Somewhere I belong: phylogenetic comparative methods and machine learning to investigate the evolution of a species-rich lineage of parasites AO_SCPLOWBSTRACTC_SCPLOWA substantial portion of biodiversity evolved through adaptive radiation. However, the effects of explosive speciation on species interactions remain poorly understood. Metazoan parasites infecting radiating host lineages could improve our knowledge because of their intimate host relationships. Yet limited molecular, phenotypic, and ecological data discourage multivariate analyses of evolutionary patterns and encourage the use of discrete characters. Here, we assemble new molecular, morphological, and host range data widely inferred from a species-rich lineage of parasites (Cichlidogyrus, Platyhelminthes: Monogenea) infecting cichlid fishes to address data scarcity. We infer a multi-marker (28S/18S rDNA, ITS1, COI mtDNA) phylogeny of 58/137 species and characterise major lineages through synapomorphies inferred from mapping morphological characters. We predict the phylogenetic position of species without DNA data through shared character states, a combined molecular-morphological phylogenetic analysis, and a classification analysis with support vector machines. Based on these predictions and a cluster analysis, we assess the systematic informativeness of continuous characters, search for continuous equivalents for discrete characters, and suggest new characters for morphological traits not analysed to date. We also model the attachment/reproductive organ and host range evolution using the data of 136/137 described species and multivariate phylogenetic comparative methods (PCMs). We show that discrete characters can mask phylogenetic signals but can be key for characterising species groups. Regarding the attachment organ morphology, a divergent evolutionary regime for at least one lineage was detected and a limited morphological variation indicates host and environmental parameters affecting its evolution. However, moderate success in predicting phylogenetic positions, and a low systematic informativeness and high multicollinearity of morphological characters call for a revaluation of characters included in species characterisations.
Analysis of diverse eukaryotes suggests the existence of an ancestral 1 mitochondrial apparatus derived from the bacterial type II secretion system The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.
Probing effects of the SARS-CoV-2 E protein on membrane curvature and intracellular calcium SARS-CoV-2 contains four structural proteins in its genome. These proteins aid in the assembly and budding of new virions at the ER-Golgi intermediate compartment (ERGIC). Current fundamental research efforts largely focus on one of these proteins - the spike (S) protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other structural proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length envelope (E) protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening. Co-infection of Candidatus Piscichlamydia trichopodus (order Chlamydiales) and Henneguya sp (Myxosporea, Myxobolidae) in snakeskin gourami Trichopodus pectoralis (Regan 1910) The present study describes a simultaneous infection of a novel Chlamydia-like organism (CLO) with a Myxozoa parasite, Henneguya sp. in snakeskin gourami Trichopodus pectoralis in Thailand. A new CLO is proposed "Candidatus Piscichlamydia trichopodus" (CPT) based on 16S rRNA phylogenetic analysis. Systemic intracellular CPT infection was confirmed by histological examination, in situ hybridization, PCR assay, and sequencing of 16S rRNA. This novel pathogen belongs to the order Chlamydiales but differs in certain aspects from other species. The histopathological changes associated with CPT infection were different from the typical pathological lesions of epitheliocystis caused by previously known CLO. Unlike other CLO, CPT localized in the connective tissue rather than in the epithelial cells and formed smaller clumps of intracellular bacteria that stained dark blue with hematoxylin. On the other hand, typical myxospores of the genus Henneguya with tails were observed in the gill sections. Infection with Henneguya sp. resulted in extensive destruction of the gill filaments, most likely leading to respiratory distress. Due to the frequency of co-infections and the unavailability of culture methods for CLO and Henneguya sp, it was difficult to determine which pathogens were directly responsible for the associated mortality. However, co-infections may increase the negative impact on the host and the severity of the disease. Given the commercial importance of the snakeskin gourami and its significant aquaculture potential, the findings of this study are important for further studies on disease prevention. Designing viral diagnostics with model-based optimization Diagnostics, particularly for rapidly evolving viruses, stand to benefit from a principled, measurement-driven design that harnesses machine learning and vast genomic data--yet the capability for such design has not been previously built. Here, we develop and extensively validate an approach to designing viral diagnostics that applies a learned model within a combinatorial optimization framework. Concentrating on CRISPR-based diagnostics, we screen a library of 19,209 diagnostic-target pairs and train a deep neural network that predicts, from RNA sequence alone, diagnostic signal better than contemporary techniques. Our model then makes it possible to design assays that are maximally sensitive over the spectrum of a viruss genomic variation. We introduce ADAPT (https://adapt.guide), a system for fully-automated design, and use ADAPT to design optimal diagnostics for the 1,933 vertebrate-infecting viral species within 2 hours for most species and 24 hours for all but 3. We experimentally show ADAPTs designs are sensitive and specific down to the lineage level, including against viruses that pose challenges involving genomic variation and specificity. ADAPTs designs exhibit significantly higher fluorescence and permit lower limits of detection, across a viruss entire variation, than the outputs of standard design techniques. Our model-based optimization strategy has applications broadly to viral nucleic acid diagnostics and other sequence-based technologies, and, paired with clinical validation, could enable a critically-needed, proactive resource of assays for surveilling and responding to pathogens. IL-21 signaling promotes the establishment of KSHV infection in human tonsil lymphocytes by increasing early targeting of plasma cells Factors influencing Kaposis sarcoma-associated herpesvirus (KSHV) transmission and the early stages of KSHV infection in the human immune system remain poorly characterized. KSHV is known to extensively manipulate the host immune system and the cytokine milieu, and cytokines are known to influence the progression of KSHV-associated diseases. Here, using our unique model of KSHV infection in tonsil lymphocytes, we investigate the influence of host cytokines on the establishment of KSHV infection in human B cells. Our data demonstrate that KSHV manipulates the host cytokine microenvironment during early infection and susceptibility is generally associated with downregulation of multiple cytokines. However, we show that IL-21 signaling promotes KSHV infection by promoting both plasma cell numbers and increasing KSHV infection in plasma cells as early as 3 days post-infection. Our data reveal that this phenotype is dependent upon a specific milieu of T cells, that includes IL-21 producing Th17, Tc17 and CD8+ central memory T cells. These results suggest that IL-21 plays a significant role in the early stages of KSHV infection in the human immune system and that specific immunological states favor the initial establishment of KSHV infection by increasing infection in plasma cells. Embryonic exposure to cannabidiol disrupts active neural circuits, an effect increased by Δ-9-tetrahydrocannabinol and involving CB1R and CB2R in zebrafish In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development. "Neighbourhood watch" model: embryonic epiblast cells assess positional information in relation to their neighbours In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name "neighbourhood watch" model) illuminates the classic "French Flag Problem" and how positional information is interpreted by a sheet of cells in a large developing system.

Summary statementIn a large developing system, the chick embryo before gastrulation, cells interpret gradients of positional signals relative to their neighbours to position the primitive streak, establishing bilateral symmetry. 3D Reconstruction of Bird Flight Trajectories Using a Single Video Camera Video cameras are finding increasing use in the study and analysis of bird flight over short ranges. However, reconstruction of flight trajectories in three dimensions typically requires the use of multiple cameras and elaborate calibration procedures. We present an alternative approach that uses a single video camera and a simple calibration procedure for the reconstruction of such trajectories. The technique combines prior knowledge of the wingspan of the bird with a camera calibration procedure that needs to be used only once in the lifetime of the system. The system delivers the exact 3D coordinates of the position of the bird at the time of every full wing extension and uses interpolated height estimates to compute the 3D positions of the bird in the video frames between successive wing extensions. The system is inexpensive, compact and portable, and can be easily deployed in the laboratory as well as the field. 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. Depletion of Sun1/2 induces heterochromatin accrual in mesenchymal stem cells during adipogenesis Critical to the mechano-regulation of mesenchymal stem cells (MSC), Linker of the Nucleoskeleton and Cytoskeleton (LINC) complex transduces cytoskeletal forces to the nuclei. The LINC complex contains outer nuclear membrane Nesprin proteins that associate with the cytoskeleton and their inner nuclear membrane couplers, Sun proteins. In addition to coupling Nesprin-associated cytoskeletal elements to inner nuclear membrane, Sun proteins also function in regulating nuclear mechanics and chromatin tethering to inner nuclear membrane. This suggests that release of LINC-mediated cytoskeletal connections from cell nuclei may have different effects on chromatin organization and MSC differentiation than those due to ablation of intra-nuclear Sun proteins. To test this hypothesis we compared Sun1/2 depletion with expression of the dominant-negative KASH domain (dnKASH) that inhibits Nesprin-Sun association. In cells cultured under adipogenic conditions, disconnecting LINC from the cytoskeleton through dnKASH expression, increased adipogenic gene expression and fat droplet formation; heterochromatin H3K27 and H3K9 methylation was unaltered. In contrast, Sun1/2 depletion inhibited adipogenic gene expression and fat droplet formation; as well the anti-adipogenic effect of Sun1/2 depletion was accompanied by increased H3K9me3, which was enriched on Adipoq, silencing this fat locus. We conclude that releasing the nucleus from cytoskeletal constraints via dnKASH accelerates adipogenesis while depletion of Sun1/2 increases heterochromatin accrual on adipogenic genes in a fashion independent of LINC complex function. Therefore, while these two approaches both disable LINC functions, their divergent effects on the epigenetic landscape indicate they cannot be used interchangeably to study mechanical regulation of cell differentiation. Liberation from fibrogenesis or tumorigenesis via cellular senescence by a novel small molecule BackgroundUncontrolled proliferative diseases such as fibrosis or cancer can be fatal. We previously found that a compound containing a chromone scaffold, ONG41008, had potent anti-fibrotic effects in diseased human lung myofibroblasts but not normal human lung fibroblasts.

MethodsWe investigated the effects of ONG41008 on tumor cells, and compared these effects with those in pathogenic myofibrotic cells and normal fibroblasts cells.

FindingsStimulation of A549 lung carcinoma epithelial cells with ONG41008 resulted in cellular senescence, indicating that dysregulated cell proliferation is common to fibrotic cells and tumor cells. Replicative senescence of A549 cells resulted in multinucleation, which was followed by oncogene-induced senescence. There was significant upregulation of expression and nuclear translocation of p-TP53 and p16 in ONG41008-treated A549 cells, and all cells died after 72 hr. Similar effects occurred after ONG41008 treatment in several human aggressive cancer cell lines such as PANC1, MCF7, PC3, or primary non-small cell lung carcinoma cells. Unlike cisplatin, ONG41008 was not toxic to normal human lung fibroblasts or primary prostate epithelial cells, suggesting ONG41008 can distinguish the intracellular microenvironment between normal cells and aged or diseased cells. This effect might occur as a result of the increased NAD/NADH ratio or increased lactate dehydrogenase levels in aged or diseased cells.

InterpretationTo our best knowledge, this is the first study to show that a small molecule can arrest uncontrolled proliferation during fibrogenesis or tumorigenesis in vitro. ONG41008 could be a potential drug for a broad range of fibrotic or tumorigenic diseases.

Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSThe notion that aging is a disease and that diseases occur as a consequence of aging was first put forward by David Sinclair and colleagues (Aging Cell;14(4):497-510). There are biological systems that provide evidence for this notion; for example, somatic cells can revert to embryonic cells, producing younger somatic cells. This phenomenon underlies induced pluripotent stem cells. Another example is that some types of jellyfish can live forever. These examples suggest that a counter-aging program exists in animals. Human diseases are the manifestations of cell aging generated by the accumulation of somatic mutations. Aged and pathogenic cells are senescent, so a drug that specifically targeted senescent cells might initiate a cellular program that could ameliorate age-associated disease. Indeed, the kinase inhibitor dasatinib induces cellular senescence (Clin Ther 2007 29:2289-2308). In 2017, two drugs that target senescent cells were identified: quercetin and fisetin. These drugs selectively kill senescent cells, and are referred to as senolytic drugs or senotherapeutics (Aging 2017 8;9(3):955-963). Although it is established that senescent cells accumulate in cancer and idiopathic pulmonary fibrosis (Nat Commun 2017 23;8 :14532), the effect of senolytic drugs in these diseases is largely unknown.

Added value of this studyThis study characterized a novel drug, termed ONG41008, which was found to have both senogenic and senolytic effects in cell-based assays. ONG41008 induced senescence in myofibroblasts and several cancer cell lines representative of aggressive human cancers, which was followed by cell death. Importantly, ONG41008 exhibited essentially no toxicity on normal human lung fibroblasts or primary prostate epithelial cells.

Implication of all the evidenceBased on our results, we believe that ONG41008 is a potent inducer of cellular senescence (replicative senescence and oncogene-induced senescence) and causes arrest of uncontrolled, pathogenic proliferation of myofibroblasts or cancer cells. De-noising Spatial Transcriptomics Data Based on Position and Image Information Spatial transcriptomics (ST) technologies provide gene expression close to or even superior to single-cell resolution while retaining the physical locations of sequencing and often also providing matched pathology images. However, the expression data captured by ST technologies suffer from high noise levels, as a result of the shallow coverage in each sequencing unit. The extra experimental steps for preserving the spatial locations of sequencing could result in even more severe noises, compared to regular single-cell RNA-sequencing (scRNA-seq). Fortunately, such noises could be largely removed by leveraging information from the physical locations of sequencing, and the tissue and cellular organization reflected by corresponding pathology images. In this work, we demonstrated the extensive levels of noise in ST data. We developed a mathematical model, named Sprod, to impute accurate ST gene expression based on latent space and graph learning of matched location and imaging data. We comprehensively validated Sprod and demonstrated its advantages over prior methods for removing drop-outs in scRNA-seq data. We further showed that, after adequate imputation by Sprod, differential expression analyses, pseudotime analyses, and cell-to-cell interaction inferences yield significantly more informative results. Overall, we envision denoising by Sprod to become a key first step to empower ST technologies for biomedical discoveries and innovations. 2,3-cAMP treatment mimics stress molecular response in Arabidopsis thaliana The role of the RNA degradation product 2,3-cyclic adenosine monophosphate (2,3-cAMP) is poorly understood. Recent studies have identified 2,3-cAMP in plant material and determined its role in stress signaling. The level of 2,3-cAMP increases upon wounding, dark, and heat, and 2,3-cAMP by binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insight into 2,3-cAMP function, we used a multi-omics approach combining transcriptomics, metabolomics, and proteomics to dissect Arabidopsis response to 2,3-cAMP treatment. We demonstrated that 2,3-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide-adenosine pathway from human cells might also exist in plants. Transcriptomic analysis revealed only minor overlap between 2,3-cAMP-and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2,3-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses including protein and RNA degradation products, glucosinolates, chaperones and SG components. Finally, we demonstrated that 2,3-cAMP treatment influences the movement of processing bodies, supporting the role of 2,3-cAMP in the formation and motility of membraneless organelles. Cytosolic aggregation of mitochondrial proteins disrupts cellular homeostasis by stimulating the aggregation of other proteins Mitochondria are organelles with their own genomes, but they rely on the import of nuclear-encoded proteins that are translated by cytosolic ribosomes. Therefore, it is important to understand whether failures in the mitochondrial uptake of these nuclear-encoded proteins can cause proteotoxic stress and identify response mechanisms that may counteract it. Here, we report that upon impairments in mitochondrial protein import, high-risk precursor and immature forms of mitochondrial proteins form aberrant deposits in the cytosol. These deposits then cause further cytosolic accumulation and consequently aggregation of other mitochondrial proteins and disease-related proteins, including -synuclein and amyloid {beta}. This aggregation triggers a cytosolic protein homeostasis imbalance that is accompanied by specific molecular chaperone responses at both the transcriptomic and protein levels. Altogether, our results provide evidence that mitochondrial dysfunction, specifically protein import defects, contributes to impairments in protein homeostasis, thus revealing a possible molecular mechanism by which mitochondria are involved in neurodegenerative diseases. Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. A rationalized definition of tumor suppressor microRNAsexcludes miR-34a While several microRNAs (miRNAs) have been proposed to act as tumor suppressors, a consensual definition of tumor suppressing miRNAs is still missing. Similarly to coding genes, we propose that tumor suppressor miRNAs must show evidence of genetic or epigenetic inactivation in cancers, and exhibit an anti-tumorigenic (e.g., anti-proliferative) activity under endogenous expression levels. Here we observe that this definition excludes the most extensively studied tumor suppressor candidate miRNA, miR-34a. In analyzable cancer types, miR-34a does not appear to be down-regulated in primary tumors relatively to normal adjacent tissues. Deletion of miR-34a is occasionally found in human cancers, but it does not seem to be driven by an anti-tumorigenic activity of the miRNA, since it is not observed upon smaller, miR-34a-specific alterations. Its anti-proliferative action was observed upon large, supra-physiological transfection of synthetic miR-34a in cultured cells, and our data indicates that endogenous miR-34a levels do not have such an effect. Our results therefore argue against a general tumor suppressive function for miR-34a, providing an explanation to the lack of efficiency of synthetic miR-34a administration against solid tumors. Integrative small and long RNA-omics analysis of human healing and non-healing wounds discovers cooperating microRNAs as therapeutic targets MicroRNAs (miR) are important posttranscriptional regulators and exhibit a high potential to be utilized in diagnosis and therapy. However, our insufficient knowledge of the miR-mediated gene regulation in human skin wound healing severely hinders the identification of clinically relevant miRs. Here, we performed paired small RNA and long RNA sequencing in human tissue samples, including matched skin and acute wounds collected at each healing stage and chronic non-healing venous ulcers (VU). With integrative small and long RNA-omics analysis, we developed a compendium (https://www.xulandenlab.com/humanwounds-mirna-mrna), which will be an open, comprehensive resource to broadly aid wound healing research. With this first clinical, wound-centric resource of miRs and mRNAs, we identified 17 pathologically relevant miRs that exhibited abnormal VU expression and displayed their targets enriched explicitly in the VU gene signature. Intermeshing regulatory networks controlled by these miRs revealed their high cooperativity in contributing to chronic wound pathology characterized by persistent inflammation and proliferative phase initiation failure. Furthermore, we demonstrated that miR-34a, miR-424, and miR-516, upregulated in VU, cooperatively suppressed keratinocyte growth while promoting inflammatory response. Collectively, our study opens the possibility of developing innovative wound treatment that targets pathologically relevant cooperating miRs to attain higher therapeutic efficacy and specificity. Hippocampal Interneuronal Dysfunction and Hyperexcitability in a Porcine Model of Concussion Cognitive impairment is a common symptom following mild traumatic brain injury (mTBI or concussion) and can persist for years in some individuals. While the underlying mechanisms driving these impairments remain unknown, structural changes in the hippocampus post-mTBI have been reported in human patients, as well as electrophysiological changes in rodents following impact models of TBI. In addition, slice preparations from a closed-head, rotational acceleration injury (RAI) in swine showed reduced axonal function and hippocampal circuitry disruption. However, electrophysiological changes in neurons and their subtypes have not been examined. Using in vivo electrophysiology techniques, we examined the laminar oscillatory field potentials and single unit activity in the intact hippocampal network at 7 days post-RAI in anesthetized minipigs. Concussion altered the electrophysiological properties of pyramidal cells and interneurons differently in area CA1. Specifically, while firing rate and burst occurrence of CA1 interneurons were significantly decreased post-mTBI, these parameters were unchanged in putative CA1 pyramidal cells. However, pyramidal CA1 cells in TBI animals were significantly less entrained to hippocampal gamma (40 - 80 Hz) oscillations. In addition, stimulation of the Schaffer collaterals revealed hyperexcitability in the laminar CA1 response post-mTBI. Finally, computational simulations suggest that the reported changes in interneuronal firing rate and action potentials may be due to alterations in voltage-gated sodium channels. These data demonstrate that a single concussion can lead to significant neuronal and circuit level changes in the hippocampus, and that the loss of pyramidal gamma entrainment and changes in interneuronal firing may be important contributors to cognitive dysfunction following mTBI. 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. Distinct SARS-CoV-2 antibody reactivity patterns elicited by natural infection and mRNA Vaccination We analyzed data from two ongoing COVID-19 longitudinal serological surveys in Orange County, CA., between April 2020 and March 2021. A total of 8,476 finger stick blood specimens were collected before and after an aggressive mRNA vaccination campaign. IgG levels were determined using a multiplex antigen microarray containing 10 SARS-CoV-2 antigens, 4 SARS, 3 MERS, 12 Common CoV, and 8 Influenza antigens. Twenty-six percent of 3,347 specimens from unvaccinated Orange County residents in December 2020 were SARS-CoV-2 seropositive. The Ab response was predominantly against nucleocapsid (NP), full length spike and the spike S2 domain. Anti-receptor binding domain (RBD) reactivity was low and there was no cross-reactivity against SARS S1 or SARS RBD. An aggressive mRNA vaccination campaign at the UCI Medical Center started on December 16, 2020 and 6,724 healthcare workers were vaccinated within 3 weeks. Seroprevalence increased from 13% in December to 79% in January, 93% in February and 99% in March. mRNA vaccination induced much higher Ab levels especially against the RBD domain and significant cross-reactivity against SARS RBD and S1 was also observed. Nucleocapsid protein Abs can be used to distinguish individuals in a population of vaccinees to classify those who have been previously infected and those who have not, because nucleocapsid is not in the vaccine. Previously infected individuals developed higher Ab titers to the vaccine than those who have not been previously exposed. These results indicate that mRNA vaccination rapidly induces a much stronger and broader Ab response than SARS-CoV-2 infection. Decreased cell stiffness facilitates detachment and migration of breast cancer cells in 3D collagen matrices: An exploratory study BackgroundTumour-cell detachment is a critical early event in the metastatic cascade. However, the role of the cells mechanical properties in detachment and migration is not well understood. This exploratory study aimed to assess how intracellular stiffness changes these processes.

MethodsMDA-MB-231 cells were embedded as 10,000-cell spheroids in 2 and 4 mg/ml collagen matrices. Intracellular stiffness was assessed with mitochondria tracking microrheology of cells that migrated distances equivalent to four and six times the cell diameter (dC) from the spheroid and compared to cells at the spheroid surface (0dC), representing medium, high and no migration, respectively.

Results and discussionThe mitochondrial mean square displacement and intracellular stiffness decreased during detachment and migration for both collagen concentrations (i.e. rigidities). The mean square displacement of 4dC and 6dC cells was similar, whereas cell stiffness was lower for 4dC than for 6dC cells. With increasing matrix rigidity, the intracellular stiffness decreased for 0dC cells and did not change for 4dC and 6dC cells. It is proposed that decreased cell stiffness drives detachment and migration and increased matrix rigidity physically hinders migration, and cells need to become softer or remodel the environment to migrate. The independence of the stiffness of migrated cells from matrix rigidity suggests that cells remodel their environment through matrix proteins cleavage to migrate.

ConclusionsThe study revealed the collective effects of enhanced migratory conditions and increased matrix rigidity on the mechanical properties of the cells. The expression of matrix metalloproteinases and transforming growth factor {beta} and the role of cell volume on detachment and migration in matrices with varying pore sizes are proposed targets for further studies on metastatic cancer cells. A network approach to genetic circuit designs As genetic circuits become more sophisticated, the size and complexity of data about their designs increases. This data captured goes beyond monolithic genetic sequences and towards circuit modularity and functional details, which are beneficial for analyzing circuit performance and establishing design automation techniques. However, the accessibility, visualisation and usability of design data (and metadata) have received relatively little attention to date. Here, we present a method to turn circuit designs into networks and showcase its potential to enhance the utility of design data. Since networks are dynamic structures, initial graphs can be interactively shaped into sub-networks of relevant information based on requirements such as abstraction, hierarchy and protein interactions. Additionally, several visual changes can be applied, such as colouring or clustering nodes based on types (e.g., genes or promoters), resulting in easier comprehension from a user perspective. This approach allows circuit designs to be coupled to other networks, such as metabolic pathways or implementation protocols captured in graph-like formats. Therefore, we advocate using networks to structure, access and improve synthetic biology information. Structural basis for Nirmatrelvir in vitro efficacy against SARS-CoV-2 variants The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). We evaluated the in vitro catalytic activity and in vitro potency of nirmatrelvir against the main protease (Mpro) of prevalent variants of concern (VOC) or variants of interest (VOI): Alpha (, B.1.1.7), Beta ({beta}, B.1.351), Delta ({delta}, B1.617.2), Gamma ({gamma}, P.1), Lambda ({lambda}, B.1.1.1.37/C37), Omicron (o, B.1.1.529) as well as the original Washington or wildtype strain. These VOC/VOI carry prevalent mutations at varying frequencies in the Mpro specifically for: , {beta},{gamma} (K90R), {lambda} (G15S) and o (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrate that they are catalytically comparable to wildtype. Nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the o, {lambda} and {beta} Mpro at 1.63 - 2.09 [A] resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells (1). Late-acting self-incompatible system, preferential allogamy and delayed selfing in the heteromorphic invasive populations of Ludwigia grandiflora subsp. hexapetala AO_SCPLOWBSTRACTC_SCPLOWBreeding system influences local population genetic structure, effective size, offspring fitness and functional variation. Determining the respective importance of self- and cross-fertilization in hermaphroditic flowering plants is thus important to understand their ecology and evolution. The worldwide invasive species, Ludwigia grandiflora subsp. hexapetala (Lgh) presents two floral morphs: one self-compatible short-styled morph (S-morph) and one self-incompatible long-styled morph (L-morph). In this study, we identified the breeding systems of western European experimental and natural populations of Lgh by comparing structural characteristics of pollen and style, by studying self- and cross-pollen tube elongations and the viability of the resulting seeds and seedlings in both floral morphs. Our results showed no differences in pollen shape and stigma surfaces no matter the floral morph. In the self-incompatible L-morph flowers, self-pollen tubes were stopped tardily, in the ovarian area, and were unable to fertilize the ovules. This first formal identification of a late-acting, prezygotic self-incompatible system (LSI) in Ludwigia genus contributes a case of LSI in an additional family within the Myrtales order. In the self-compatible S-morph flowers, self-pollen always succeeded to self-fertilize the ovules that nearly all developed into viable seedlings. However, cross-pollen tubes always elongated faster than self-pollen tubes in both morphs. S-morph individuals may thus advantage preferential allogamy over selfing when cross-pollen is available despite its self-compatibility. As expected in late-acting self-incompatible systems, L-morph flowers authorised 0.2{per thousand} of selfed seeds during the uppermost flowering season, that increased to 1{per thousand} at the end of the flowering season. Such delayed selfing resulted in a significant quantity of viable floating seeds. They may contribute to the local regeneration, seed bank and propagation of the L-morph, which may contribute to explain its invasion success worldwide. Management plans of Lgh would gain to consider the breeding systems we identified. Quantifying the potential for red blood cell β-adrenergic sodium-proton exchangers to protect oxygen transport in hypoxic and hypercapnic white seabass White seabass (Atractoscion nobilis) are increasingly experiencing periods of low oxygen (O2; hypoxia) and high carbon dioxide (CO2, hypercapnia) due to climate change and eutrophication of the coastal waters of California. Hemoglobin (Hb) is the principal O2 carrier in the blood and in many teleost fishes Hb-O2 binding is compromised at low pH; however, the red blood cells (RBC) of some species regulate intracellular pH with adrenergically-stimulated sodium-proton-exchangers ({beta}-NHE). We hypothesized that RBC {beta}-NHEs in white seabass are an important mechanism that can protect the blood O2-carrying capacity during hypoxia and hypercapnia. We determined the O2-binding characteristics of white seabass blood, the response of RBCs to adrenergic stimulation, and quantified the protective effect of {beta}-NHE activity on Hb-O2 saturation. White seabass had typical teleost Hb characteristics, with a moderate O2 affinity (PO2 at half-saturation; P50 2.9 kPa) that was highly pH-sensitive (Bohr coefficient -0.92; Root effect 52%). The presence of RBC {beta}-NHEs was confirmed by functional, molecular and bioinformatic data and super-resolution imaging revealed, for the first time, the subcellular location of {beta}-NHE protein in vesicle-like structures and on the RBC membrane, and its translocation after adrenergic stimulation. The activation of RBC {beta}-NHEs increased Hb-O2 saturation by [~]8% in normoxia at 1 kPa PCO2, and by up to 20% in hypoxia. Our results confirm that RBC {beta}-NHE activity in white seabass has great potential to protect arterial O2 transport in environmentally relevant conditions of hypoxia and hypercapnia, but also reveal a potential vulnerability of fish to combinations of these stressors.