP, and it is negatively correlated with the binding of the GABP transcription factor, establishing at least two clusters of RPGs that could be coordinately regulated. However, DYRK1A silencing leads to a global reduction of RPGs, mRNA pointing at DYRK1A activities beyond those dependent on its chromatin association. Significantly, cells in which DYRK1A is depleted have reduced RP levels, fewer ribosomes, reduced global protein synthesis and smaller size. We therefore propose a novel role for DYRK1A in coordinating the expression of genes encoding RPs, thereby controlling cell growth in mammals. A novel 5 prime DNA binding site in RFC facilitates PCNA loading for gap DNA repair RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases {delta} and {gamma}. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures revealing a novel 2nd DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located in the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. It appears 5' DNA binds after 3' DNA binding to RFC. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. We propose that the 5' site facilitates RFCs PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 50-nt gap, suggesting a potential role of the RFC 5' DNA site in longer gaps including during lagging strand DNA synthesis. Testing for phylogenetic signal in single-cell RNA-seq data AO_SCPLOWBSTRACTC_SCPLOWPhylogenetic methods are emerging as a useful tool to understand cancer evolutionary dynamics, including tumor structure, heterogeneity, and progression. Most currently used approaches utilize either bulk whole genome sequencing (WGS) or single-cell DNA sequencing (scDNA-seq) and are based on calling copy number alterations and single nucleotide variants (SNVs). Here we explore the potential of single-cell RNA sequencing (scRNA-seq) to reconstruct cancer evolutionary dynamics. scRNA-seq is commonly applied to explore differential gene expression of cancer cells throughout tumor progression. The method exacerbates the single-cell sequencing problem of low yield per cell with uneven expression levels. This accounts for low and uneven sequencing coverage and makes SNV detection and phylogenetic analysis challenging. In this paper, we demonstrate for the first time that scRNA-seq data contains sufficient evolutionary signal and can be utilized in phylogenetic analyses. We explore and compare results of such analyses based on both expression levels and SNVs called from scRNA-seq data. Both techniques are shown to be useful for reconstructing phylogenetic relationships between cells, reflecting the clonal composition of a tumor. Both standardized expression values and SNVs appear to be equally capable of reconstructing a similar pattern of phylogenetic relationship. This pattern is stable even when phylogenetic uncertainty is taken in account. Our results open up a new direction of somatic phylogenetics based on scRNA-seq data. Further research is required to refine and improve these approaches to capture the full picture of somatic evolutionary dynamics in cancer. Collective polymerase dynamics emerge from DNA supercoiling during transcription All biological processes ultimately come from physical interactions. The mechanical properties of DNA play a critical role in transcription. RNA polymerase can over or under twist DNA (referred to as DNA supercoiling) when it moves along a gene resulting in mechanical stresses in DNA that impact its own motion and that of other polymerases. For example, when enough supercoiling accumulates, an isolated polymerase halts and transcription stops. DNA supercoiling can also mediate non-local interactions between polymerases that shape gene expression fluctuations. Here, we construct a comprehensive model of transcription that captures how RNA polymerase motion changes the degree of DNA supercoiling which in turn feeds back into the rate at which polymerases are recruited and move along the DNA. Surprisingly, our model predicts that a group of three or more polymerases move together at a constant velocity and sustain their motion (forming what we call a polymeton) whereas one or two polymerases would have halted. We further show that accounting for the impact of DNA supercoiling on both RNA polymerase recruitment and velocity recapitulates empirical observations of gene expression fluctuations. Finally, we propose a mechanical toggle switch whereby interactions between genes are mediated by DNA twisting as opposed to proteins. Understanding the mechanical regulation of gene expression provides new insights into how endogenous genes can interact and informs the design of new forms of engineered interactions.

PACS numbers: Design, immunogenicity and efficacy of a Pan-SARS-CoV-2 synthetic DNA vaccine Here we have employed SynCon(R) design technology to construct a DNA vaccine expressing a pan-Spike immunogen (INO-4802) to induce broad immunity across SARS-CoV-2 variants of concern (VOC). Compared to WT and VOC-matched vaccines which showed reduced cross-neutralizing activity, INO-4802 induced potent neutralizing antibodies and T cell responses against WT as well as B.1.1.7, P.1, and B.1.351 VOCs in a murine model. In addition, a hamster challenge model demonstrated that INO-4802 conferred superior protection following intranasal B.1.351 challenge. Protection against weight loss associated with WT, B.1.1.7, P.1 and B.1.617.2 challenge was also demonstrated. Vaccinated hamsters showed enhanced humoral responses against VOC in a heterologous WT vaccine prime and INO-4802 boost setting. These results demonstrate the potential of the pan-SARS-CoV-2 vaccine, INO-4802 to induce cross-reactive immune responses against emerging VOC as either a standalone vaccine, or as a potential boost for individuals previously immunized with WT-matched vaccines. Rapid evolution of seed dormancy during sunflower de-domestication Hybridization between crops and their wild relatives may promote the evolution of de-domesticated (feral) weeds. Wild sunflower (Helianthus annuus L.) is typically found in ruderal environments, but crop-wild hybridization may facilitate the evolution of weedy populations. Using one crop-specific mitochondrial marker (CMS-PET1) and 14 nuclear SSR markers, we studied the origin and genetic diversity of a recently discovered weedy population of sunflower (named BRW). Then, using a resurrection approach, we tested for rapid evolution of weedy traits (seed dormancy, herbicide resistance, and competitive ability) by sampling weedy and wild populations 10 years apart (2007 and 2017). All the weedy plants present the CMS-PET1 cytotype, confirming their feral origin. At the nuclear markers, BRW showed higher genetic diversity than the cultivated lines and low differentiation with one wild population, suggesting that wild hybridization increased their genetic diversity. We found support for rapid evolution towards higher seed dormancy, but not for higher competitive ability or herbicide resistance. Our results highlight the importance of seed dormancy during the earliest stages of adaptation and show that crop-wild hybrids can evolve quickly in agricultural environments. Leveraging a surrogate outcome to improve inference on a partially missing target outcome Sample sizes vary substantially across tissues in the Genotype-Tissue Expression (GTEx) project, where considerably fewer samples are available from certain inaccessible tissues, such as the substantia nigra (SSN), than from accessible tissues, such as blood. This severely limits power for identifying tissue-specific expression quantitative trait loci (eQTL) in undersampled tissues. Here we propose Surrogate Phenotype Regression Analysis (SO_SCPLOWPRAYC_SCPLOW) for leveraging information from a correlated surrogate outcome (e.g. expression in blood) to improve inference on a partially missing target outcome (e.g. expression in SSN). Rather than regarding the surrogate outcome as a proxy for the target outcome, SO_SCPLOWPRAYC_SCPLOW jointly models the target and surrogate outcomes within a bivariate regression framework. Unobserved values of either outcome are treated as missing data. We describe and implement an expectation conditional maximization algorithm for performing estimation in the presence of bilateral outcome missingness. SO_SCPLOWPRAYC_SCPLOW estimates the same association parameter estimated by standard eQTL mapping and controls the type I error even when the target and surrogate outcomes are truly uncorrelated. We demonstrate analytically and empirically, using simulations and GTEx data, that in comparison with marginally modeling the target outcome, jointly modeling the target and surrogate outcomes increases estimation precision and improves power. The obstacles and potential clues of prime editing applications in tomato, a dicot plant Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement. Cell Cycle Delay, Pro-metaphase Arrest and C-metaphase Inducing Effects of Petroleum Ether Fraction of Leaf Aqueous Extract of Clerodendrum viscosum Vent. Clerodendrum viscosum is a traditionally used medicinal plant. The present study aimed to analyze cell cycle delay, pro-metaphase arrest, and c-metaphase inducing effects of the petroleum ether fraction (AQPEF) of leaf aqueous extract of C. Viscosum (LAECV). The LAECV was fractionated with petroleum ether and its metaphase arrest, cell cycle delay, and c-metaphase inducing activities were tested on A. cepa root tip cells. The AQPEF induced cell cycle delay, and colchicine like metaphase, c-metaphase, in A. cepa root tip cells. Thus, the present study explores AQPEF as an active fraction of LAECV having metaphase arresting, cell cycle delay, and c-metaphase inducing potentials. A northern range extension of a Canadian species of Special Concern, Dielis pilipes (Hymenoptera: Scoliidae), in the Okanagan Valley of British Columbia The only known Canadian records of the yellow scarab hunter wasp, Dielis pilipes (Saussure) (Hymenoptera: Scoliidae), are from the southern Okanagan and Similkameen valleys of British Columbia. We report a 25-km northern range extension of the species into the Ponderosa Pine Biogeoclimatic Ecosystem Classification zone, collected in an unmanaged agricultural field in Summerland, British Columbia. This finding is of conservation importance and has implications for natural biological control of ten-lined June beetles, Polyphylla decemlineata (Say) and P. crinita LeConte (Coleoptera: Scarabaeidae), incidental agricultural pests in the Okanagan. Pax6 loss alters the morphological and electrophysiological development of mouse prethalamic neurons Pax6 is a well-known regulator of early neuroepithelial progenitor development. Its constitutive loss has a particularly strong effect on the developing prethalamus, causing it to become extremely hypoplastic. To overcome this difficulty in studying the long-term consequences of Pax6 loss for prethalamic development, we used conditional mutagenesis to delete Pax6 at the onset of neurogenesis and studied the developmental potential of the mutant prethalamic neurons in vitro. We found that Pax6 loss affected their rates of neurite elongation, the location and length of their axon initial segments and their electrophysiological properties. Our results broaden our understanding of the long-term consequences of Pax6 deletion in the developing mouse forebrain, suggesting that it can have cell autonomous effects on the structural and functional development of some neurons.

SUMMARY STATEMENTPax6 impacts neurite extension, axon initial segment properties and ability to fire normal action potentials in maturing neurons, revealing actions extending beyond those previously characterized in progenitors. ALLOPURINOL BLOCKS THE AORTIC ANEURYSM IN A MOUSE MODEL OF MARFAN SYNDROME VIA REDUCING AORTIC OXIDATIVE STRESS BackgroundThe pathogenesis and progression of aortic aneurysm in Marfan syndrome (MFS) involves dysregulated TGF-{beta} and nitric oxide signaling, altered hemodynamics, and biomechanical forces. Increasing evidence indicates that redox stress participates in MFS aortopathy, though its contribution is not well established. We reported elevated reactive oxygen species (ROS) formation and NADPH oxidase NOX4 upregulation in MFS patients and mice aortic samples. Here we address the contribution of xanthine oxidoreductase (XOR), which is transcribed as dehydrogenase (XDH) and then post-translationally modified to oxidase (XO), catabolizing mainly purines into uric acid and ROS.

Methods and ResultsWe report that in MFS patients, XOR protein levels increased in aortic samples. In MFS mice (Fbn1C1041G/+), XOR mRNA transcripts augmented in the aorta of young but not older MFS mice. Moreover, the enzymatic activity of XO form increased with respect to XDH. The administration of the XOR inhibitor allopurinol (ALO) blocked the progression of aortic root aneurysm in MFS mice. ALO was also protective when administrated before the appearance of aneurysm. MFS-induced elastic fiber fragmentation, fibrotic remodeling, nuclear translocation of pNRF2, and increased 3-nitrotyrosine levels in the aortic tunica media as well as endothelial dysfunction were all prevented by allopurinol. The large overproduction of H2O2 in MFS aorta did not occur in allopurinol-treated mice whose uric acid plasma levels remained unaltered.

ConclusionsThis study strengthens the concept that redox stress is an important determinant of aortic aneurysm formation and progression in MFS and supports a clinical trial for ALO in the pharmacological treatment of MFS aortopathy.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/464182v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@eab02aorg.highwire.dtl.DTLVardef@1d3b39eorg.highwire.dtl.DTLVardef@1b54d0forg.highwire.dtl.DTLVardef@1d61ad0_HPS_FORMAT_FIGEXP  M_FIG C_FIG Computationally scalable regression modeling for ultrahigh-dimensional omics data with ParProx Statistical analysis of ultrahigh-dimensional omics scale data has long depended on univariate hypothesis testing. With growing data features and samples, the obvious next step is to establish multivariable association analysis as a routine method for understanding genotype-phenotype associations. Here we present ParProx, a state-of-the-art implementation to optimize overlapping group lasso regression models for time-to-event and classification analysis, guided by biological priors through coordinated variable selection. ParProx not only enables model fitting for ultrahigh-dimensional data within the architecture for parallel or distributed computing, but also allows users to obtain interpretable regression models consistent with known biological relationships among the independent variables, a feature long neglected in statistical modeling of omics data. We demonstrate ParProx using three different omics data sets of moderate to large numbers of variables, where we use genomic regions and pathways to arrive at sparse regression models comprised of biologically related independent variables. ParProx is naturally applicable to a wide range of studies using ultrahigh-dimensional omics data, ranging from genome-wide association analysis to single cell sequencing studies where multivariable modeling is computationally intractable. Comparison of hybrid learning and remote education in the implementation of the 'Adopt a Microorganism' methodology The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. Construction of a new chromosome-scale, long-read reference genome assembly for the Syrian hamster, Mesocricetus auratus BackgroundThe Syrian hamster (Mesocricetus auratus) has been suggested as a useful mammalian model for a variety of diseases and infections, including infection with respiratory viruses such as SARS-CoV-2. The MesAur1.0 genome assembly was generated in 2013 using whole-genome shotgun sequencing with short-read sequence data. Current more advanced sequencing technologies and assembly methods now permit the generation of near-complete genome assemblies with higher quality and greater continuity.

FindingsHere, we report an improved assembly of the M. auratus genome (BCM_Maur_2.0) using Oxford Nanopore Technologies long-read sequencing to produce a chromosome-scale assembly. The total length of the new assembly is 2.46 Gbp, similar to the 2.50 Gbp length of a previous assembly of this genome, MesAur1.0. BCM_Maur_2.0 exhibits significantly improved continuity with a scaffold N50 that is 6.7 times greater than MesAur1.0. Furthermore, 21,616 protein coding genes and 10,459 noncoding genes are annotated in BCM_Maur_2.0 compared to 20,495 protein coding genes and 4,168 noncoding genes in MesAur1.0. This new assembly also improves the unresolved regions as measured by nucleotide ambiguities, where approximately 17.11% of bases in MesAur1.0 were unresolved compared to BCM_Maur_2.0 in which the number of unresolved bases is reduced to 3.00%.

ConclusionsAccess to a more complete reference genome with improved accuracy and continuity will facilitate more detailed, comprehensive, and meaningful research results for a wide variety of future studies using Syrian hamsters as models. Updated vaccine protects from infection with SARS-CoV-2 variants, prevents transmission and is immunogenic against Omicron in hamsters Current first-generation COVID-19 vaccines are based on prototypic spike sequences from ancestral 2019 SARS-CoV-2 strains. However, the ongoing pandemic is fueled by variants of concern (VOC) that threaten to escape vaccine-mediated protection. Here we show in a stringent hamster model that immunization using prototypic spike expressed from a potent YF17D viral vector (1) provides vigorous protection against infection with ancestral virus (B lineage) and VOC Alpha (B.1.1.7), however, is insufficient to provide maximum protection against the Beta (B.1.351) variant. To improve vaccine efficacy, we created a revised vaccine candidate that carries an evolved spike antigen. Vaccination of hamsters with this updated vaccine candidate provides full protection against intranasal challenge with all four VOCs Alpha, Beta, Gamma (P.1) and Delta (B.1.617.2) resulting in complete elimination of infectious virus from the lungs and a marked improvement in lung pathology. Vaccinated hamsters did also no longer transmit the Delta variant to non-vaccinated sentinels. Hamsters immunized with our modified vaccine candidate also mounted marked neutralizing antibody responses against the recently emerged Omicron (B.1.1.529) variant, whereas the old vaccine employing prototypic spike failed to induce immunity to this antigenically distant virus. Overall, our data indicate that current first-generation COVID-19 vaccines need to be urgently updated to cover newly emerging VOCs to maintain vaccine efficacy and to impede virus spread at the community level.

Significance StatementSARS-CoV-2 keeps mutating rapidly, and the ongoing COVID-19 pandemic is fueled by new variants escaping immunity induced by current first-generation vaccines. There is hence an urgent need for universal vaccines that cover variants of concern (VOC). In this paper we show that an adapted version of our vaccine candidate YF-S0* provides full protection from infection, virus transmission and disease by VOCs Alpha, Beta, Gamma and Delta, and also results in markedly increased levels of neutralizing antibodies against recently emerged Omicron VOC in a stringent hamster model. Our findings underline the necessity to update COVID-19 vaccines to curb the pandemic, providing experimental proof on how to maintain vaccine efficacy in view of an evolving SARS-CoV-2 diversity. Artificial seawater based long-term culture of colonial ascidians Tunicates are highly diverse marine invertebrate filter-feeders that are vertebrates closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually by budding, typically leading to the formation of colonies where animals, called zooids, are interconnected through an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions.

To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates.

Herein we present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 km away from their natural habitat. We show that colonies adapt well to in-lab culturing provided that a suitable marine microbiome is present, and that a specific strain can be isolated, propagated and efficiently used for research over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species, such as Botryllus schlosseri, as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.

HighlightsO_LIFirst artificial seawater based recirculating aquaculture for colonial ascidians
C_LIO_LIOver 3 years of continuous breeding
C_LIO_LISemi-automated setup with minimized maintenance
C_LIO_LIGood biomass production for strain propagation
C_LIO_LI4 different culture conditions for optimized breeding for species of interest
C_LI Exosomal TAR DNA binding protein 43 profile in canine model of amyotrophic lateral sclerosis: A preliminary study in developing blood-based biomarker for neurodegenerative diseases ObjectiveBlood-based biomarkers provide a crucial information in progress of neurodegenerative diseases with minimally invasive sampling method. Validated blood-based biomarker application in people with amyotrophic lateral sclerosis would derive numerous benefits. Canine degenerative myelopathy is a naturally occurring animal disease model to study the biology of human amyotrophic lateral sclerosis. Serum derived exosomes are potential carriers for cell-specific cargoes making them ideal venue to study biomarkers for a variety of diseases and biological processes. This study assessed the exosomal proteins that may be assigned as surrogate biomarker that may reflect biochemical changes in central nervous system.

MethodsExosomes were isolated from canine serum using commercial exosome isolation reagents. Exosomes target proteins contents were analysed by Western blotting method.

ResultsThe profiles of potential biomarker candidates in spinal cord homogenate and that of serum-derived exosomes were found elevated in dogs with degenerative myelopathy as compare to control subjects.

ConclusionsSerum-derived exosomal biomolecules can serve as surrogate biomarkers in neuro degenerative diseases.

Key MessagesO_LIA canine with degenerative myelopathy can serve as a model animal to study human amyotrophic lateral sclerosis.
C_LIO_LISerum-derived exosomes contains Transactive Response DNA Binding Protein 43 (TDP-43), potential biomarker candidate.
C_LIO_LIThe levels of spinal cord TDP-43 proteins and that of serum-derived exosomes exhibited a similar profiling. Therefore, serum derived exosomes may be used as a venue for establishing blood-based biomarkers for neurodegenerative diseases.
C_LI Electrical Stimulated GLUT4 Signaling Attenuates Critical Illness-Associated Muscle Wasting BackgroundCritical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a byproduct of critical care, attributed to impaired recovery, long-term complications, and mortality. CIM pathophysiology is complex, heterogeneous and remains incompletely understood, however loss of mechanical stimuli contributes to critical illness associated muscle atrophy and weakness. Passive mechanical loading (ML) and electrical stimulation (ES) therapies augment muscle mass and function. While having beneficial outcomes, the mechanistic underpinning of these therapies is less known. Therefore, here we aimed to assess the mechanism by which chronic supramaximal ES ameliorates CIM in a unique experimental rat model of critical care.

MethodsRats were subjected to 8 days critical care conditions entailing deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES. Muscle size and function were assessed at the single cell level. RNAseq and Western blotting were employed to understand the mechanisms driving ES muscle outcomes in CIM.

ResultsFollowing 8 days of controlled mechanical ventilation and immobilization, soleus muscle mass, Myosin:Actin ratio and single muscle fiber maximum force normalized to cross-sectional area (specific force) were reduced by 40-50% (p< 0.0001). ES significantly reduced the loss of soleus muscle fiber cross-sectional area (CSA) and Myosin:Actin ratio by approximately 30% (p< 0.05) yet failed to effect specific force. RNAseq pathway analysis revealed downregulation of insulin signaling in the soleus muscle following critical care and GLUT4 trafficking was reduced by 55% leading to an 85% reduction of muscle glycogen content (p< 0.01). ES promoted phosphofructokinase and insulin signaling pathways to control levels (p< 0.05), consistent with the maintenance of GLUT4 translocation and glycogen levels. AMPK, but not AKT, signaling pathway was stimulated following ES, where the downstream target TBC1D4 increased 3 logFC (p= 0.029) and AMPK-specific P-TBC1D4 levels were increased approximately 2-fold (p= 0.06). Reduction of muscle protein degradation rather than protein synthesis promoted soleus CSA, as ES reduced E3 ubiquitin proteins, Atrogin-1 (p= 0.006) and MuRF1 (p= 0.08) by approximately 50%, downstream of AMPK-FoxO3.

ConclusionsES maintained GLUT4 translocation through increased AMPK-TBC1D4 signaling leading to improved muscle glucose homeostasis. Soleus CSA and myosin content was promoted through reduced protein degradation via AMPK-FoxO3 E3 ligases, Atrogin-1 and MuRF1. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signaling and reduced muscle protein degradation in CIM. 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. Ecological interactions in breast cancer: Cell facilitation promotes growth and survival under drug pressure The interplay of positive and negative interactions between drug sensitive and resistant cells determines the efficacy of treatment in heterogeneous cancer cell populations. We developed isogenic estrogen receptor positive (ER+) breast cancer cell lineages sensitive and resistant to ribociclib-induced CDK4/6 inhibition to investigate cell interactions under selective pressure. During treatment with ribociclib alone or in combination with endocrine therapies, sensitive cells survive and proliferate better when grown together with resistant cells than when grown in monoculture. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays detected estrone, a less active metabolite, in medium taken from each lineage but detected highly active estradiol only from resistant cells. Because sensitive cells are more dependent on estrogen signaling than resistant cells, they benefit from the increase in local estradiol from resistant cells, leading to facilitated growth when grown under treatment in coculture. We developed a suite of mathematical models to quantify the strength of competition and facilitation between cell lines and evaluate the effects of treatments that modify these interactions. These analyses support the conclusion that resistant cell lineages facilitate growth of sensitive lineages during therapy through the increased production and sharing of local estradiol and themselves suffer from increased competition and suppression as a result. These models show that blocking facilitation has the potential to control both resistant and sensitive cell populations and inhibit the emergence of a refractory population. This combination of experiments and models shows that interactions within heterogeneous cancer cell populations can combine positive and negative interactions to shape the dynamics of resistance. Soluble ECM promotes organotypic formation in lung alveolar model Micropatterned suspension culture creates consistently sized and shaped cell aggregates but has not produced organotypic structures from stable cells, thus restricting its use in accurate disease modeling. Here, we show that organotypic structure is achieved in hybrid suspension culture via supplementation of soluble extracellular matrix (ECM). We created a viable lung organoid from epithelial, endothelial, and fibroblast human stable cell lines in suspension culture. We demonstrate the importance of soluble ECM in organotypic patterning with the emergence of lumen-like structures with airspace showing feasible gas exchange units, formation of branching, perfusable vasculature, and long-term 70-day maintenance of lumen structure. Our results show a dependent relationship between enhanced fibronectin fibril assembly and the incorporation of ECM in the organoid. We successfully applied this technology in modeling lung fibrosis via bleomycin induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell interactions in lung tissue. Our human fluorescent lung organoid (hFLO) model represents features of pulmonary fibrosis which were ameliorated by fasudil treatment. We also demonstrate a 3D culture method with potential of creating organoids from mature cells, thus opening avenues for disease modeling and regenerative medicine, enhancing understanding of lung cell biology in health and lung disease. Asymmetrical dose-responses shape the evolutionary trade-off between antifungal resistance and nutrient use Antimicrobial resistance is an emerging threat for public health. The success of resistance mutations depends on the trade-off between the benefits and costs they incur. This trade-off is largely unknown and uncharacterized for antifungals. Here, we systematically measure the effect of all amino acid substitutions in the yeast cytosine deaminase Fcy1, the target of the antifungal 5-FC (flucytosine). We identify over 900 missense mutations granting resistance to 5-FC, a large fraction of which appear to act through destabilisation of the protein. The relationship between 5-FC resistance and growth sustained by cytosine deamination is characterized by a sharp trade-off, such that small gains in resistance universally lead to large losses in canonical enzyme function. We show that this steep relationship can be explained by differences in the dose-response functions of 5-FC and cytosine. Finally, we observe the same trade-off shape for the ortholog of FCY1 in Cryptoccocus neoformans, a human pathogen. Our results provide a powerful resource and platform for interpreting drug target variants in fungal pathogens as well as unprecedented insights into resistance-function trade-offs.