In Vivo Multi-Day Calcium Imaging of CA1 Hippocampus in Freely Moving Rats Reveals a High Preponderance of Place Cells with Consistent Place Fields Calcium imaging using GCaMP indicators and miniature microscopes has been used to image cellular populations during long timescales and in different task phases, as well as to determine neuronal circuit topology and organization. Because the hippocampus (HPC) is essential for tasks of memory, spatial navigation, and learning, calcium imaging of large populations of HPC neurons can provide new insight on cell changes over time during these tasks. All reported HPC in vivo calcium imaging experiments have been done in mouse. However, rats have many behavioral and physiological experimental advantages over mice. In this paper, we present the first (to our knowledge) in vivo calcium imaging from CA1 hippocampus in freely moving male rats. Using the UCLA Miniscope, we demonstrate that, in rat, hundreds of cells can be visualized and held across weeks. We show that calcium events in these cells are highly correlated with periods of movement, with few calcium events occurring during periods without movement. We additionally show that an extremely large percent of cells recorded during a navigational task are place cells (77.3{+/-}5.0%, surpassing the percent seen during mouse calcium imaging), and that these cells enable accurate decoding of animal position and can be held over days with consistent place fields in a consistent spatial map. A detailed protocol is included, and implications of these advancements on in vivo imaging and place field literature are discussed.

Significance statementIn vivo calcium imaging in freely moving animals allows the visualization of cellular activity across days. In this paper, we present the first in vivo Ca2+ recording from CA1 hippocampus in freely moving rats. We demonstrate that hundreds of cells can be visualized and held across weeks, and that calcium activity corresponds to periods of movement. We show that a high percentage (77.3{+/-}5.0%) of imaged cells are place cells, and that these place cells enable accurate decoding and can be held stably over days with little change in field location. Because the hippocampus is essential for many tasks involving memory, navigation, and learning, imaging of large populations of HPC neurons can shed new insight on cellular activity changes and organization.
Linking individual differences in human primary visual cortex to contrast sensitivity around the visual field A central question in neuroscience is how the organization of cortical maps relates to perception, for which human primary visual cortex (V1) is an ideal model system. V1 nonuniformly samples the retinal image, with greater cortical magnification (surface area per degree of visual field) at the fovea than periphery and at the horizontal than vertical meridian. Moreover, the size and cortical magnification of V1 varies greatly across individuals. Here, we used fMRI and psychophysics in the same observers to quantify individual differences in V1 cortical magnification and contrast sensitivity at the four polar angle meridians. Across observers, the overall size of V1 and localized cortical magnification positively correlated with contrast sensitivity. Moreover, greater cortical magnification and higher contrast sensitivity at the horizontal than the vertical meridian were strongly correlated. These data reveal a link between cortical anatomy and visual perception at the level of individual observer and stimulus location.
Adult fibroblasts use aggresomes only in distinct cell-states The aggresome is a protein turnover system in which proteins are trafficked along microtubules to the centrosome for degradation. Despite extensive focus on aggresomes in immortalized cell lines, it remains unclear if the aggresome is conserved in all primary cells and all cell-states. Here we examined the aggresome in primary adult mouse dermal fibroblasts shifted into four distinct cell-states. We found that in response to proteasome inhibition, quiescent and immortalized fibroblasts formed aggresomes whereas proliferating and senescent fibroblasts did not. Using this model, we generated a resource to better understand the aggresome, providing a characterization of the proteostasis networks in which the aggresome is used and transcriptomic features associated with the presence or absence of aggresome formation. Using this resource, we validate a previously reported role for p38 MAPK signaling in aggresome formation and identify TAK1 as a novel driver of aggresome formation upstream of p38 MAPKs. Together, our data demonstrate that the aggresome is a non-universal protein degradation system which can be used cell-state specifically and provide a resource for better understanding aggresome formation and function. Structural insights into p300 regulation and acetylation-dependent genome organisation Histone modifications are deposited by chromatin modifying enzymes and read out by proteins that recognize the modified state. BRD4-NUT is an oncogenic fusion protein of the acetyl lysine reader BRD4 that binds to the acetylase p300 and enables formation of long-range intra- and interchromosomal interactions. We here examine how acetylation reading and writing enable formation of such interactions. We show that NUT contains an acidic transcriptional activation domain that binds to the TAZ2 domain of p300. We use NMR to investigate the structure of the complex and found that the TAZ2 domain has an autoinhibitory role for p300. NUT-TAZ2 interaction or mutations found in cancer that interfere with autoinhibition by TAZ2 allosterically activate p300. p300 activation results in a self-organizing, acetylation-dependent feed-forward reaction that enables long-range interactions by bromodomain multivalent acetyl-lysine binding. We discuss the implications for chromatin organisation, gene regulation and dysregulation in disease. Natural variations in reproductive timing of brood size and X-chromosome nondisjunction in Caenorhabditis elegans Reproductive timing underlies self-sperm production and X-chromosome nondisjunction rate in Caenorhabditis elegans. These traits can be measured as brood size and male-production rate, and brood size decreases and male-production rate increases as worms age. These phenotypic changes occur simultaneously; however, whether these changes share common underlying genetic architectures still remains unclear. To enhance our understanding of reproductive timing in C. elegans, we measured and compared these two reproductive timing-related traits of 96 wild strains during early, late and total reproductive periods. We found that the two traits exhibited natural phenotypic variation with few outliers and a similar reproductive timing pattern as previous reports. The brood size and the male-production rate were not genetically correlated, implying that they might have different genetic architectures and that meiosis quality can be maintained despite increased progeny-production in the late reproductive period caused by more self-sperm. We also identified loci and candidate genetic variants significantly associated with male-production rate in the late and total reproductive periods. Our results provide an insight into life history traits in wild C. elegans strains. Effect of tag attachment on the flight performance of five raptor species Bio-logging devices play a fundamental and indispensable role in movement ecology studies, particularly in the wild. However, researchers are becoming increasingly aware of the influence that attaching devices can have on animals, particularly on their behaviour, energy expenditure and survival. The way a device is attached to an animals body has also potential consequences for the collected data, and quantifying the type and magnitude of such potential effects is fundamental to enable researchers to combine and compare data from different studies, as much as it is to improve animal welfare.

For over two decades, large terrestrial birds have been in the focus of long-term movement ecology research, employing bio-logging devices attached with different types of harnesses. However, comparative studies investigating the effects of different harness types used on these species are scarce.

In this study, we tested for potential differences in data collected by two commonly used harness types, backpack and leg-loop, on the flight performance of 10 individuals from five raptor species, equipped with high resolution bio-logging devices, in the same area and time. We explored the effect of harness type on vertical speed, horizontal speed, glide ratio, height above sea level, distance travelled, proportion of soaring and flapping behaviour, and VeDBA (a proxy for energy expenditure) between and within individuals, all used as fine-scale measures of flight performance. Birds equipped with leg-loops climbed up to 0.65 ms-1 faster, reached 19% greater heights while soaring, and spent less time in active flight compared to birds equipped with backpacks, suggesting that backpack harnesses, compared to leg-loops, might cause additional drag affecting the birds flight performance. A lower rate of sinking while gliding, a slightly higher glide ratio, higher horizontal speed while soaring, and lower VeDBA, were also indicative of less drag using leg-loops. Our results add to the existing literature highlighting the design-related advantages of leg-loops, and support the use of leg-loops as a better alternative to backpack harnesses for large soaring birds, when possible. Our study also highlights how apparently small changes in device attachment can lead to notable improvements in tagging practice, with implications for animal welfare, data interpretation and comparability. Generation of a microsporidia species attribute database and analysis of the extensive ecological and phenotypic diversity of microsporidia Microsporidia are a large group of fungal-related obligate intracellular parasites. Though many microsporidia species have been identified over the past 160 years, there is a lacking depiction of the full diversity of this phylum. To systematically describe the characteristics of these parasites, we created a database of 1,440 species and their attributes, including the hosts they infect and spore characteristics. We find that microsporidia have been reported to infect 16 metazoan and four protozoan phyla, with smaller phyla being underrepresented. Most species are only reported to infect a single host, but those that are generalists are also more likely to infect a broader set of host tissues. Strikingly, polar tubes are 3-fold longer in species that infect tissues besides the intestine, suggesting that polar tube length is a determinant of tissue specificity. Phylogenetic analysis revealed four clades which each contain microsporidia infecting hosts from all major habitats. Although related species are more likely to infect similar hosts, we observe examples of changes in host specificity and convergent evolution. Taken together, our results show that microsporidia display vast diversity in their morphology and the hosts they infect, illustrating the flexibility of these parasites to evolve new traits. Embedded 3D printing in self-healing annealable composites for precise patterning of functionally mature human neural constructs Human in vitro models of neural tissue with controllable cellular identity, tunable microenvironment, and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing in jammed microgel supports (i.e., granular gels) holds great promise as it allows precise and programmable patterning of extremely soft and compliant tissue constructs. However, in contrast to the vast material landscape available for bulk hydrogels, granular printing support formulations are restricted to a handful of materials without the ability for facile adjustment of biofunctional properties of the cellular microenvironment. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high-fidelity support for embedded printing akin to granular gels. To address this need, we present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular-matrix solution to enable precise freeform patterning of human stem cells and consequent generation and long-term maintenance of mature subtype-specific neurons that extend projections within the volume of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular channels. Due to its modularity, SHAPE biomanufacturing toolbox not only offers a solution for functional modeling of mechanically sensitive neural constructs, but also has potential to be applied to a wide range of biomaterials with different crosslinking mechanisms to model tissues and diseases where recapitulation of complex architectural features and topological cues is essential. 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. Single-nucleus RNA Sequencing and Spatial Transcriptomics Reveal the Immunological Microenvironment of Cervical Squamous Cell Carcinoma Effective treatment of advanced invasive cervical cancer remains challenging nowadays. Herein, single-nucleus RNA sequencing (snRNA-seq) and SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq) technology are used to investigate the immunological microenvironment of cervical squamous cell carcinoma (CSCC), a major type of cervical cancers. The expression levels of most immune checkpoint genes in tumor and inflammation areas of CSCC were not significantly higher than those in the non-cancer samples except for LGALS9 and IDO1. Stronger signals of CD56+ NK cells and immature dendritic cells are found in the hypermetabolic tumor areas, while more eosinophils, immature B cells, and Treg cells are found in the hypometabolic tumor areas. Moreover, a cluster of cancer-associated fibroblasts (CAFs) are identified around some tumors, which highly expressed ACTA2, POSTN, ITGB4, and FAP. The CAFs might support the growth and metastasis of tumors by inhibiting lymphocyte infiltration and remodeling the tumor extracellular matrix. Furthermore, CAFs are associated with poorer survival probability in CSCC patients and might be present in a small fraction ([~]20%) of advanced cancer patients. Collectively, these findings might enhance understanding of the CSCC immunological microenvironment and shed some light on the treatment of advanced CSCC. Modulations in motor unit discharge are related to changes in fascicle length during isometric contractions The integration of electromyography (EMG) and ultrasound imaging has provided important information about the mechanisms of muscle activation and contraction. Unfortunately, EMG does not allow an accurate assessment of the interplay between the neural drive received by muscles, changes in fascicle length (FL) and the force/torque produced. We aimed to assess the relationship between modulations in tibialis anterior (TA) motor unit (MU) firing rate, FL and dorsiflexion torque (DT) using ultrasound-transparent high-density EMG electrodes. EMG and ultrasound images were recorded simultaneously from TA, using a 32-electrode silicon matrix, while performing isometric dorsiflexion, at diverse ankle joint positions (0{degrees} and 30{degrees} plantar flexion) and torques (20% and 40% of maximum). EMG signals were decomposed into individual MUs and changes in FL were assessed with a fascicle-tracking algorithm. MU firings were converted into a cumulative spike train (CST) that was cross-correlated with DT (CST-DT) and FL (CST-FL). High cross-correlations were found for CST-FL, 0.60 (range: 0.31-0.85) and CST-DT 0.71 (range: 0.31-0.88). Cross-correlation lags revealed that the delay between CST-FL (~75ms) was significantly smaller than CST-DT (~150ms, p<0.001). These delays affected the interpretation of MU recruitment/de-recruitment thresholds, with FL showing similar lengths for both recruitment and de-recruitment. This study is the first to demonstrate that changes in TA FL are closely related to both modulations in MU firing frequency and DT. These relationships allow assessment of the interplay between neural drive, muscle contraction and resultant torque, thereby providing a better understanding of the mechanisms responsible for the generation of muscle force.

NEW AND NOTEWORTHYBy employing ultrasound-transparent high-density surface EMG electrodes, we show that modulations in tibialis anterior motor unit discharge rate were closely related to both changes in its fascicle length and resultant torque. These relationships allowed quantifying delays between neural drive and muscle shortening as well as muscle shortening and torque during submaximal isometric contractions, providing an accurate estimation of the time required to generate muscle force and subsequent production of torque via the tendon. A novel Boolean network inference strategy to model early hematopoiesis aging Hematopoietic stem cell (HSC) aging is a multifactorial event that leads to changes in HSC properties and function. These changes are intrinsically coordinated and affect the early hematopoiesis, involving hematopoietic stem and progenitor cells (HSPCs). The objective of this work is to better understand the mechanisms and factors controlling these changes. We have therefore developed an original strategy to construct a Boolean network of genes explaining the priming and homeostasis of HSCs (graphical abstract). Based on our previous scRNA-seq data, we performed an exhaustive analysis of the transcriptional network and identified active transcription modules or regulons along the differentiation trajectory of selected HSPC states. This global view of transcriptional regulation led us to focus on 15 components, 13 selected TFs (Tal1, Fli1, Gata2, Gata1, Zfpm1, Egr1, Junb, Ikzf1, Myc, Cebpa, Bclaf1, Klf1, Spi1) and 2 complexes regulating the ability of HSC to cycle (CDK4/6 - Cyclin D and CIP/KIP). We then defined the connections controlling the differentiation dynamics of HSC states and constructed an influence graph between the TFs involved in the dynamics by mixing observations from our scRNA-seq data and knowledge from the literature. Then, using answer set programming (ASP) and in silico perturbation analysis, we obtained a Boolean model which is the solution of a Boolean satisfiability problem. Finally, perturbation of the model based on age-related changes revealed important regulations, such as the overactivation of Egr1 and Junb or the loss of Cebpa activation by Gata2, which were found to be relevant for the myeloid bias of aged HSC. Our work shows the efficiency of the combination of manual and systematic methods to elaborate a Boolean model. The developed strategy led to the proposal of new regulatory mechanisms underlying the differentiation bias of aged HSCs, explaining the decreased transcriptional priming of HSCs to all mature cell types except megakaryocytes. 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. Robust and annotation-free analysis of alternative splicing using short-read scRNA-seq data Although alternative splicing is a fundamental and pervasive aspect of gene expression in higher eukaryotes, it is often omitted from single-cell studies due to quantification challenges inherent to commonly used short-read sequencing technologies. Here, we undertake the analysis of alternative splicing across numerous diverse murine cell types from two large-scale single-cell datasets--the Tabula Muris and BRAIN Initiative Cell Census Network--while accounting for understudied technical artifacts and unannotated isoforms. We find strong and general cell-type-specific alternative splicing, complementary to total gene expression but of similar discriminatory value, and identify a large volume of novel isoforms. We specifically highlight splicing variation across different cell types in primary motor cortex neurons, bone marrow B cells, and various epithelial cells; and show that the implicated transcripts include many genes which do not display total expression differences. To elucidate the regulation of alternative splicing, we build a custom predictive model based on splicing factor activity, recovering several known interactions while generating new hypotheses, including potential regulatory roles for novel alternative splicing events in critical genes including Khdrbs3 and Rbfox1. We make our results available using public interactive browsers to spur further exploration by the community. Embryo development in Carica papaya Linn. Papaya (Carica papaya Linn.) is a tropical plant whose draft genome has been sequenced. Papaya produces large fruits rich in vitamins A and C and is an important cash crop in developing countries. Nonetheless, little is known about how the female gametophyte develops, how it is fertilized and how it develops into a mature seed containing an embryo and an endosperm. The Papaya female gametophyte displays a Polygonum-type architecture consisting of two synergid cells, an egg cell, a central cell, and three antipodal cells. Reports are available of the presumed existence of varieties in which cross fertilization is bypassed and autonomous development of embryos occurs (e.g., apomixis). In this study, we analyzed the development of female gametophytes in a commercial Hawaiian parental line and in the presumed apomictic Costa Rican line L1. Samples were collected before and after anthesis to compare the overall structure, size and transcriptional patterns of several genes that may be involved in egg and endosperm cell fate and proliferation. These genes were the putative papaya homologs of ARGONAUTE9 (AGO9), MEDEA (MEA), RETINOBLASTOMA RELATED-1 (RBR1), and SLOW WALKER-1 (SWA1). Our results suggest that its feasibly to identify structural features of Polygonum-type development, and that in bagged female flowers of line L1 we might have observed autonomous development of embryo-like structures. Possible downregulation of papaya homologs for AGO9, MEA, RBR1 and SWA1 was observed in embryo sacs from line L1 before and after anthesis, which may suggest a link between suspected apomixis and transcriptional downregulation of genes for RNA-directed DNA methylation, histone remodelers, and rRNA processing. Most notably, the large size of the papaya embryo sac suggests that it could be a cytological alternative to Arabidopsis thaliana for study. Significant variation in embryo sac size was observed between the varieties under study, suggesting wide differences in the genetic regulation of anatomical features. Constraints on the evolution of toxin-resistant Na,K-ATPases have limited dependence on sequence divergence A growing body of theoretical and experimental evidence suggests that intramolecular epistasis is a major determinant of rates and patterns of protein evolution and imposes a substantial constraint on the evolution of novel protein functions. Here, we examine the role of intramolecular epistasis in the recurrent evolution of resistance to cardiotonic steroids (CTS) across tetrapods, which occurs via specific amino acid substitutions to the -subunit family of Na,K-ATPases (ATP1A). After identifying a series of recurrent substitutions at two key sites of ATP1A that are predicted to confer CTS resistance in diverse tetrapods, we then performed protein engineering experiments to test the functional consequences of introducing these substitutions onto divergent species backgrounds. In line with previous results, we find that substitutions at these sites can have substantial background-dependent effects on CTS resistance. Globally, however, these substitutions also have pleiotropic effects that are consistent with additive rather than background-dependent effects. Moreover, the magnitude of a substitutions effect on activity does not depend on the overall extent of ATP1A sequence divergence between species. Our results suggest that epistatic constraints on the evolution of CTS-resistant forms of Na,K-ATPase likely depend on a small number of sites, with little dependence on overall levels of protein divergence. We propose that dependence on a limited number sites may account for the observation of convergent CTS resistance substitutions observed among taxa with highly divergent Na,K-ATPases.

Significance StatementIndividual amino acid residues within a protein work in concert to produce a functionally coherent structure that must be maintained even as orthologous proteins in different species diverge over time. Given this dependence, we expect identical mutations to have more similar effects on protein function in more closely related species. We tested this hypothesis by performing protein-engineering experiments on ATP1A, an enzyme mediating target-site insensitivity to cardiotonic steroids (CTS) in diverse animals. These experiments reveal that the phenotypic effects of substitutions can sometimes be background-dependent, but also that the magnitude of these phenotypic effects does not correlate with overall levels of ATP1A sequence divergence. Our results suggest that epistatic constraints are determined by states at a small number of sites, potentially explaining the frequent convergent CTS resistance substitutions among Na,K-ATPases of highly divergent taxa. Embryonic exposure to cannabidiol disrupts active neural circuits, an effect increased by Δ9-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. Thermodynamic constraints on the assembly and diversity ofmicrobial ecosystems are different near to and far fromequilibrium Non-equilibrium thermodynamics has long been an area of substantial interest to ecologists because most fundamental biological processes, such as protein synthesis and respiration, are inherently energy-consuming. However, most of this interest has focused on developing coarse ecosystem-level maximisation principles, providing little insight into underlying mechanisms that lead to such emergent constraints. Microbial communities are a natural system to decipher this mechanistic basis because their interactions in the form of substrate consumption, metabolite production, and cross-feeding can be described explicitly in thermodynamic terms. Previous work has considered how thermodynamic constraints impact competition between pairs of species, but restrained from analysing how this manifests in complex dynamical systems. To address this gap, we develop a thermodynamic microbial community model with fully reversible reaction kinetics, which allows direct consideration of free-energy dissipation. This also allows species to interact via products rather than just substrates, increasing the dynamical complexity, and allowing a more nuanced classification of interaction types to emerge. Using this model, we find that community diversity increases with substrate lability, because greater free-energy availability allows for faster generation of niches. Thus, more niches are generated in the time frame of community establishment, leading to higher final species diversity. We also find that allowing species to make use of near-to-equilibrium reactions increases diversity in a low free-energy regime. In such a regime, two new thermodynamic interaction types that we identify here reach comparable strengths to the conventional (competition and facilitation) types, emphasising the key role that thermodynamics plays in community dynamics. Our results suggest that accounting for realistic thermodynamic constraints is vital for understanding the dynamics of real-world microbial communities.

Author summaryThere is a growing interest in microbial communities due to their important role in biogeochemical cycling as well as plant and animal health. Although our understanding of thermodynamic constraints on individual cells is rapidly improving, the impact of these constraints on complex microbial communities remains largely unexplored theoretically and empirically. Here, we develop a new microbial community model which allows thermodynamic efficiency and entropy production to be calculated directly. We find that availability of substrates with greater free-energy allows for a faster rate of niche generation, leading to higher final species diversity. We also show that when the free-energy availability is low, species with reactions close to thermodynamic equilibrium are favoured, leading to more diverse and efficient communities. In addition to the conventional interaction types (competition and facilitation), our model reveals the existence of two novel interaction types mediated by products rather than substrates. Though the conventional interactions are generally the strongest, the novel interaction types are significant when free-energy availability is low. Our results suggest that non-equilibrium thermodynamics need to be considered when studying microbial community dynamics. An interplay between cellular growth and atypical fusion defines morphogenesis of a modular glial niche Neural stem cells (NSCs) are embedded in a multi-layered, intricate cellular microenvironment supporting their activity, the niche. Whilst shape and function are inseparable, the morphogenetic aspects of niche development are poorly understood. Here, we use the formation of the glial network of a NSC niche to investigate acquisition of architectural complexity. Cortex glia (CG) in Drosophila regulate neurogenesis and build a reticular structure around NSCs. We first show that individual CG cells grow tremendously to ensheath several NSC lineages, eventually spanning the entire tissue while partitioning the NSC population. Elaborate proliferative mechanisms convert these cells into syncytia rich in cytoplasmic bridges. Unexpectedly, CG syncytia further undergo homotypic cell-cell fusion, relying on defined molecular players of cell fusion such as cell surface receptors and actin regulators. Exchange of cellular components is however dynamic in space and time, a previously unreported unique mechanism. This atypical cell fusion remodels cellular borders, restructuring the CG syncytia. Ultimately, the coordination of growth and fusion builds the multi-level architecture of the niche, and creates a modular, spatial partition of the NSC population. Our findings provide novel insights into how a niche forms and organises while developing intimate contacts with a stem cell population. 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. A Bayesian method to cluster single-cell RNA sequencing data using Copy Number Alterations MotivationCancers are composed by several heterogeneous subpopulations, each one harbouring different genetic and epigenetic somatic alterations that contribute to disease onset and therapy response. In recent years, copy number alterations leading to tumour aneuploidy have been identified as potential key drivers of such populations, but the definition of the precise makeup of cancer subclones from sequencing assays remains challenging. In the end, little is known about the mapping between complex copy number alterations and their effect on cancer phenotypes.

ResultsWe introduce CONGAS, a Bayesian probabilistic method to phase bulk DNA and single-cell RNA measurements from independent assays. CONGAS jointly identifies clusters of single cells with subclonal copy number alterations, and differences in RNA expression. The model builds statistical priors leveraging bulk DNA sequencing data, does not require a normal reference and scales fast thanks to a GPU backend and variational inference. We test CONGAS on both simulated and real data, and find that it can determine the tumour subclonal composition at the single-cell level together with clone-specific RNA phenotypes in tumour data generated from both 10x and Smart-Seq assays.

AvailabilityCONGAS is available as 2 packages: CONGAS (https://github.com/caravagnalab/congas), which implements the model in Python, and RCONGAS (https://caravagnalab.github.io/rcongas/), which provides R functions to process inputs, outputs, and run CONGAS fits. The analysis of real data and scripts to generate figures of this paper are available via RCONGAS; code associated to simulations is available at https://github.com/caravagnalab/rcongas_test.

Contactgcaravagna@units.it

Supplementary informationSupplementary data are available at Bioinformatics online. Robust retrieval of data stored in DNA by de Bruijn graph-based de novo strand assembly DNA data storage is a rapidly developing technology with great potential due to its high density, long-term durability, and low maintenance cost. The major technical challenges include various errors, such as the strand breaks, rearrangements, and indels that frequently arise during DNA synthesis, amplification, sequencing, and preservation. Through a de novo assembly strategy, we developed an algorithm based on the de Bruijn graph and greedy path search (DBGPS) to address these issues. DBGPS shows distinct advantages in handling DNA breaks, rearrangements, and indels. The robustness of DBGPS is demonstrated by accelerated aging, multiple independent data retrievals, deep error-prone PCR, and large data scale simulations. Remarkably, 6.8 MB of data can be retrieved accurately from a seriously corrupted sample that has been treated at 70 {degrees}C for 70 days. With DBGPS, we were able to achieve a logical density of 1.30 bits/cycle and a physical density of 295 PB/g.

One-Sentence SummaryA de Bruijn graph-based de novo assembly algorithm for DNA data storage enables fast and robust data readouts even with DNA samples that have been severely corrupted. Changes in Behavior and Salivary Serum Amyloid A in cows with Subclinical Mastitis The aim of this study was to identify detailed changes in behavior, and in salivary serum amyloid A (SAA), associated with subclinical mastitis. This included standard sickness behaviors (which form part of an adaptive response to conserve energy, minimize heat loss and facilitate recovery following infection and inflammation) and lesser-studied behaviors, that are biologically predicted to change at low-level infection, and therefore particularly relevant for detecting subclinical mastitis (here labelled  luxury behaviors). SAA is a physiological marker of systemic inflammation, with levels in milk and serum already known to increase during subclinical mastitis. We investigated whether the same was true for SAA in cow saliva. Data were collected for n = 34 commercial barn-housed dairy cows, tested in 17 matched-pairs. Each pair comprised a cow with subclinical mastitis (SCM) and a healthy control (CTRL), identified using somatic cell count (SCC) (SCM: SCC >200 x1000 cells/ml; CTRL: SCC <100 x1000 cells/ml). SCM cows were selected for study ad-hoc, at which point they were paired with a CTRL cow, based upon parity and calving date; consequently, the full data set was accrued over several months. Data were collected for each pair over 3 days: SCC (l4:00-l5:00h) Day 1; behavior (24h from 00:00h) Day 2; salivary serum amyloid-A (SAA) Day 3. We report, for the first time, that an increase in salivary SAA occurs during subclinical mastitis; SAA was higher in SCM cows and demonstrated a positive (weak) correlation with SCC. The behavioral comparisons revealed that SCM cows had reductions in activity (behavioral transitions and distance moved), social exploration, social reactivity (here: likelihood to move away/be displaced following receipt of agonism), performance of social grooming and head butts, and the receipt of agonistic non-contact challenges. In addition, SCM cows received more head swipes, and spent a greater proportion of time lying with their head on their flank than CTRL cows. SCM cows also displayed an altered feeding pattern; they spent a greater proportion of feeding time in direct contact with two conspecifics, and a lower proportion of feeding time at self-locking feed barriers, than CTRL cows. Behavioral measures were found to correlate, albeit loosely, with serum SAA in a direction consistent with predictions for sickness behavior. These included positive correlations with both lying duration and the receipt of all agonistic behavior, and negative correlations with feeding, drinking, the performance of all social and all agonistic behavior, and social reactivity. We conclude that changes in salivary SAA, social behavior, and activity offer potential in the detection of subclinical mastitis and recommend further investigation to substantiate and refine our findings. Reduction of protein disulfide isomerase results in open conformations and stimulates dynamic exchange between structural ensembles Protein disulfide isomerase (PDI) is a ubiquitous redox-regulated enzyme that interacts with hundreds of client proteins intracellularly and extracellularly. It comprises two redox-sensitive domains, each hosting the conserved catalytic motif CxxC, two redox-insensitive protein-binding domains, and three linkers. Snapshots of oxidized and reduced PDI have been obtained by X-ray crystallography. Yet, how PDIs structure dynamically changes in response to the redox microenvironment and ligand binding remain unknown. Here, we used multiparameter confocal single-molecule Forster resonance energy transfer (smFRET) and multiple FRET pairs to track the movements of the two catalytic domains with high temporal resolution. Our studies document that, at equilibrium, PDI visits three structurally distinct conformational ensembles, two "open" (O1 and O2) and one "closed" (C). We show that the redox environment dictates the time spent in each ensemble and the rate at which they exchange. While oxidized PDI samples O1, O2 and C more evenly and in a slower fashion, reduced PDI predominantly populates O1 and O2, and exchanges between them more rapidly, on the sub-millisecond timescale. These findings were not expected based on crystallographic data. Using mutational analyses, we further demonstrate that the two active sites are structurally nonequivalent and that ligands targeting the active sites of reduced PDI shift the equilibrium towards closed conformations of the enzyme. This work introduces a new structural framework that challenges current views of PDI dynamics, helps rationalize the multifaced role of PDI in biology and may assist drug development. Synthetic Antigen Presenting cells reveal the diversity and functional specialisation of extracellular vesicles composing the fourth signal of T cell immunological synapses. The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four.

Graphical abstract

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org.highwire.dtl.DTLVardef@100f3aorg.highwire.dtl.DTLVardef@57adfforg.highwire.dtl.DTLVardef@6052fcorg.highwire.dtl.DTLVardef@1e85c21_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles.
C_LIO_LIBSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV.
C_LIO_LItSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs.
C_LI Chromatin remodelling by INO80 at promoter proximal pause sites promotes premature termination of mRNA synthesis How co-transcriptional RNA quality control is regulated remains poorly understood. Here, we report that in S. cerevisiae premature transcription termination of mRNAs is regulated by the evolutionarily conserved ATP-dependent chromatin remodeling INO80 complex. Loss of INO80 leads to an increase in promoter-proximally paused RNA Polymerase II and defective progression into the gene body. We show that promoter-proximal transcriptional pausing correlates with loading of RNA surveillance and transcription termination factors to mRNA transcripts. Cells lacking INO80 are defective for the Nrd1-Nab3-Sen1 (NNS)-dependent pathway for transcription termination at snRNA genes and promoter-proximally sites of mRNA genes. We demonstrate that INO80 promotes the association of the RNA surveillance and termination factor Nab2 with short promoter-proximal mRNA transcripts. We provide evidence that co-transcriptional recruitment of Nab2 to chromatin is regulated by INO80, which enables the interaction of Nab2 with the histone variant H2A.Z. Our work suggests a chromatin mechanism for premature transcription termination at promoter-proximally pausing sites, linking RNA quality control to the transcriptional process.