GPI-anchored FGF directs cytoneme-mediated bidirectional signaling to self-regulate tissue-specific dispersion How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and activates target-specific bidirectional FGF-FGFR signaling through cytonemes. FGF-source and recipient ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through CAM-like FGF-FGFR binding. Contact-mediated FGF-FGFR binding induces bidirectional signaling, which, in turn, promotes ASP and source cells to polarize cytonemes toward each other and reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source cell membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion and signaling.
HYBRID REACTION-DIFFUSION AND CLOCK-AND-WAVEFRONT MODEL FOR THE ARREST OF OSCILLATIONS IN THE SOMITOGENESIS SEGMENTATION CLOCK AO_SCPLOWBSTRACTC_SCPLOWThe clock and wavefront paradigm is arguably the most widely accepted model for explaining the embryonic process of somitogenesis. According to this model, somitogenesis is based upon the interaction between a genetic oscillator, known as segmentation clock, and a differentiation wavefront, which provides the positional information indicating where each pair of somites is formed. Shortly after the clock and wavefront paradigm was introduced, Meinhardt presented a conceptually different mathematical model for morphogenesis in general, and somitogenesis in particular. Recently, Cotterell et al. rediscovered an equivalent model by systematically enumerating and studying small networks performing segmentation. Cotterell et al. called it a progressive oscillatory reaction-diffusion (PORD) model. In the Meinhardt-PORD model, somitogenesis is driven by short-range interactions and the posterior movement of the front is a local, emergent phenomenon, which is not controlled by global positional information. With this model, it is possible to explain some experimental observations that are incompatible with the clock and wavefront model. However the Meinhardt-PORD model has some important disadvantages of its own. Namely, it is quite sensitive to fluctuations and depends on very specific initial conditions (which are not biologically realistic). In this work, we propose an equivalent Meinhardt-PORD model, and then amend it to couple it with a wavefront consisting of a receding morphogen gradient. By doing so, we get a hybrid model between the Meinhardt-PORD and the clock-and-wavefront ones, which overcomes most of the deficiencies of the two originating models.

Somitogenesis, the process by which somites are formed, is an essential developmental stage in many vertebrates. This process occurs with a strikingly regular periodicity, that is preserved among embryos of a single species. The clock and wavefront paradigm is arguably the most widely accepted model for explaining somitogenesis. However, it is incapable of explaining some experimental facts, like the appearance of somites in the absence of an external wavefront (i.e. a receding morphogen gradient). Shortly after the clock and wavefront paradigm was introduced, Meinhardt presented a conceptually different mathematical model for morphogenesis in general, and somitogenesis in particular. Recently, Cotterell et al. rediscovered an equivalent model by systematically enumerating and studying small networks performing segmentation, and called it a progressive oscillatory reaction-diffusion (PORD) model. The Meinhardt-PORD model tackles some of the deficiencies of the clock and wave-front models, but it has some serious issues of its own. In the present work, we introduce an equivalent Meinhardt-PORD model, and then amend it to couple it with a receding morphogen gradient. By doing so, we get a hybrid model that incorporates characteristics of the Meinhardt-PORD and clock-and-wavefront models. We show that this hybrid model under-goes a bifurcation, from a stable to an unstable limit cycle, as the value of the parameter accounting for a background regulatory input (associated to the receding morphogen gradient) decreases. This bifurcation allows the model to explain why somites can form in the absence of an external wavefront, reassesses the role of the receding morphogen gradient as a conductor for somitogenesis, and makes the model behavior robust to random fluctuations, as well as independent from specific initial conditions (the latter, are two of the weak points of the Meinhardt-PORD model). We argue that this findings provide convincing evidence that reaction-diffusion and positional information (receding morphogen gradient) mechanisms could work together in somitogenesis.
An IPI based immune prognostic model for diffuse large B-cell lymphoma BackgroundInternational Prognostic Index (IPI) was widely used to better discriminate prognosis of patients with diffuse large B-cell lymphoma (DLBCL). However, there is a significant need to identify novel valuable biomarkers in the context of targeted therapies, such as immune checkpoint blockade (ICB) therapy.

MethodsGene expression data and clinical information of DLBCL were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. 371 immune-related hub genes in DLBCL patients with different IPI levels were identified by weighted gene co-expression network analysis (WGCNA), and 8 genes were selected to construct an IPI-based immune prognostic model (IPI-IPM). Afterward, the genetic, somatic mutational and molecular profiles of IPI-IPM subgroups were analyzed, as well as the potential clinical response of ICB in different IPI-IPM subgroups.

ResultsThe IPI-IPM was constructed based on the expression of CMBL, TLCD3B, SYNDIG1, ESM1, EPHA3, HUNK, PTX3 and IL12A, where high-risk patients had shorter overall survival (OS) than low-risk patients, consistent with the results in the GEO cohorts. The comprehensive results showed that high IPI-IPM risk scores were correlated with immune-related signaling pathways, high KMT2D and CD79B mutation rates, as well as up-regulation of inhibitory immune checkpoints including PD-L1, BTLA and SIGLEC7, indicating more potential response to ICB therapy.

ConclusionThe IPI-IPM has independent prognostic significance for DLBCL patients, which provides an immunological perspective to elucidate the mechanisms on tumor progression, also sheds a light on developing immunotherapy for DLBCL. Optimized detection of allelic imbalances specific for homologous recombination deficiency improves the prediction of clinical outcomes in cancer Homologous recombination DNA-repair deficiency (HRD) is a common driver of genomic instability and confers a therapeutic vulnerability in human cancer. Using a systematic pan- cancer characterization, we revealed distinct patterns of somatic allelic imbalances (AIs) in high-grade serous ovarian cancer (HGSC). Using machine learning on a multi-omics dataset, we generated an optimized algorithm to detect HRD in HGSC (ovaHRDscar). ovaHRDscar improved the prediction of clinical outcomes in three independent validation cohorts. Characterization of 98 spatiotemporally distinct tumor samples indicated ovary/adnex as the preferred site to assess HRD. Cancer-specific optimization improved the prediction of clinical outcomes also in triple-negative breast cancer (tnbcHRDscar). In conclusion, our systematic approach based on multi-omics data improves the detection of HRD with the premise to improve patient selection for HR-targeted therapies. Cell-specific Bioorthogonal Tagging of Glycoproteins Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function. Organ- and function-specific anatomical organization and bioelectronic modulation of the vagus nerve Afferent and efferent fibers in the vagus nerve travel inside fascicles and form branches to innervate organs and regulate organ functions. The spatial organization of fibers and fascicles, with respect to the functions they mediate and the organs they innervate, is unknown. Accordingly, it is unknown whether such organization can be leveraged by bioelectronic devices for function- and organ-specific neurostimulation. To characterize the functional microscopic anatomy of the vagus, we developed a pipeline consisting of micro-computed tomography imaging of fascicles and quantitative immunohistochemistry of single fibers. We found that, in swine, the vagus contains clusters of fascicles, organized along 2 overlapping functional axes: one axis specific to sensory vs. motor somatic and visceral functions, with clusters increasingly separating in the cephalad direction, and a second axis specific to innervated organs, including larynx, lungs and heart, with increasing separation in the caudad direction. Large myelinated afferents and efferents, and small unmyelinated efferents occupy large areas of specific clusters of fascicles; small unmyelinated afferents are numerous and occupy small areas across every fascicle. To test whether fiber populations can be selectively modulated, we developed a multi-contact cuff electrode accommodating the nerve fascicular structure. Stimulation targeting separate nerve sections evokes compound action potentials from fibers of distinct functional types and elicits differential organ responses, including laryngeal muscle contraction, cough reflex, and changes in breathing and heart rate. Our results indicate that vagus fibers are anatomically organized according to functions they mediate and organs they innervate and can be differentially modulated by spatially selective nerve stimulation. Environmental control of Pub1 (NEDD4 family E3 ligase) in S. pombe is regulated byTORC2 and Gsk3. Cells respond to changing nutrient environments by adjusting the abundance of surface nutrient transporters and receptors. This can be achieved by modulating ubiquitin-dependent endocytosis, which in part is regulated by the NEDD4 family of E3 ligases. Here we report novel regulation of Pub1, a fission yeast Schizosaccharomyces pombe member of the NEDD4-family of E3 ligases. We show that nitrogen stress inhibits Pub1 function, thereby increasing the abundance of the amino acid transporter Aat1 at the plasma membrane and enhancing sensitivity to the toxic arginine analogue canavanine. We show that TOR complex 2 (TORC2) signalling negatively regulates Pub1, thus TORC2 mutants under nutrient stress have decreased Aat1 at the plasma membrane and are resistant to canavanine. Inhibition of TORC2 signalling increases Pub1 phosphorylation, and this is dependent on Gsk3 activity. Addition of the Tor inhibitor Torin1 increases phosphorylation of Pub1 at serine 199 (S199) by 2.5-fold, and Pub1 protein levels in S199A phospho-ablated mutants are reduced. S199 is conserved in NEDD4 and is located immediately upstream of a WW domain required for protein interaction. Together, we describe how the major TORC2 nutrient-sensing signalling network regulates environmental control of Pub1 to modulate the abundance of nutrient transporters. Ineffectual AEC1 differentiation from KRT8hi transitional state without fibrosis is associated with fatal COVID-19 ARDS ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. Hidden dynamics of proteasome autoregulation discovered by cryo-EM data-driven deep learning The cellular functions are executed by macromolecular complexes in nonequilibrium dynamic processes, which exhibit a vast diversity of conformational states. Solving conformational continuum of important complexes at atomic level remains a key challenge in the methodology of computational biophysics. Here we introduce a deep-learning framework, named AlphaCryo4D, which enables atomic-level cryogenic electron microscopy reconstructions of nonequilibrium conformational continuum. AlphaCryo4D integrates 3D deep residual learning with manifold embedding of energy landscapes, which simultaneously improves 3D classification accuracy and reconstruction resolution via an energy-based particle-voting algorithm. Applications of this methodology to six experimental datasets demonstrated its generality in breaking resolution barrier of visualizing dynamic components of functional complexes, in choreographing continuous inter-subunit motions and in exploring their  hidden conformational space. Importantly, AlphaCryo4D identified 64 conformers of the substrate-bound human 26S proteasome and reconstituted single-nucleotide-exchange dynamics of proteasomal AAA-ATPase motor during translocation initiation, which together illuminates a grand hierarchical allostery for proteasome autoregulation. Human inhalable antibody fragments neutralizing SARS-CoV-2 variants for COVID-19 therapy As of October 2021, coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global emergency, and novel therapeutics are urgently needed. Here we describe human single chain variable fragment (scFv) antibodies (76clAbs) that block an epitope of the SARS-CoV-2 spike protein essential for ACE2-mediated entry into cells. 76clAbs neutralize the delta variant and other variants being monitored (VBMs) and inhibit spike-mediated pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. In two independent animal models, intranasal administration counteracted the infection. Due to high efficiency, remarkable stability, resilience to nebulization and low production cost, 76clAbs may become a relevant tool for rapid, self-administrable early intervention in SARS-CoV-2-infected subjects independently of their immune status. Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies. Hybridization alters the shape of the genotypic fitness landscape, increasing access to novel fitness peaks during adaptive radiation Estimating the complex relationship between fitness and genotype or phenotype (i.e. the adaptive landscape) is one of the central goals of evolutionary biology. However, adaptive walks connecting genotypes to organismal fitness, speciation, and novel ecological niches are still poorly understood and processes for surmounting fitness valleys remain controversial. One outstanding system for addressing these connections is a recent adaptive radiation of ecologically and morphologically novel pupfishes (a generalist, molluscivore, and scale-eater) endemic to San Salvador Island, Bahamas. We leveraged whole-genome sequencing of 139 hybrids from two independent field fitness experiments to identify the genomic basis of fitness, estimate genotypic fitness networks, and measure the accessibility of adaptive walks on the fitness landscape. We identified 132 SNPs that were significantly associated with fitness in field enclosures. Six out of the 13 regions most strongly associated with fitness contained differentially expressed genes and fixed SNPs between trophic specialists; one gene (mettl21e) was also misexpressed in lab-reared hybrids, suggesting a potential intrinsic genetic incompatibility. We then constructed genotypic fitness networks from adaptive alleles and show that scale-eating specialists are the most isolated of the three species on these networks. Intriguingly, introgressed and de novo variants reduced fitness landscape ruggedness as compared to standing variation, increasing the accessibility of genotypic fitness paths from generalist to specialists. Our results suggest that adaptive introgression and de novo mutations alter the shape of the fitness landscape, providing key connections in adaptive walks circumventing fitness valleys and triggering the evolution of novelty during adaptive radiation. Vapor exposure to Δ9-tetrahydrocannabinol (THC) slows locomotion of the Maine Lobster (Homarus americanus) RationaleDespite a long history of use in synaptic physiology, the lobster has been a neglected model for behavioral pharmacology. A restaurateur proposed that exposing lobster to cannabis smoke reduces anxiety and pain during the cooking process. It is unknown if lobster gill respiration in air would result in significant {Delta}9-tetrahydrocannabinol (THC) uptake and whether this would have any detectable behavioral effects.

ObjectiveThe primary goal was to determine tissue THC levels in the lobster after exposure to THC vapor. Secondary goals were to determine if THC vapor altered locomotor behavior or nociception.

MethodsTissue samples were collected (including muscle, brain and hemolymph) from Homarus americanus (N=3 per group) following 30 or 60 minutes of exposure to vapor generated by an e-cigarette device using THC (100 mg/mL in a propylene glycol vehicle). Separate experiments assessed locomotor behavior and hot water nociceptive responses following THC vapor exposure.

ResultsTHC vapor produced duration-related THC levels in all tissues examined. Locomotor activity was decreased (distance, speed, time-mobile) by 30 min inhalation of THC. Lobsters exhibit a temperature-dependent withdrawal response to immersion of tail, antennae or claws in warm water; this is novel evidence of thermal nociception for this species. THC exposure for 60 minutes had only marginal effect on nociception under the conditions assessed.

ConclusionsVapor exposure of lobsters, using an e-cigarette based model, produces dose-dependent THC levels in all tissues and reduces locomotor activity. Hot water nociception was temperature dependent, but only minimal anti-nociceptive effect of THC exposure was confirmed. Expression and subcellular localization of USH1C/harmonin in the human retina provides insights into pathophysiology and therapy of Usher syndrome Usher syndrome (USH) is the most common form of hereditary deafness-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retina uncovered harmonin a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Muller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones, and outer segments of rods as well as at adhesive junctions of MGCs and photoreceptors in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in MGCs and photoreceptors and rhodopsin in photoreceptors. Subcellular expression and colocalization of harmonin correlates with the clinical phenotype observed in USH1C patients. In addition, primary cilia defects in USH1C patient-derived fibroblasts could be reverted by delivery of harmonin a1 transcript isoform. Our data provide novel insights into photoreceptor cell biology, USH1C pathophysiology and for developing gene therapy treatment. African Swine Fever Virus and host response - transcriptome profiling of the Georgia 2007/1 strain and porcine macrophages African swine fever virus (ASFV) has a major global economic impact. With a case fatality in domestic pigs approaching 100%, it currently presents the largest threat to animal farming. Although genomic differences between attenuated and highly virulent ASFV strains have been identified, the molecular determinants for virulence at the level of gene expression have remained opaque. Here we characterise the transcriptome of ASFV genotype II Georgia 2007/1 (GRG) during infection of the physiologically relevant host cells, porcine macrophages. In this study we applied Cap Analysis Gene Expression sequencing (CAGE-seq) to map the 5 ends of viral mRNAs at 5 and 16 hours post-infection. A bioinformatics analysis of the sequence context surrounding the transcription start sites (TSSs) enabled us to characterise the global early and late promoter landscape of GRG. We compared transcriptome maps of the GRG isolate and the lab-attenuated BA71V strain that highlighted GRG virulent-specific transcripts belonging to multigene families, including two predicted MGF 100 genes I7L and I8L. In parallel, we monitored transcriptome changes in the infected host macrophage cells. Of the 9,384 macrophage genes studied, transcripts for 652 host genes were differentially regulated between 5 and 16 hours-post-infection compared with only 25 between uninfected cells and 5 hours post-infection. NF-kB activated genes and lysosome components like S100 were upregulated, and chemokines such as CCL24, CXCL2, CXCL5 and CXCL8 downregulated.

ImportanceAfrican swine fever virus (ASFV) causes haemorrhagic fever in domestic pigs with case fatality rates approaching 100%, and no approved vaccines or antivirals. The highly-virulent ASFV Georgia 2007/1 strain (GRG) was the first isolated when ASFV spread from Africa to the Caucasus region in 2007. Then spreading through Eastern Europe, and more recently across Asia. We used an RNA-based next generation sequencing technique called CAGE-seq to map the starts of viral genes across the GRG DNA genome. This has allowed us to investigate which viral genes are expressed during early or late stages of infection and how this is controlled, comparing their expression to the non-virulent ASFV-BA71V strain to identify key genes that play a role in virulence. In parallel we investigated how host cells respond to infection, which revealed how the ASFV suppresses components of the host immune response to ultimately win the arms race against its porcine host. PIN-FORMED1 polarity in the shoot is insensitive to the polarity of neighbouring cells In plants, epidermal cells are planar-polarized along an axis marked by the asymmetric localization patterns of several proteins including PIN-FORMED1 (PIN1), which facilitates the directional efflux of the plant hormone auxin to pattern phyllotaxis (Heisler et al., 2010; Mansfield et al., 2018; Reinhardt et al., 2003). While PIN1 polarity is known to be regulated non-cell autonomously via the MONOPTEROS (MP) transcription factor, how this occurs has not been determined (Bhatia et al., 2016). Here we use mosaic expression of the serine threonine kinase PINOID (PID) to test whether PIN1 polarizes according to the polarity of neighbouring cells. Our findings reveal that PIN1 is insensitive to the polarity of PIN1 in neighbouring cells arguing against auxin flux or extracellular auxin concentrations acting as a polarity cue, in contrast to previous model proposals (Abley et al., 2016; Stoma et al., 2008). 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. Determinants of Murine Skin Microbiota Composition in Homeostasis and Wound Healing Animal microbiota have complex interactions with hosts and environment that determines its composition. Yet the ability of hosts to determine their microbiota composition is less well studied. In this study, to investigate the role host genetics in determining skin microbiota, we used 30 different mouse strains from the recombinant inbred panel, the Collaborative Cross. Murine skin microbiota composition was strongly dependent on murine strain with > 50% of the variation explained by murine strain. In particular, a quantitative trait locus on chromosome 4 associates both with Staphylococcus abundance and principal-component multi-trait analyses. Additionally, excisional wound associated changes in microbiota composition were not uniform across mouse strains and were host-specific, the genetic background accounting for about 40% of the variation in microbiota. Genetic background also had the highest effect on the healing speed of wounds accounting for over 50% of the variation while mouse age and microbiota composition change accounted only for 20% and 5% of the healing speed despite reaching statistical significance. In conclusion, host genetics has a significant impact on the skin microbiota composition during both homeostasis and wound healing. These findings have long reaching implications in our understanding of associations between microbiota dysbiosis and disease. Balancing sensitivity and specificity in preclinical research The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low. Foraging with friends, moving with anyone: effects of the social environment on movement-integrated habitat selection O_LIMovement links the distribution of habitats with the social environment of animals using those habitats; yet integrating movement, habitat selection, and socioecology remains an opportunity for further study.
C_LIO_LIHere, our objective was to disentangle the roles of habitat selection and social association as drivers of collective movement in a gregarious ungulate. To accomplish this objective, we (1) assessed whether socially familiar individuals form discrete social communities and whether social communities have high spatial, but not necessarily temporal, overlap; and (2) we modelled the relationship between collective movement and selection of foraging habitats using socially informed integrated step selection analysis.
C_LIO_LIWe used social network analysis to assign individuals to social communities and determine short and long-term social preference among individuals. Using integrated step selection functions (iSSF), we then modelled the effect of social processes, i.e., nearest neighbour distance and social preference, and movement behaviour on patterns of habitat selection.
C_LIO_LIBased on assignment of individuals to social communities and home range overlap analyses, individuals assorted into discrete social communities, and these communities had high spatial overlap. By unifying social network analysis with iSSF, we identified movement-dependent social association, where individuals foraged with more familiar individuals, but moved collectively with any between foraging patches.
C_LIO_LIOur study demonstrates that social behaviour and space use are inter-related based on spatial overlap of social communities and movement-dependent habitat selection. Movement, habitat selection, and social behaviour are linked in theory. Here, we put these concepts into practice to demonstrate that movement is the glue connecting individual habitat selection to the social environment.
C_LI 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. Reference-free cell-type deconvolution of multi-cellular pixel-resolution spatially resolved transcriptomics data Recent technological advancements have enabled spatially resolved transcriptomic profiling but at multi-cellular pixel resolution, thereby hindering the identification of cell-type-specific spatial patterns and gene expression variation. To address this challenge, we developed STdeconvolve as a reference-free approach to deconvolve underlying cell-types comprising such multi-cellular pixel resolution spatial transcriptomics (ST) datasets. Using simulated as well as real ST datasets from diverse spatial transcriptomics technologies comprising a variety of spatial resolutions such as Spatial Transcriptomics, 10X Visium, DBiT-seq, and Slide-seq, we show that STdeconvolve can effectively recover cell-type transcriptional profiles and their proportional representation within pixels without reliance on external single-cell transcriptomics references. STdeconvolve provides comparable performance to existing reference-based methods when suitable single-cell references are available, as well as potentially superior performance when suitable single-cell references are not available. STdeconvolve is available as an open-source R software package with the source code available at https://github.com/JEFworks-Lab/STdeconvolve. Tardigrade CAHS Proteins Act as Molecular Swiss Army Knives to Mediate Desiccation Tolerance Through Multiple Mechanisms Tardigrades, also known as water bears, make up a phylum of small but extremely hardy animals, renowned for their ability to survive extreme stresses, including desiccation. How tardigrades survive desiccation is one of the enduring mysteries of animal physiology. Here we show that CAHS D, an intrinsically disordered protein belonging to a unique family of proteins possessed only by tardigrades, undergoes a liquid-to-gel phase transition in a concentration dependent manner. Unlike other gelling proteins, such as gelatin, our data support a mechanism in which gel formation of CAHS D is driven by intermolecular {beta}-{beta} interactions. We find that gel formation corresponds with strong coordination of water and slowing of water diffusion. The degree of water coordination correlates with the ability of CAHS D to protect lactate dehydrogenase from unfolding when dried. This implies that the mechanism for unfolding protection can be attributed to a combination of hydration and slowed molecular motion. Conversely, rapid diffusion leading to efficient molecular shielding appears to be the predominant mechanism preventing protein aggregation. Our study demonstrates that distinct mechanisms are required for holistic protection during desiccation, and that protectants, such as CAHS D, can act as molecular  Swiss Army Knives capable of providing protection through several different mechanisms simultaneously. ZIKV disrupts placental ultrastructure and drug transporter expression in mice Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome. Long-term conservation effects of protected areas in stochastic population dynamics Terrestrial and marine protected areas are essential tools in mitigating anthropogenic impacts and promoting population persistence and resource sustainability. Adequately implemented protected areas (PAs) promote long-term conservation effects. Stochasticity causes fluctuations in the conservation effects of PAs, and so it is important to investigate the variabilities of these conservation effects to inform their long-term conservation effects. To investigate long-term conservation effects, I develop and analyze new models of stochastic processes that encompass the fluctuations generated by demographic or environmental stochasticity in PAs management. The stochastic model is built upon individual processes. In the model, density-independent mortality, migration between PAs and non-PAs, and site preferences characterize the features of the PA. The effect of PAs size is also examined. The long-term conservation effects are quantified using the coefficient of variation (CV) of population size in PAs, where a lower CV indicates higher robustness in stochastic variations. Typically, the results from this study demonstrate that sufficiently reduced density-independent mortality in PAs and high site preference and immigration rate of PA are likely to decrease the CV. However, different types of stochasticity induce rather different consequences: under demographic stochasticity, the CV is always reduced when PAs increase the population size therein, but an increased population size by PAs does not always decrease the CV under environmental stochasticity. The deterministic dynamics of the model are investigated, facilitating effective management decisions. Changes to the mtDNA copy number during yeast culture growth We show that the mtDNA copy number in growing cultures of the yeast Saccharomyces cerevisiae increases by a factor of up to 4, being lowest ([~]10 per haploid genome) and stable during rapid fermentative growth, and highest at the end of the respiratory phase. When yeast are grown on glucose, the onset of the mtDNA copy number increase coincides with the early stages of the diauxic shift, and the increase continues through respiration. A lesser yet still substantial copy number increase occurs when yeast are grown on a nonfermentable carbon source, i.e. when there is no diauxic shift. The mtDNA copy number increase during and for some time after the diauxic shift is not driven by an increase in cell size. The copy number increase occurs in both haploid and diploid strains, but is markedly attenuated in a diploid wild isolate that is a ready sporulator. Strain-to-strain differences in mtDNA copy number are least apparent in fermentation and most apparent in late respiration or stationary phase. While changes in mitochondrial morphology and function were previously known to accompany changes in physiological state, it had not been previously shown that the mtDNA copy number changes substantially over time in a clonal growing culture. The mtDNA copy number in yeast is therefore a highly dynamic phenotype.