Drosophila nicotinic acetylcholine receptors and their interactions with insecticidal peptide toxins Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of receptor subunit composition is needed for effective design of insecticides. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles we determined the effects of -Bungarotoxin (-Btx) and {omega}-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm -Btx interactions with the D5, D6, D7 subunits. Finally, we report the localization of fluorescent tagged endogenous D6 during nervous system development. Taken together this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.
Human gaze is systematically offset from the center of cone topography The small physical depression of the human retina, the fovea, is the retinal locus of prime visual resolution, achieved by a peaking topography of the light sensitive cone photoreceptor outer segments 1-3 and a post-receptor wiring scheme preserving high-density sampling 4,5. Humans dynamically direct their gaze such that the retinal images of objects of interest fall onto the foveola, the central one-degree diameter of the fovea 6-8, but it is yet unclear if a relationship between the individual photoreceptor topography at this location and visual fixation behavior exists 9,10. By employing adaptive optics in vivo imaging and micro-stimulation 11-13, we created topographical maps of the complete foveolar cone mosaics in both eyes of 20 healthy participants while simultaneously recording the retinal location of a fixated visual object in a psychophysical experiment with cellular resolution. We found that the locus of fixation was systematically shifted away from the topographical centers towards a naso-superior quadrant on the retina, about 5 minutes of arc of visual angle on average, with a mirror symmetrical trend between fellow eyes. In cyclopean view, the topographical centers were superior to the fixated target, corresponding to areas in the visual field usually more distant 14,15 and thus containing higher spatial frequencies. Given the large variability in foveal topography between individuals, and the surprising precision with which fixation is repeatedly directed to just a small bouquet of cones in the foveola, these findings demonstrate a finely tuned, functionally relevant link between the development of the cellular mosaic of photoreceptors and visual behavior.
Nafamostat-interferon-alpha combination suppresses SARS-CoV-2 infection by targeting cooperatively host TMPRSS2 in vitro and in vivo SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNa) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNa and that both Serpin E1 and camostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world. An alignment free approach confirms semantic properties of species proteomes Alignment-based methods dominate molecular biology. However, by primarily allowing one-to-one comparisons, these methods are focused on a gene-centered viewpoint and lack the broad context essential to analyze how complex biological systems function and evolve. In actuality, a gene is part of genome where more than one sequence contributes to the functional network and evolutionary trajectory of the cell. The need for conservation of established interactions, is arguably more important to the evolutionary success of species than conservation of individual function. To test whether such contextual information exists, a distributional semantics method - Latent Semantic Analysis (LSA), was applied to thousands of species proteomes. Using natural language processing, Latent Taxonomic Signatures (LTSs) were identified that outperformed existing alignment-based BLAST methods when random protein sequences were being mapped to annotated taxonomy according to GenBank. LTSs are a novel proteome distributed feature, suggesting the existence of evolutionary constraints imposed on individual proteins by their proteome context. Even orphan proteins are exhibiting LTSs, which makes their uniqueness linked to a specific taxonomic level questionable. Unlike more simple bias, LTSs represent a self-similarity pattern, where random sets of species proteins show the same statistical properties of a complete proteome at many scales. Natural language processing and machine learning provide insights not easily discernable using alignment based methods suggestive there is more to species related differences than just translational optimization. Inference of disease-associated microbial biomarkers based on metagenomic and metatranscriptomic data The identification of disease-associated microbial biomarkers is crucial for disease diagnosis and therapy. However, the heterogeneity, high dimensionality, and large amounts of microbial data present tremendous challenges for the discovery of biomarkers. In this paper, we present IDAM, a novel method for disease-associated biomarker inference from metagenomic and metatranscriptomic data, which does not require prior metadata. This method integrates gene context conservation (uber-operon) and regulatory mechanisms (gene co-expression patterns), using a mathematical graph model. We demonstrated the excellent performance of IDAM in biomarker inference, through the datasets from inflammatory bowel disease, melanoma, type 1 diabetes mellitus, and irritable bowel syndrome. The software of IDAM is freely available at https://github.com/OSU-BMBL/IDAM. Contribution of arm movements to balance recovery after tripping in older adults. Falls are common in daily life, often caused by trips and slips and, particularly in older adults, with serious consequences. Although arm movements play an important role in balance control, there is limited research into the role of arm movements during balance recovery after tripping in older adults. We investigated how older adults use their arms to recover from a trip and the difference in the effects of arm movements between fallers (n=5) and non-fallers (n=11).

Sixteen older males and females (69.7{+/-}2.3 years) walked along a walkway and were occasionally tripped over suddenly appearing obstacles. We analysed the first trip using a biomechanical model based on full-body kinematics and force-plate data to calculate whole body orientation during the trip and recovery phase. With this model, we simulated the effects of arm movements at foot-obstacle impact and during trip recovery on body orientation.

Apart from an increase in sagittal plane forward body rotation at touchdown in fallers, we found no significant differences between fallers and non-fallers in the effects of arm movements on trip recovery. Like earlier studies in young adults, we found that arm movements during the recovery phase had most favourable effects in the transverse plane: by delaying the transfer of angular momentum of the arms to the body, older adults rotated the tripped side more forward thereby allowing for a larger recovery step. Older adults that are prone to falling might improve their balance recovery after tripping by learning to prolong ongoing arm movements. A flexible electronic strain sensor for the real-time monitoring of tumor regression Assessing the efficacy of cancer therapeutics in mouse models is a critical step in treatment development. However, low resolution measurement tools and small sample sizes make determining drug efficacy in vivo a difficult and time-intensive task. Here we present a commercially scalable wearable electronic sensor that automates the in vivo testing of cancer therapeutics by continuously monitoring micrometer-scale tumor volume progression or regression in real-time. Histology, caliper measurements, and bioluminescence imaging over a one-week treatment period validated the sensors recordings, but the sensors higher time and length scale resolutions provided the only measurement technique capable of continuously monitoring the immediate hour-scale pharmacodynamic response of a given drug. In mice with two subcutaneously implanted tumor models our sensors recorded significant volume reductions in tumors just 5 hours after small molecule or immunotherapy treatment initiation. We anticipate that real-time tumor regression datasets could help expedite and automate the process of screening cancer therapies in vivo. Variation in white spruce needle respiration at the species range limits: a potential impediment to northern expansion White spruce (Picea glauca) spans a massive range from arctic treeline to temperate forests, yet the variability in respiratory physiology and related implications for tree carbon balance at the extremes of this distribution remain as enigmas. Working at both the most northern and southern extents of the white spruce distribution range more than 5000 km apart, we measured the short- term temperature response of dark respiration (R/T) at upper and lower canopy positions. R/T curves were fit to both polynomial and thermodynamic models so that model parameters could be compared among locations, canopy positions, and with previously published data. Respiration measured at 25{degrees}C (R25) was 68% lower at the southern location than at the northern location (0.73{+/-}0.15 vs. 2.27{+/-}0.02 mol m-2 s-1), resulting in a significantly lower (p< 0.01) intercept in R/T response in temperate trees. Only at the southern location did upper canopy leaves have a steeper temperature response than lower canopy leaves, likely reflecting steeper canopy gradients in light. No differences were observed in the maximum temperature of respiration. At the northern range limit respiration is nearly twice that of the average R25 reported in a global leaf respiration database. This large carbon cost likely challenges tree survival and contributes to restricting the location of the northern treeline. We predict that without significant thermal acclimation, foliage respiration will increase with projected end-of-the-century warming and will likely constrain the future range limits of this important boreal species.

Summary StatementWhite spruce (Picea glauca) needle respiration at the northern limit of the species range is three times higher than at the southern range limit (when measured at 25 {degrees}C). This high carbon cost likely challenges tree survival and contributes to the location of the northern treeline. A general theory for temperature-dependence in biology At present, there is no simple, complete, and first principles-based model for quantitatively describing the full range of observed biological temperature responses. Here, we derive a theory exhibiting these features based on the Eyring-Evans-Polanyi theory governing chemical reaction rates, and which is applicable across all scales from the micro to the macro. Assuming only that the conformational entropy of molecules changes with temperature, we derive a theory for the temperature dependence which takes the form of an exponential function modified by a power-law. Our framework leads to six deductions applicable to any biological trait that depends on temperature, and elucidates novel aspects of universal temperature responses across the tree of life, from quantum to classical scales. All predictions are well supported by data for a wide variety of biological rates and steady states, from molecular to ecological scales and across multiple taxonomic groups. In addition, we provide novel explanations of several empirical relationships including optimal values in temperature response curves.

One-Sentence SummaryWe derive a simple and universal formulae to characterize temperature responses of biological processes across the tree of life. Endometriotic Organoids: A Novel In Vitro Model of Endometriotic Lesion Development. The development and progression of endometriotic lesions are poorly understood, but immune cell dysfunction and inflammation are closely associated with the pathophysiology of endometriosis. A lack of suitable 3D in vitro models permitting the study of interactions between cell types and the microenvironment is a contributing factor. To address this limitation, we developed endometriotic organoids (EO) to explore the role of epithelial-stromal interactions and model peritoneal cell invasion associated with lesion development. Using a non-adherent microwell culture system, spherical organoids were generated with endometriotic epithelial cells (12Z) combined with immortalized endometriotic stromal cells (iEc-ESC) or immortalized uterine stromal cells (iHUF). Organoids self-organized with stromal cells occupying the center and epithelial cells on the periphery of the organoid. Endometriotic organoids (EO), containing iEc-ESC, resulted in the development of stratified 12Z epithelial cells compared to those with iHUF where the 12Z cells developed as a single layered epithelium. Transcriptomic analysis found 4,522 differentially expressed genes (DEG) between EO and 12Z/iHUF organoids, and the top DEG included increased expression of interleukins and prostaglandin synthase enzymes. An overlap of the EO DEG with baboon endometriotic lesions was highly significant. Finally, to mimic invasion of endometrial tissue into the peritoneum, a model was developed using EO and extracellular matrix containing human peritoneal mesothelial cells (LP9). Invasion of EO into the extracellular matrix-LP9 layer was increased in presence of estrogen or THP1-derived proinflammatory macrophages. Taken together, our results strongly support the concept that EO are an appropriate model for dissecting mechanisms that contribute to endometriotic lesion development.

One Sentence SummaryEndometriotic organoids are an appropriate model to study epithelial-stromal interactions and model cell invasion associated with lesion development. Lower limb in sprinters with larger relative mass but not larger normalized moment of inertia Inhomogeneous muscle hypertrophy in response to daily motor executions is a morphological adaptation in humans. Inhomogeneous morphological adaptation would change the mass distribution, which in turn may affect the mechanical difficulty in moving from an inertia perspective; however, the morphological adaptation from the inertia perspective has not been examined. Here we show no corresponding differences in the normalized mass and normalized moment of inertia between the sprinters and the untrained non-sprinters. We analyzed the fat- and water-separated magnetic resonance images from the lower limbs of 11 male sprinters (100 m best time of 10.44-10.83 s) and 12 untrained non-sprinters. The lower-limb relative mass was significantly larger in sprinters (18.7{+/-}0.7% body mass) than in non-sprinters (17.6{+/-}0.6% body mass), while the normalized moment of inertia of the lower limb around the hip in the anatomical position was similar between them (0.044{+/-}0.002 vs. 0.042{+/-}0.002 [a. u.]). The thigh relative mass in sprinters (12.9{+/-}0.4% body mass) was significantly larger than that in non-sprinters (11.9{+/-}0.4% body mass), whereas the shank and foot relative masses were similar. We revealed that the adaptation to sprinting in humans does not relatively increase the mechanical difficulty to swing the lower limb in terms of inertia, even though the lower-limb mass increases, reflecting muscular hypertrophy. This is suggested to be one of the significant advantages of human morphology for adaptability to explosive motor tasks.

NEW & NOTEWORTHYWe compared the lower-limb inertia properties between male sprinters and untrained non-sprinters by using the fat- and water-separated magnetic resonance images. Sprinters had lower limbs with a larger relative mass but not a larger normalized moment of inertia than non-sprinters. We suggest one of the practical advantages of human morphological characteristics for their adaptation to explosive motor tasks, which provides a novel perspective to the literature for understanding human morphological adaptability. The Functional Nanopore Screen: A Versatile High-throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the Functional Nanopore (FuN) screen now provides a quantitative and dynamic read-out of nanopore assembly and function in E. coli. The assay is based on genetically-encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta- and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology and synthetic biology. Ongoing recombination in SARS-CoV-2 revealed through genealogical reconstruction AO_SCPLOWBSTRACTC_SCPLOWThe evolutionary process of genetic recombination has the potential to rapidly change the properties of a viral pathogen, and its presence is a crucial factor to consider in the development of treatments and vaccines. It can also significantly affect the results of phylogenetic analyses and the inference of evolutionary rates. The detection of recombination from samples of sequencing data is a very challenging problem, and is further complicated for SARS-CoV-2 by its relatively slow accumulation of genetic diversity. The extent to which recombination is ongoing for SARS-CoV-2 is not yet resolved. To address this, we use a parsimony-based method to reconstruct possible genealogical histories for samples of SARS-CoV-2 sequences, which enables us to pinpoint specific recombination events that could have generated the data. We propose a statistical framework for disentangling the effects of recurrent mutation from recombination in the history of a sample, and hence provide a way of estimating the probability that ongoing recombination is present. We apply this to samples of sequencing data collected in England and South Africa, and find evidence of ongoing recombination. Neutralization of SARS-CoV-2 Omicron BA.2 by Therapeutic Monoclonal Antibodies Monoclonal antibody therapy for the treatment of SARS-CoV-2 infection has been highly successful in decreasing disease severity; however, the recent emergence of the heavily mutated Omicron variant has posed a challenge to this treatment strategy. The Omicron variant BA.1 has been found to evade neutralization by several of the therapeutic monoclonal antibodies authorized for emergency use, while Vir-7831 and a cocktail consisting of monoclonal antibodies AZD8895+AZD1061 retain significant neutralizing activity. A newly emerged variant, Omicron BA.2, containing some of the BA.1 mutations plus an additional 6 mutations and 3 deletions, 3 of which lie in the receptor binding domain, has been found to be spreading with increased transmissibility. We report here, using spike protein-pseudotyped lentiviruses, decreased neutralization of BA.2 by several therapeutic monoclonal antibodies but that the mixture of AZD8895+AZD1061 retained substantial neutralizing activity against BA.2. SARS-CoV-2 infection induces soluble platelet activation markers and PAI-1 in the early moderate stage of COVID-19 IntroductionCoagulation dysfunction and thromboembolism emerge as strong comorbidity factors in severe COVID-19. However, it is unclear when particularly platelet activation markers and coagulation factors dysregulated during the pathogenesis of COVID-19. Here, we sought to assess the levels of coagulation and platelet activation markers at moderate and severe stages of COVID-19 to understand the pathogenesis.

MethodsTo understand this, hospitalized COVID-19 patients with (severe cases that required intensive care) or without pneumonia (moderate cases) were recruited. Phenotypic and molecular characterizations were performed employing basic coagulation tests including PT, APTT, D-Dimer and TFPI. The flow cytometry-based multiplex assays were performed to assess FXI, anti-thrombin, prothrombin, fibrinogen, FXIII, P-selectin, sCD40L, plasminogen, tissue-plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and D-Dimer.

ResultsThe investigations revealed induction of plasma P-selectin and CD40 ligand (sCD40L) in moderate COVID-19 cases which were significantly abolished with the progression of COVID-19 severity. Moreover, a profound reduction in plasma tissue factor pathway inhibitor (TFPI) and FXIII were identified particularly in the severe COVID-19. Further analysis revealed fibrinogen induction in both moderate and severe patients. Interestingly, an elevated PAI-1 more prominently in moderate, and tPA particularly in severe COVID-19 cases were observed. Particularly, the levels of fibrinogen and tPA directly correlated with the severity of the disease.

ConclusionsIn summary, induction of soluble P-selectin, sCD40L, fibrinogen and PAI-1 in moderate COVID-19 cases suggests the activation of platelets and coagulation system before patients require intensive care. These findings would help in designing better thromboprophylaxis to limit the COVID-19 severity. Notch signaling via Hey1 and Id2b regulates Muller glia's regenerative response to retinal injury Zebrafish Muller glia (MG) respond to retinal injury by suppressing Notch signaling and producing progenitors for retinal repair. A certain threshold of injury-derived signal must be exceeded in order to engage MG in a regenerative response (MGs injury-response threshold). Pan-retinal Notch inhibition expands the zone of injury-responsive MG at the site of focal injury, suggesting that Notch signaling regulates MGs injury-response threshold. We found that Notch signaling enhanced chromatin accessibility and gene expression at a subset of regeneration-associated genes in the uninjured retina. Two Notch effector genes, hey1 and id2b, were identified that reflect bifurcation of the Notch signaling pathway, and differentially regulate MGs injury-response threshold and proliferation of MG-derived progenitors. Furthermore, Notch signaling component gene repression in the injured retina suggests a role for Dll4, Dlb, and Notch3 in regulating Notch signaling in MG and epistasis experiments confirm that the Dll4/Dlb-Notch3-Hey1/Id2b signaling pathway regulates MGs injury-response threshold and proliferation. Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ-9-tetrahydrocannabinol 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. 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. HaloTag9: an engineered protein tag to improve fluorophore performance HaloTag9 is an engineered variant of HaloTag7 with up to 40% higher brightness and increased fluorescence lifetime when labeled with fluorogenic rhodamines. Moreover, combining HaloTag9 with HaloTag7 and other fluorescent probes enabled live-cell multiplexing using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. The increased brightness of HaloTag9 and its use in fluorescence lifetime multiplexing makes it a powerful tool for live-cell imaging. Conserved organ-specific microbial assemblages in different populations of a terrestrial crab Brachyuran crabs originated in the oceans and evolved specific morphological and physiological adaptations to live in freshwater, intertidal and even terrestrial habitats but the role of a selection mechanism involving symbiotic microorganisms long these colonization processes are not known. In this work we investigated the associated microbiota of three populations of a terrestrial brachyuran crab, Chiromantes haematocheir, to find evidence of a conserved crab-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. In spite of the presence of truly marine larval stages and no gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated to the gut and the gills of the crabs (with more than 15% of the genera detected specifically enriched only in one organ). Our results suggest an early establishment of a new common, stable microbiota in the transition from water to land. TeamTree analysis: a new approach to evaluate scientific production Advances in science and technology depend on the work of research teams and the publication of results through peer-reviewed articles representing a growing socio-economic resource. Current methods to mine the scientific literature regarding a field of interest focus on content, but the workforce credited by authorship remains largely unexplored, and appropriate measures of scientific production are debated. Here, a new bibliometric approach named TeamTree analysis is introduced that visualizes the development and composition of the workforce driving a field. A new citation-independent measure that scales with the H index estimates impact based on publication record, genealogical ties and collaborative connections. This author-centered approach complements existing tools to mine the scientific literature and to evaluate research across disciplines. A platform of patient-derived microtumors identifies individual treatment responses and therapeutic vulnerabilities in ovarian cancer. BackgroundIn light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease with complex microenvironment and high recurrence rates, we developed a 3D preclinical ovarian cancer model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) for identification of treatment vulnerabilities and validation of anti-cancer drug efficacy using immunohistochemistry, immune cell phenotyping, functional assays and protein profiling analyses.

MethodsPDM and TILs were isolated from fresh primary ovarian cancer tissue specimen using mechanical disruption and limited enzymatic digestion and were subsequently cultured in suspension in defined media in the absence of serum. The heterogeneous cellular composition of isolated PDM as well as autologous TILs was analyzed by FFPE immunohistochemistry and multi-color flow cytometry, respectively. For in-depth protein profiling of PDM we established Reverse Phase Protein Array (RPPA) analyses of >110 total and phospho-proteins. Treatment efficacy in response to chemotherapeutics as well as immunotherapeutic compounds was assessed in PDM and PDM-TIL co-cultures using a functional viability assay in microplate format.

ResultsThe enzymatic digestion of primary ovarian cancer tissue and suspension culture in defined serum-free media allowed fast and efficient recovery of patient-derived microtumors (PDM). Immunohistochemical analyses demonstrated histopathological comparability of ovarian cancer PDM with corresponding patient tumor tissue. Reverse Phase Protein Array (RPPA)-based analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Finally, combining PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated the potential for patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach.

Conclusion3D patient-derived ovarian cancer microtumors represent a preclinical, ex vivo tumor model that reflects intertumoral heterogeneity and represent the cellular complexity of individual patient tumors. Combining protein pathway analysis and anti-cancer drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision-making and personalizing cancer treatment. The "Alu-ome" shapes the epigenetic environment of regulatory elements controlling cellular defense Promoters and enhancers are sites of transcription initiation (TSSs) and carry active histone modifications, including H3K4me1, H3K4me3, and H3K27ac. Yet, the principles governing the boundaries of such regulatory elements are still poorly characterized. Alu elements are good candidates for a boundary function, being highly abundant in gene-rich regions, while essentially excluded from regulatory elements. Here, we show that the interval from the TSS to the first upstream Alu accommodates all H3K4me3 and most H3K27ac marks, while excluding DNA methylation. Remarkably, the average length of this intervals greatly varies in-between tissues, being longer in stem-and shorter in immune-cells. The very shortest TSS-to-Alu intervals were observed at promoters active in T cells, particularly at immune genes, correlating with RNA polymerase II transcription through the first Alu and accumulation of H3K4me1 signal on this first Alu. Finally, DNA methylation at first-Alus was found to evolved with age, regressing from young to middle-aged, then recovering later in life. Thus, the first Alus upstream of TSSs appear as dynamic boundaries marking the transition from DNA methylation to active histone modifications at regulatory elements, while also participating in the recording of immune gene transcriptional events by positioning H3K4me1-modified nucleosomes. 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. An hidden Markov model to estimate homozygous-by-descent probabilities associated with nested layers of ancestors Inbreeding results from the mating of related individuals and has negative consequences because it brings together deleterious variants in one individual. Genomic estimates of the inbreeding coefficients are preferred to pedigree-based estimators as they measure the realized inbreeding levels and they are more robust to pedigree errors. Several methods identifying homozygous-by-descent (HBD) segments with hidden Markov models (HMM) have been recently developed and are particularly valuable when the information is degraded or heterogeneous (e.g., low-fold sequencing, low marker density, heterogeneous genotype quality or variable marker spacing). We previously developed a multiple HBD class HMM where HBD segments are classified in different groups based on their length (e.g., recent versus old HBD segments) but we recently observed that for high inbreeding levels with many HBD segments, the estimated contributions might be biased towards more recent classes (i.e., associated with large HBD segments) although the overall estimated level of inbreeding remained unbiased. We herein propose a new model in which the HBD classification is modeled in successive nested levels with decreasing expected HBD segment lengths, the underlying exponential rates being directly related to the number of generations to the common ancestor. The non-HBD classes are now modeled as a mixture of HBD segments from later generations and shorter non-HBD segments (i.e., both with higher rates). The new model has improved statistical properties and performs better on simulated data compared to our previous version. We also show that the parameters of the model are easier to interpret and that the model is more robust to the choice of the number of classes. Overall, the new model results in an improved partitioning of inbreeding in different HBD classes and should be preferred. P2X2 receptor subunit interfaces are missense variant hotspots where mutations tend to increase apparent ATP affinity Background and PurposeP2X receptors (P2XRs) are trimeric ligand-gated ion channels (LGICs) that open a cation-selective pore in response to ATP binding to their large extracellular domain (ECD). The seven known P2XR subtypes can assemble as homo- or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2XRs is well established, but little is known about the spectrum and prevalence of human genetic variations and the functional implications in specific domains.

Experimental ApproachHere we examine the impact of P2X2 receptor (P2X2R) inter-subunit interface missense variants identified in the human population or through structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches.

Key resultsWe demonstrate that predicted ECD inter-subunit interfaces display a higher-than-expected density of missense variations and that the majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity.

Conclusions and ImplicationsWe provide the first structural mapping of the mutational burden across the human population in a LGIC and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other LGICs, P2X2Rs have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.

Bullet point summary What is already known: O_LIP2X2 receptors are ATP-activated ion channels implicated in hearing and nociceptice pathways
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 What this study adds: O_LIA structural mapping of missense variants observed in the human population
C_LIO_LIWe identify the intersubunit-interface as a variant hotspot and decipher functional impact of mutations
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 Clinical significance: O_LIThe development of both inhibitors and activators of P2X2 receptor function may be required
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