title: Bmper is required for morphogenesis of the anterior and posterior semicircular canal ducts in the developing zebrafish inner ear

abstract: BMP signalling is known to have a conserved function in development of the semicircular canal system of the vertebrate inner ear, but its regulation, target genes and effects on cell behaviour during otic morphogenesis are not fully understood. We have characterised the effects of mutations in the zebrafish gene bmper, which codes for a regulator of BMP signalling with both pro- and anti-BMP roles in different developmental contexts. The inner ears of bmper mutant embryos develop with truncations of the anterior and posterior semicircular canal ducts. To image the developing ear in live embryos, we have exploited a new transgenic line, Tg(smad6b:EGFP), which exhibits strong GFP expression in the otic epithelium. Morphometric analysis indicates defects in the bmper mutant ear from early stages of semicircular canal formation, correlating with a specific reduction in BMP signalling activity and specific loss of dlx5a expression in dorsal otic epithelium. Subsequent changes to cell shape occur at the truncation site and the dorsolateral septum. The bmper mutations that we describe are adult viable; truncation of the anterior and posterior semicircular canal ducts persists into adulthood. Our results argue against a major role for Bmper in specification of the pre-placodal region, induction of the otic placode, or development of the neural crest, processes in which Bmper function has previously been implicated. Instead, we conclude that a key requirement for Bmper function in the zebrafish is to promote BMP signalling during patterning and morphogenesis of the semicircular canal system.


title: Where there's smoke, there's fuel: predicting Great Basin rangeland wildfire

abstract: Wildfires are a growing management concern in western US rangelands, where invasive annual grasses have altered fire regimes and contributed to an increased incidence of catastrophic large wildfires. Fire activity in arid, non-forested ecosystems is thought to be largely controlled by interannual variation in fuel amount, which in turn is controlled by antecedent weather. Thus, long-range forecasting of fire activity in rangelands should be feasible given annual estimates of fuel quantity. Using a 32 yr time series of spatial data, we employed machine learning algorithms to predict the relative probability of large (>405 ha) wildfire in the Great Basin based on fine-scale annual and 16-day estimates of cover and production of vegetation functional groups, weather, and multitemporal scale drought indices. We evaluated the predictive utility of these models with a leave-one-year-out cross-validation, building spatial hindcasts of fire probability for each year that we compared against actual footprints of large wildfires. Herbaceous aboveground biomass production, bare ground cover, and long-term drought indices were the most important predictors of burning. Across 32 fire seasons, 88% of the area burned in large wildfires coincided with the upper 3 deciles of predicted fire probabilities. At the scale of the Great Basin, several metrics of fire activity were moderately to strongly correlated with average fire probability, including total area burned in large wildfires, number of large wildfires, and maximum fire size. Our findings show that recent years of exceptional fire activity in the Great Basin were predictable based on antecedent weather-driven growth of fine fuels and reveal a significant increasing trend in fire probability over the last three decades driven by widespread changes in fine fuel characteristics.

category: ecology


title: Where there's smoke, there's fuel: dynamic vegetation data improve predictions of wildfire hazard in the Great Basin


title: Host-parasite coevolution promotes innovation through deformations in fitness landscapes

abstract: During the struggle for survival, populations occasionally evolve new functions that give them access to untapped ecological opportunities. Theory suggests that coevolution between species can promote the evolution of such innovations by deforming fitness landscapes in ways that open new adaptive pathways. We directly tested this idea by using high throughput gene editing-phenotyping technology (MAGE-Seq) to measure the fitness landscape of a virus, bacteriophage {lambda}, as it coevolved with its host, the bacterium Escherichia coli. Through computer simulations of {lambda}s evolution on the empirical fitness landscape, we showed that {lambda} was more likely to evolve to use a new receptor if it experienced a shift in its fitness landscape caused by coevolution. This result was further validated by additional laboratory experiments. This study provides direct evidence for the role of coevolution in driving evolutionary novelty and provides a quantitative framework for predicting evolution in coevolving ecological communities.

category: evolutionary biology


title: Mechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens

abstract:
Several bacterial human pathogens secrete nucleotidyl cyclase toxins, that are activated by interaction with actin of the eukaryotic host cells. However, the underlying molecular mechanism of this process which protects bacteria from self-intoxication is unclear. Here, we report structures of ExoY from Pseudomonas aeruginosa and Vibrio vulnificus in complex with their corresponding activators F-actin and profilin-G-actin. The structures reveal that in contrast to the apo state, two flexible regions become ordered and interact strongly with actin. The specific stabilization of these regions results in an allosteric stabilization of the distant nucleotide binding pocket and thereby to an activation of the enzyme. Differences in the sequence and conformation of the actin-binding regions are responsible for the selective binding to either F- or G-actin. This specificity can be biotechnologically modulated by exchanging these regions from one toxin to the other.

Other bacterial nucleotidyl cyclases, such as the anthrax edema factor and CyaA from Bortedella pertussis, that bind to calmodulin undergo a similar disordered-to-ordered transition during activation, suggesting that the allosteric activation-by-stabilization mechanism of ExoY is paradigmatic for all bacterial nucleotidyl cyclase toxins.

category: biochemistry


title: Prediction of antibiotic resistant strains of bacteria from their beta-lactamases protein

abstract:
Number of beta-lactamase variants have ability to deactivate ceftazidime antibiotic, which is the most commonly used antibiotic for treating infection by Gram-negative bacteria. In this study an attempt has been made to develop a method that can predict ceftazidime resistant strains of bacteria from amino acid sequence of beta-lactamases. We obtained beta-lactamases proteins from the {beta}-lactamase database, corresponding to 87 ceftazidime-sensitive and 112 ceftazidime-resistant bacterial strains. All models developed in this study were trained, tested, and evaluated on a dataset of 199 beta-lactamases proteins. We generate 9149 features for beta-lactamases using Pfeature and select relevant features using different algorithms in scikit-learn package. A wide range of machine learning techniques (like KNN, DT, RF, GNB, LR, SVC, XGB) has been used to develop prediction models. Our random forest-based model achieved maximum performance with AUROC of 0.80 on training dataset and 0.79 on the validation dataset. The study also revealed that ceftazidime-resistant beta-lactamases have amino acids with non-polar side chains in abundance. In contrast, ceftazidime-sensitive beta-lactamases have amino acids with polar side chains and charged entities in abundance. Finally, we developed a webserver "ABCRpred", for the scientific community working in the era of antibiotic resistance to predict the antibiotic resistance/susceptibility of beta-lactamase protein sequences. The server is freely available at (http://webs.iiitd.edu.in/raghava/abcrpred/).

Key PointsO_LICeftazidime is commonly used to treat infection caused by Gram-negative bacteria.
C_LIO_LIBeta-lactamase is responsible for lysing ceftazidime, make it resistant to bacteria.
C_LIO_LIComparison of resistant and sensitive variants of beta-lactamase.
C_LIO_LIClassification of sensitive and resistant strain of bacteria based on beta-lactamase.
C_LIO_LIPrediction models have been developed using different machine learning techniques.
C_LI

category: bioinformatics


title: Macroscale structural changes of thylakoid architecture during high light acclimation in Chlamydomonas reinhardtii

abstract: Photoprotection mechanisms are ubiquitous among photosynthetic organisms. The photoprotection capacity of the green alga Chlamydomonas reinhardtii is correlated with protein levels of stress-related light-harvesting complex (LHCSR) proteins, which are strongly induced by high light (HL). However, the dynamic response of overall thylakoid structure during acclimation to growth in HL has not been characterized. Here, we combined live-cell super-resolution microscopy and analytical membrane subfractionation to investigate macroscale structural changes of thylakoid membranes during HL acclimation in C. reinhardtii. Subdiffraction-resolution bioimaging revealed that overall thylakoid structures became thinned and shrunken during HL acclimation. The stromal space around the pyrenoid also became enlarged. Analytical density-dependent membrane fractionation indicated that the structural changes were partly a consequence of membrane unstacking. The analysis of both an LHCSR loss-of-function mutant, npq4 lhcsr1, and a regulatory mutant that over-expresses LHCSR, spa1-1, showed that structural changes occurred independently of LHCSR protein levels, demonstrating that LHCSR was neither necessary nor sufficient to induce the thylakoid structural changes associated with HL acclimation. In contrast, stt7-9, a mutant lacking a kinase of major light-harvesting antenna proteins, had a distinct thylakoid structural response during HL acclimation relative to all other lines tested. Thus, while LHCSR and the antenna protein phosphorylation are core features of HL acclimation, it appears that only the latter acts as a determinant for thylakoid structural rearrangements. These results indicate that two independent mechanisms occur simultaneously to cope with HL conditions. Possible scenarios for HL-induced thylakoid structural changes are discussed.

category: plant biology


title: Herbicidal activity of fluoroquinolone derivatives

abstract: Development of herbicides with novel modes of action are crucial for weed control and to hinder herbicide resistance. An attractive novel herbicidal target is plant DNA gyrase, which has been demonstrated to be effectively inhibited by the known antimicrobial ciprofloxacin. Despite this good herbicidal activity ciprofloxacin is not suitable as a herbicide due to its antimicrobial activity, therefore, a diverse library of analogues was analysed to gain insight into the aspects required for herbicidal activity. This analysis revealed significant structural modifications were tolerated and that the fluoride at C-6 and a cyclic amino group at C-7 were not crucial for herbicidal activity. The analysis also revealed that these modifications also affected the antibacterial activity with one compound demonstrating good herbicidal activity and weak antibacterial activity, against both Gram-positive and Gram-negative bacteria.


title: Metabolic resilience is encoded in genome plasticity

abstract: Metabolism plays a central role in evolution, as resource conservation is a selective pressure for fitness and survival. Resource-driven adaptations offer a good model to study evolutionary innovation more broadly. It remains unknown how resource-driven optimization of genome function integrates chromatin architecture with transcriptional phase transitions. Here we show that tuning of genome architecture and heterotypic transcriptional condensates mediate resilience to nutrient limitation. Network genomic integration of phenotypic, structural, and functional relationships reveals that fat tissue promotes organismal adaptations through metabolic acceleration chromatin domains and heterotypic PGC1A condensates. We find evolutionary adaptations in several dimensions; low conservation of amino acid residues within protein disorder regions, nonrandom chromatin location of metabolic acceleration domains, condensate-chromatin stability through cis-regulatory anchoring and encoding of genome plasticity in radial chromatin organization. We show that environmental tuning of these adaptations leads to fasting endurance, through efficient nuclear compartmentalization of lipid metabolic regions, and, locally, human-specific burst kinetics of lipid cycling genes. This process reduces oxidative stress, and fatty-acid mediated cellular acidification, enabling endurance of condensate chromatin conformations. Comparative genomics of genetic and diet perturbations reveal mammalian convergence of phenotype and structural relationships, along with loss of transcriptional control by diet-induced obesity. Further, we find that radial transcriptional organization is encoded in functional divergence of metabolic disease variant-hubs, heterotypic condensate composition, and protein residues sensing metabolic variation. During fuel restriction, these features license the formation of large heterotypic condensates that buffer proton excess, and shift viscoelasticity for condensate endurance. This mechanism maintains physiological pH, reduces pH-resilient inflammatory gene programs, and enables genome plasticity through transcriptionally driven cell-specific chromatin contacts. In vivo manipulation of this circuit promotes fasting-like adaptations with heterotypic nuclear compartments, metabolic and cell-specific homeostasis. In sum, we uncover here a general principle by which transcription uses environmental fluctuations for genome function, and demonstrate how resource conservation optimizes transcriptional self-organization through robust feedback integrators, highlighting obesity as an inhibitor of genome plasticity relevant for many diseases.

category: systems biology


title: Automated movement assessment in stroke rehabilitation

abstract: We are developing a system for long term Semi-Automated Rehabilitation At the Home (SARAH) that relies on low-cost and unobtrusive video-based sensing. We present a cyber-human methodology used by the SARAH system for automated assessment of upper extremity stroke rehabilitation at the home. We propose a hierarchical model for automatically segmenting stroke survivors movements and generating training task performance assessment scores during rehabilitation. The hierarchical model fuses expert therapist knowledge-based approaches with data-driven techniques. The expert knowledge is more observable in the higher layers of the hierarchy (task and segment) and therefore more accessible to algorithms incorporating high level constraints relating to activity structure (i.e. type and order of segments per task). We utilize an HMM and a Decision Tree model to connect these high level priors to data driven analysis. The lower layers (RGB images and raw kinematics) need to be addressed primarily through data driven techniques. We use a transformer based architecture operating on low-level action features (tracking of individual body joints and objects) and a Multi-Stage Temporal Convolutional Network(MS-TCN) operating on raw RGB images. We develop a sequence combining these complimentary algorithms effectively, thus encoding the information from different layers of the movement hierarchy. Through this combination, we produce a robust segmentation and task assessment results on noisy, variable and limited data, which is characteristic of low cost video capture of rehabilitation at the home. Our proposed approach achieves 85% accuracy in per-frame labeling, 99% accuracy in segment classification and 93% accuracy in task completion assessment. Although the methodology proposed in this paper applies to upper extremity rehabilitation using the SARAH system, it can potentially be used, with minor alterations, to assist automation in many other movement rehabilitation contexts (i.e. lower extremity training for neurological accidents).

category: neuroscience


title: Sleep as a random walk - A superstatistical analysis of EEG data across sleep stages

abstract: In clinical practice, human sleep is classified into stages, each associated with different levels of muscular activity and marked by characteristic patterns in the EEG signals. It is however unclear whether this subdivision into discrete stages with sharply defined boundaries is truly reflecting the dynamics of human sleep. To address this question, we consider one-channel EEG signals as heterogeneous random walks: stochastic processes controlled by hyper-parameters that are themselves time-dependent. We first demonstrate the heterogeneity of the random process by showing that each sleep stage has a characteristic distribution and temporal correlation function of the raw EEG signals. Next, we perform a superstatistical analysis by computing  hyper-parameters, such as the standard deviation, kurtosis and skewness of the raw signal distributions, within subsequent 30-second epochs. It turns out that also the hyper-parameters have characteristic, sleep-stage-dependent distributions, which can be exploited for a simple Bayesian sleep stage detection. Moreover, we find that the hyper-parameters are not piece-wise constant, as the traditional hypnograms would suggest, but show rising or falling trends within and across sleep stages, pointing to an underlying continuous rather than subdivided process that controls human sleep.


title: Feedback processing in attack and defense conflicts: a neurophysiological study

abstract: Despite being extensively modelled to explore decision making processes in economic tasks, there are no studies exploring the neurophysiological correlates of the Predator-Prey Game, a game theory paradigm designed to operationalize attack and defence conflicts. In the present study, we explored the relationship between the ERP components associated with feedback, namely feedback-related negativity (FRN) and feedback -elated P3b, and investment in an adapted version of the Predator-Prey Game (PPG), operationalizing attack and defence. Forty-seven (28 female) community-dwelling volunteers without history of neurological disease were recruited. Participants played the PPG game both as attackers and defenders while EEG signal was recorded with a 128 channels sensor net. Behavioural results showed that individuals tend to invest more and decide faster when playing in defence, rather than in attack. Electrophysiological data showed FRN to be sensitive to the valence of feedback, with increased amplitudes of FRN loss feedbacks. On the other hand, the P3b amplitudes were sensitive to the role, with increased amplitude for attack when compared with defence. The integration of the behavioural and ERP findings supports the theoretical model positing that attack elicits more deliberate and less automatic brain processes than defence.


title: Bacterial filamentation is an in vivo mechanism for cell-to-cell spreading

abstract: Intracellular pathogens are challenged with limited space and resources while replicating in a single host cell. Mechanisms for direct invasion of neighboring host cells have been discovered in cell culture, but we lack an understanding of how bacteria directly spread from cell-to-cell in vivo. Here, we describe the discovery of a bacterial species that uses filamentation as an in vivo mechanism for intracellular spreading between the intestinal epithelial cells of its host, the rhabditid nematode Oscheius tipulae. In vitro and in vivo filamentation by this bacterium, Bordetella atropi, requires a highly conserved nutrient-sensing pathway used by divergent bacteria to detect rich conditions and inhibit the divisome. Thus, B. atropi uses a distinct mechanism for cell-to-cell spreading by coopting a pathway that normally regulates bacterial cell size to trigger filamentation inside host cells.

category: microbiology


title: HIV-2/SIV Vpx antagonises NF-B activation by targeting p65

abstract:
The NF-{kappa}B family of transcription factors and associated signalling pathways are abundant and ubiquitous in human immune responses. Activation of NF-{kappa}B transcription factors by viral pathogen-associated molecular patterns, such as viral RNA and DNA, is fundamental to anti-viral innate immune defences and pro-inflammatory cytokine production that steers adaptive immune responses. Diverse non-viral stimuli, such as lipopolysaccharide and cytokines, also activate NF-{kappa}B and the same anti-pathogen gene networks. Viruses adapted to human cells often encode multiple proteins aimed at varied NF-{kappa}B pathway targeted to mitigate the anti-viral effects of NF-{kappa}B-dependent host immunity. In this study we have demonstrated using numerous assays, in a number of different cell types, that plasmid-encoded or virus-delivered Simian Immunodeficiency Virus (SIV) accessory protein Vpx is a broad antagonist of NF-{kappa}B signalling active against diverse innate NF-{kappa}B agonists. Using targeted Vpx mutagenesis, we showed that this novel Vpx phenotype is independent of known Vpx cofactor DCAF1 and other cellular binding partners, including SAMHD1, STING and the HUSH complex. We found that Vpx co-immunoprecipitated with canonical NF-{kappa}B transcription factor p65 and not NF-{kappa}B transcription factor proteins p50 or p100, preventing nuclear translocation of p65, a novel mechanism of NF-{kappa}B antagonism by lentiviruses. We found that broad antagonism of NF-{kappa}B activation by Vpx was conserved across distantly related lentiviruses as well as for Vpr from SIV Mona monkey (SIVmon), which has Vpx-like SAMHD1-degradation activity.

ImportanceBroad antagonism of NF-{kappa}B activation has been described for HIV-1, but not for Vpx-encoding lentiviruses such as HIV-2. Here we extend our understanding of lentiviral antagonism by identifying an interaction between Vpx and transcription factor NF-{kappa}B p65, leading to inhibition of its nuclear translocation and broad NF-{kappa}B antagonism. This further evidences a requirement for lentiviruses to target universal regulators of immunity, including NF-{kappa}B, to avoid the anti-viral sequelae of pro-inflammatory gene expression stimulated by both viral and extra-viral agonists, such as lipopolysaccharide translocation, during disruption of the gut microbiome barrier during HIV-1 infection. Further structural studies of p65 targeting by Vpx may yield translational insights in the form of novel pan-NF-{kappa}B inhibitors for pathologies characterised by excessive NF-{kappa}B activity. Our findings are also relevant to the gene therapy field where virus-like particle associated Vpx is routinely used to enhance vector transduction through antagonism of SAMHD1, and perhaps also through manipulation of other pathways such as NF-{kappa}B.

category: molecular biology


title: Genetic basis and dual adaptive role of floral pigmentation in sunflowers

abstract: Variation in floral displays, both between and within species, has been long known to be shaped by the mutualistic interactions that plants establish with their pollinators. However, increasing evidence suggests that abiotic selection pressures influence floral diversity as well. Here we analyze the genetic and environmental factors that underlie patterns of floral pigmentation in wild sunflowers. While sunflower inflorescences appear invariably yellow to the human eye, they display extreme diversity for patterns of ultraviolet pigmentation, which are visible to most pollinators. We show that this diversity is largely controlled by cis-regulatory variation at a single MYB transcription factor, HaMYB111, through accumulation of UV-absorbing flavonol glycosides. As expected, different patterns of ultraviolet pigments in flowers have a strong effect on pollinator preferences. However, variation for floral ultraviolet patterns is also associated with environmental variables, especially relative humidity, across populations of wild sunflowers. Larger ultraviolet patterns, which are found in drier environments, limit transpiration, therefore reducing water loss. The dual role of floral UV patterns in pollination attraction and abiotic responses reveals the complex adaptive balance underlying the evolution of floral traits.


title: Probing Specificities of Alcohol Acyltransferases for Designer Ester Biosynthesis with a High-Throughput Microbial Screening Platform

abstract: Alcohol acyltransferases (AATs) enables microbial biosynthesis of a large space of esters by condensing an alcohol and an acyl CoA. However, substrate promiscuity of AATs prevents microbial biosynthesis of designer esters with high selectivity. Here, we developed a high-throughput microbial screening platform that facilitates rapid identification of AATs for designer ester biosynthesis. First, we established a microplate-based culturing technique with in situ fermentation and extraction of esters. We validated its capability in rapid profiling of the alcohol substrate specificity of 20 chloramphenicol acetyltransferase variants derived from Staphylococcus aureus (CATSa) for microbial biosynthesis of acetate esters with various exogeneous alcohol supply. By coupling the microplate-based culturing technique with a previously established colorimetric assay, we developed a high-throughput microbial screening platform for AATs. We demonstrated that this platform could not only confirm CATSa F97W with enhanced isobutyl acetate synthesis but also identify three ATF1Sc (P348M, P348A, and P348S) variants, derived from Saccharomyces cerevisiaes AAT and engineered by model-guided protein design, for enhanced butyl acetate production. We anticipate the high-throughput microbial screening platform is a useful tool to identify novel AATs that have important roles in nature and industrial biocatalysis for designer bioester production.

category: bioengineering


title: Cryo-EM structures of human TMEM120A and TMEM120B

abstract: TMEM120A (Transmembrane protein 120A) was recently identified as a mechanical pain sensing ion channel named as TACAN, while its homologue TMEM120B has no mechanosensing property1. Here, we report the cryo-EM structures of both human TMEM120A and TMEM120B. The two structures share the same dimeric assembly, mediated by extensive interactions through the transmembrane domain (TMD) and the N-terminal coiled coil domain (CCD). However, the nearly identical structures cannot provide clues for the difference in mechanosensing between TMEM120A and TMEM120B. Although TMEM120A could mediate conducting currents in a bilayer system, it does not mediate mechanical-induced currents in a heterologous expression system, suggesting TMEM120A is unlikely a mechanosensing channel. Instead, the TMDs of TMEM120A and TMEM120B resemble the structure of a fatty acid elongase, ELOVL7, indicating their potential role of an enzyme in lipid metabolism.

category: biophysics


title: Transcriptional activation of auxin biosynthesis drives developmental reprogramming of differentiated cells

abstract: Plant cells exhibit remarkable plasticity of their differentiation states, enabling regeneration of whole plants from differentiated somatic cells. How they revert cell fate and express pluripotency, however, remains unclear. Here we show that transcriptional activation of auxin biosynthesis is crucial for reprogramming differentiated Arabidopsis leaf cells. We demonstrate that interfering with the activity of histone acetyltransferases dramatically reduces callus formation from leaf mesophyll protoplasts. Impaired histone acetylation predominantly affects transcription of auxin biosynthesis genes. Auxin biosynthesis is in turn required to accomplish initial cell division through the activation of G2/M phase genes mediated by MYB DOMAIN PROTEIN 3-RELATED (MYB3Rs). We further show that the AUXIN RESPONSE FACTOR 7 (ARF7)/ARF19 and INDOLE-3-ACETIC ACID INDUCIBLE 3 (IAA3)/IAA18-mediated auxin signaling pathway is responsible for cell cycle reactivation in protoplasts. These findings provide novel mechanistic model of how differentiated plant cells can revert their fate and reinitiate the cell cycle to become pluripotent.


title: Investigating the reliability of molecular estimates of evolutionary time when substitution rates and speciation rates vary

abstract:
BackgroundAn accurate timescale of evolutionary history is essential to testing hypotheses about the influence of historical events and processes, and the timescale for evolution is increasingly derived from analysis of DNA sequences. But variation in the rate of molecular evolution complicates the inference of time from DNA. Evidence is growing for numerous factors, such as life history and habitat, that are linked both to the molecular processes of mutation and fixation and to rates of macroevolutionary diversification. However, the most widely used methods rely on idealised models of rate variation, such as the uncorrelated and autocorrelated clocks, and molecular dating methods are rarely tested against complex models of rate change. One relationship that is not accounted for in molecular dating is the potential for interaction between molecular substitution rates and speciation, a relationship that has been supported by empirical studies in a growing number of taxa. If these relationships are as widespread as current evidence suggests, they may have a significant influence on molecular dates.

ResultsWe simulate phylogenies and molecular sequences under three different realistic rate variation models - one in which speciation rates and substitution rates both vary but are unlinked, one in which they covary continuously and one punctuated model in which molecular change is concentrated in speciation events, using empirical case studies to parameterise realistic simulations. We test three commonly used "relaxed clock" molecular dating methods against these realistic simulations to explore the degree of error in molecular dates under each model. We find average divergence time inference errors ranging from 12% of node age for the unlinked model when reconstructed under an uncorrelated rate prior using BEAST 2, to up to 91% when sequences evolved under the punctuated model are reconstructed under an autocorrelated prior using PAML.

ConclusionsWe demonstrate the potential for substantial errors in molecular dates when both speciation rates and substitution rates vary between lineages. This study highlights the need for tests of molecular dating methods against realistic models of rate variation generated from empirical parameters and known relationships.


title: Computational mechanisms for neural representation of words

abstract: A critical way for humans to acquire, represent and communicate information is through language, yet the underlying computation mechanisms through which language contributes to our word meaning representations are poorly understood. We compared three major types of word computation mechanisms from large language corpus (simple co-occurrence, graph-space relations and neural-network-vector-embedding relations) in terms of the association of words brain activity patterns, measured by two functional magnetic resonance imaging (fMRI) experiments. Word relations derived from a graph-space representation, and not neural-network-vector-embedding, had unique explanatory power for the neural activity patterns in brain regions that have been shown to be particularly sensitive to language processes, including the anterior temporal lobe (capturing graph-common-neighbors), inferior frontal gyrus, and posterior middle/inferior temporal gyrus (capturing graph-shortest-path). These results were robust across different window sizes and graph sizes and were relatively specific to language inputs. These findings highlight the role of cumulative language inputs in organizing word meaning neural representations and provide a mathematical model to explain how different brain regions capture different types of language-derived information.


title: Differentiating amyloid beta spread in autosomal dominant and sporadic Alzheimer's disease

abstract: Amyloid-beta (A{beta}) deposition is one of the hallmark pathologies in both sporadic Alzheimers disease (sAD) and autosomal dominant Alzheimers disease (ADAD), the latter of which is caused by mutations in genes involved in A{beta} processing. Despite A{beta} deposition being a centerpiece to both sAD and ADAD, some differences between these AD subtypes have been observed with respect to the spatial pattern of A{beta}. Previous work has shown that the spatial pattern of A{beta} in individuals spanning the sAD spectrum can be reproduced with high accuracy using an epidemic spreading model (ESM), which simulates the diffusion of A{beta} across neuronal connections and is constrained by individual rates of A{beta} production and clearance. However, it has not been investigated whether A{beta} deposition in the rarer ADAD can be modeled in the same way, and if so, how congruent the spreading patterns of A{beta} across sAD and ADAD are. We leverage the ESM as a data-driven approach to probe individual-level variation in the spreading patterns of A{beta} across three different large-scale imaging datasets (2 SAD, 1 ADAD). We applied the ESM separately to the Alzheimers Disease Neuroimaging initiative (N=737), the Open Access Series of Imaging Studies (N=510), and the Dominantly Inherited Alzheimers Network (N=249), the latter two of which were processed using an identical pipeline. We assessed inter- and intra-individual model performance in each dataset separately, and further identified the most likely epicenter of A{beta} spread for each individual. Using epicenters defined in previous work in sAD, the ESM provided moderate prediction of the regional pattern of A{beta} deposition across all three datasets. We further find that, while the most likely epicenter for most A{beta}-positive subjects overlaps with the default mode network, 13% of ADAD individuals were best characterized by a striatal origin of A{beta} spread. These subjects were also distinguished by being younger than ADAD subjects with a DMN A{beta} origin, despite having a similar estimated age of symptom onset. Together, our results suggest that most ADAD patients express A{beta} spreading patters similar to those of sAD, but that there may be a subset of ADAD patients with a separate, striatal phenotype.


title: Fast 3D Clear: A Fast, Aqueous-Based, Reversible Three-Day Tissue Clearing Method for Adult and Embryonic Mouse Brain and Whole Body

abstract: To date, a variety of optical clearing methods have emerged that serve as powerful tools to study intact organs and neuronal circuits. Here we describe a newly developed, aqueous clearing protocol named "Fast 3D Clear", which relies on tetrahydrofuran (THF) for tissue delipidation, and iohexol (Histodenz) for clearing, such that tissues can be imaged under immersion oil in light sheet imaging systems. Fast 3D Clear requires three days to achieve high transparency of adult and embryonic mouse tissues, while maintaining their anatomical integrity, and preserving a vast array of transgenic and viral/dye fluorophores, such as GCaMP3/6, tdTomato, Fast Blue, and IRF670. A unique advantage of Fast 3D Clear is its complete reversibility and thus compatibility with tissue sectioning and immunohistochemistry. Fast 3D Clear can be easily and quickly applied to a wide range of biomedical studies, thereby greatly facilitating the acquisition of high-resolution, two - and three -dimensional images.

category: cell biology


title: Fast 3D Clear: A Fast, Aqueous, Reversible Three-Day Tissue Clearing Method for Adult and Embryonic Mouse Brain and Whole Body


title: Genetic diversity in chimpanzee transcriptomics does not represent wild populations

abstract: Chimpanzees (Pan troglodytes) are a genetically diverse species, consisting of 4 highly distinct subspecies. As humans closest living relative they have been a key model organism in the study of human evolution, and comparisons of human and chimpanzee transcriptomes have been widely used to characterise differences in gene expression levels that could underlie the phenotypic differences between the two species. However, the subspecies from which these transcriptomic datasets have been derived is not recorded in metadata available in the public NCBI Sequence Read Archive (SRA). Furthermore, labelling of RNA-seq samples is for the most part inconsistent across studies, and the true number of individuals from whom transcriptomic data is available is difficult to ascertain. Thus we have evaluated genetic diversity at the subspecies and individual level in 486 public RNA-seq samples available in the SRA, spanning the vast majority of public chimpanzee transcriptomic data. Using multiple population genetics approaches we find that nearly all samples (96.6%) have some degree of Western chimpanzee ancestry. At the individual donor level, we identify multiple samples that have been repeatedly analysed across different studies, and identify a total of 135 genetically distinct individuals within our data, a number that falls to 89 when we exclude likely first and second-degree relatives. Altogether, our results show that current transcriptomic data from chimpanzees is capturing low levels of genetic diversity relative to what exists in wild chimpanzee populations. These findings provide important context to current comparative transcriptomics research involving chimpanzees.

category: genomics


title: Attention cueing in rivalry: insights from pupillometry

abstract:
We used pupillometry to evaluate the effects of attention cueing on perceptual bi-stability, as reported by adult human observers. Perceptual alternations and pupil diameter were measured during two forms of rivalry, generated by presenting a white and a black disk to the two eyes (binocular rivalry) or splitting the disks between eyes (interocular grouping rivalry). In line with previous studies, we found that subtle pupil size oscillations (about 0.05 mm) tracked alternations between exclusive dominance phases of the black or white disk. These oscillations were larger for perceptually stronger stimuli: presented to the dominant eye or with physically higher luminance contrast. However, cueing of endogenous attention to one of the rivaling percepts did not affect pupil oscillations during exclusive dominance phases. This was in spite of the reliable effects of endogenous attention on perceptual dominance, which shifted in favor of the cued percept by about 10%. The results were comparable for binocular and interocular grouping rivalry. Cueing only had a marginal modulatory effect on pupil size during mixed percepts in binocular rivalry. This may suggest that, rather than acting by modulating perceptual strength, endogenous attention primarily acts during periods of unresolved competition, which is compatible with attention being automatically directed to the rivaling stimuli during periods of exclusive dominance and thereby sustaining perceptual alternations.

Significance StatementBinocular rivalry depends on attention. When it is diverted away from the stimuli, perceptual alternations slow down; when it is preferentially directed to one stimulus, perception lingers more on it, consistent with attention enhancing the effective strength of the rivaling stimuli. Here we introduce pupillometry as a means to indirectly track changes in effective stimulus strength. We find that pupil size oscillates, accurately tracking perceived luminance during two forms of rivalry: binocular rivalry and interocular grouping rivalry. Both show robust effects of attention cueing on perceptual dominance, but pupil oscillations during exclusive dominance are unaffected by cueing. This suggests that endogenous attention does not affect perceptual strength during exclusive dominance, though it might do so during transition phases.


title: Disrupted choline clearance and sustained acetylcholine release in vivo by a common choline transporter coding variant associated with poor attentional control in humans

abstract:
Transport of choline via the neuronal high-affinity choline transporter (CHT; SLC5A7) is essential for cholinergic terminals to synthesize and release acetylcholine (ACh). In humans, we previously demonstrated an association between a common CHT coding substitution (rs1013940; Ile89Val) and reduced attentional control as well as attenuated frontal cortex activation. Here, we used a CRISPR/Cas9 approach to generate mice expressing the I89V substitution and assessed, in vivo, CHT-mediated choline transport, and ACh release. Relative to wild type (WT) mice, CHT-mediated clearance of choline in mice expressing one or two Val89 alleles was reduced by over 80% cortex and by over 50% in striatum. Choline clearance in CHT Val89 mice was further reduced by neuronal inactivation. Deficits in ACh release, 5 and 10 min after repeated depolarization at a low, behaviorally relevant frequency, support an attenuated reloading capacity of cholinergic neurons in mutant mice. The density of CHTs in total synaptosomal lysates and neuronal plasma-membrane-enriched fractions was not impacted by the Val89 variant, indicating a selective impact on CHT function. When challenged with a visual disruptor to reveal attentional control mechanisms, Val89 mice failed to adopt a more conservative response bias. Structural modeling revealed that Val89 may attenuate choline transport by altering conformational changes of CHT that support normal transport rates. Our findings support the view that diminished, sustained cholinergic signaling capacity underlies perturbed attentional performance in individuals expressing CHT Val89. The CHT Val89 mouse serves as a valuable model to study heritable risk for cognitive disorders arising from cholinergic dysfunction.

Significance StatementAcetylcholine (ACh) signaling depends on the functional capacity of the neuronal choline transporter (CHT). Previous research demonstrated that humans expressing the common CHT coding variant Val89 exhibit attentional vulnerabilities and attenuated fronto-cortical activation during attention. Here, we find that mice engineered to express the Val89 variant exhibit reduced CHT-mediated choline clearance and a diminished capacity to sustain ACh release. Additionally, Val89 mice lack cognitive flexibility in response to an attentional challenge. These findings provide a mechanistic and cognitive framework for interpreting the attentional phenotype associated with the human Val89 variant and establish a model that permits a more invasive interrogation of CNS effects as well as the development of therapeutic strategies for those, including Val89 carriers, with presynaptic cholinergic perturbations.


title: A universal probabilistic spike count model reveals ongoing modulation of neural variability

abstract: Neural responses are variable: even under identical experimental conditions, single neuron and population responses typically differ from trial to trial and across time. Recent work has demonstrated that this variability has predictable structure, can be modulated by sensory input and behaviour, and bears critical signatures of the underlying network dynamics and computations. However, current methods for characterising neural variability are primarily geared towards sensory coding in the laboratory: they require trials with repeatable experimental stimuli and behavioural covariates. In addition, they make strong assumptions about the parametric form of variability, rely on assumption-free but data-inefficient histogram-based approaches, or are altogether ill-suited for capturing variability modulation by covariates. Here we present a universal probabilistic spike count model that eliminates these shortcomings. Our method builds on sparse Gaussian processes and can model arbitrary spike count distributions (SCDs) with flexible dependence on observed as well as latent covariates, using scalable variational inference to jointly infer the covariate-to-SCD mappings and latent trajectories in a data efficient way. Without requiring repeatable trials, it can flexibly capture covariate-dependent joint SCDs, and provide interpretable latent causes underlying the statistical dependencies between neurons. We apply the model to recordings from a canonical non-sensory neural population: head direction cells in the mouse. We find that variability in these cells defies a simple parametric relationship with mean spike count as assumed in standard models, its modulation by external covariates can be comparably strong to that of the mean firing rate, and slow low-dimensional latent factors explain away neural correlations. Our approach paves the way to understanding the mechanisms and computations underlying neural variability under naturalistic conditions, beyond the realm of sensory coding with repeatable stimuli.


title: Electrical neuromodulation biases brightness perception of flickering light

abstract:
BackgroundVisual phenomena like brightness illusions impressively demonstrate the highly constructive nature of perception. In addition to physical illumination, the subjective experience of brightness is related to temporal neural dynamics in visual cortex.

ObjectiveHere, we asked whether biasing the temporal pattern of neural excitability in visual cortex by transcranial alternating current stimulation (tACS) modulates brightness perception of concurrent rhythmic visual stimuli.

MethodsParticipants performed a brightness discrimination task of two flickering lights, one of which was targeted by same-frequency electrical stimulation at varying phase shifts. tACS was applied with an occipital and a periorbital active control montage, based on simulations of electrical currents using finite-element head models.

ResultsExperimental results reveal that flicker brightness perception is modulated dependent on the phase shift between sensory and electrical stimulation, solely under occipital tACS. Phase-specific modulatory effects by tACS were dependent on flicker-evoked neural phase stability at the tACS-targeted frequency, recorded prior to electrical stimulation. The optimal timing of tACS application leading to enhanced brightness perception was further correlated with the neural phase delay of the cortical flicker response.

ConclusionsOur results corroborate the role of temporally coordinated neural activity in visual cortex for brightness perception of rhythmic visual input in humans. Phase-specific behavioral modulations by tACS emphasize its efficacy to transfer perceptually relevant temporal information to the cortex. These findings provide an important step towards understanding the basis of visual perception and further confirm electrical stimulation as a tool for advancing controlled modulations of neural activity and related behavior.


title: tACS phase-specifically biases brightness perception of flickering light


title: Conservation of locomotion-induced oculomotor activity through evolution in higher tetrapods

abstract:
Efference copies are neural replicas of motor outputs used to anticipate the sensory consequences of a self-generated motor action or to coordinate neural networks involved in distinct motor behaviors1. An established example of this motor-to-motor coupling is the efference copy of the propulsive motor command that supplements classical visuo-vestibular reflexes to ensure gaze stabilization during amphibian larval locomotion2. Such feedforward replica from spinal pattern-generating circuits produces a spino-extraocular motor coupled activity that evokes eye movements, spatio-temporally coordinated to tail undulation independently of any sensory signal3,4. Exploiting the evolutionary-development characteristic of the frog1, studies in metamorphing Xenopus demonstrated the persistence of this spino-extraocular motor command in adults, and its developmental adaptation to tetrapodal locomotion5,6. Here, we demonstrate for the first time the existence of a comparable locomotor-to-ocular motor coupling in the mouse. In neonates, ex vivo nerve recordings from brainstem-spinal cord preparation reveals a spino-extraocular motor coupled activity similar to the one described in Xenopus. In adult mice, trans-synaptic rabies injection in lateral rectus eye muscle labels cervical spinal cord neurons projecting directly to abducens motor neurons. Finally, treadmill-elicited locomotion in decerebrated preparations7 evokes rhythmic eye movements in synchrony with the limb gait pattern. Overall, our data are evidence for the conservation of locomotor-induced eye movements in higher tetrapods. Thus, in mammals as in amphibians, during locomotion CPG-efference copy feedforward signals might interact with sensory feedback to ensure efficient gaze control.

HighlightsO_LISpino-extraocular motor coupling is evidenced from newborn mice ex vivo preparations
C_LIO_LIAdult decerebrated mice exhibit conjugated rhythmic eye movements during treadmill locomotion
C_LIO_LILocomotor-induced oculomotor activity occurs in absence of visuo-vestibular inputs
C_LIO_LIConserved CPG-based efference copy signal in vertebrates with common features.
C_LI

eTOC blurbWe report a functional coupling between spinal locomotor and oculomotor networks in the mouse, similar to the one previously described in Amphibians. This is the first evidence for the direct contribution of locomotor networks to gaze control in mammals, suggesting a conservation of the spino-extraocular coupling in higher tetrapods during sustained locomotion.


title: In Vivo Simultaneous Nonlinear Absorption Raman and Fluorescence (SNARF) Imaging of Mouse Brain Cortical Structures

abstract: AO_SCPLOWBSTRACTC_SCPLOWTwo-photon excited fluorescence (TPEF) microscopy is a widely used optical imaging technique that has revolutionized neurophotonics through a diverse palette of dyes, specialized transgenic models, easy implementation, and straightforward data analysis. However, in vivo TPEF imaging is often limited in the number of contrasts available to distinguish different cells, structures, or functions. We propose using two label-free multiphoton microscopy techniques - stimulated Raman scattering (SRS) microscopy and transient absorption microscopy (TAM) - as complementary and orthogonal imaging modalities to TPEF for in vivo brain imaging. In this study, we construct a simultaneous nonlinear absorption, Raman, and fluorescence (SNARF) microscope and image several cortical structures up to 250-300 m below the pial surface, the highest reported in vivo imaging depth for SRS or TAM. We further demonstrate the capabilities of our SNARF microscope through the quantification of age-dependent myelination, hemodynamics, vessel structure, cell density, and cell identity in vivo. Using machine learning, we report the use of label-free SRS and TAM features to predict capillary-lining cell identities with 90% accuracy. The SNARF microscope and methodology outlined herein provide a powerful platform to study several research topics, including neurovascular coupling, blood-brain barrier, neuronal and axonal degeneration in aging, and neurodegenerative diseases.


title: Tracking and Measuring Local Protein Synthesis In Vivo

abstract: Detecting when and how much a protein molecule is synthesized is important for understanding cell function, but current methods have poor cellular or temporal resolution or are destructive to cells (Dahm et al., 2008). Here, we developed a technique to detect and quantify subcellular protein synthesis events in real time in vivo. This Protein Translation Reporting (PTR) technique uses a genetic tag that produces a stoichiometric ratio of a small peptide portion of a split fluorescent protein and the protein of interest during protein synthesis. We show that the split fluorescent protein peptide can generate fluorescence within milliseconds upon binding the larger portion of the fluorescent protein, and that the fluorescence intensity is directly proportional to the number of molecules of the protein of interest synthesized. Using PTR, we tracked and measured protein synthesis events in single cells over time in vivo. We use split red fluorescent protein to detect multiple genes or alleles in single cells simultaneously. We also split a photoswitchable fluorescent protein to photoconvert the reconstituted fluorescent protein to a different channel and arbitrarily reset the time of detection of synthesis events, continually over time.


title: Negative curvature-promoting lipids instruct nuclear ingression of low autophagic potential vacuoles

abstract: Membrane contact sites are functional nodes at which organelles exchange information through moving ions, proteins and lipids, thus driving the reorganization of metabolic pathways and the adaptation to changing cues. The nuclear-vacuole junction of Saccharomyces cerevisiae is among the most extensive and better-known organelle contact sites, described to expand in response to various metabolic stresses. While using genotoxins with unrelated purposes, we serendipitously discovered a phenomenon that we describe as the most extreme and intimate contact ever reported between nuclei and vacuoles: the vacuole becomes completely internalized in the nucleus. We define lipids supporting negative curvature, such as phosphatidic acid and sterols, as bona-fide drivers of this event. Functionally, we purport that internalized vacuoles are low efficiency ones whose removal from the cytoplasm optimizes cargo interaction with functional vacuoles. Thus, our findings also point to nucleus-vacuole interactions as important for metabolic adaptation. Yet, rather than by inter-organelle exchanges, the underlying mechanism literally concurs with vacuolar sequestration.


title: Cell-autonomous differentiation of human primed embryonic stem cells into trophoblastic syncytia through the nascent amnion-like cell state

abstract: SummeryHuman primed embryonic stem cells (pESCs) are known to be converted to cells with several trophoblast properties, but it has remained controversial whether this phenomenon represents the inherent differentiation competence of human pESCs to trophoblast lineages. In this study, we report that chemical blockage of ACTIVIN/NODAL and FGF signals is sufficient to steer human pESCs into GATA3-expressing cells that give rise to hormone-producing syncytia analogous to syncytiotrophoblasts of the post-implantation stage of the human embryo. Taking advantage of this system, we identified two distinct modes of cell-autonomous genetic programs and their coordinated actions to initiate the differentiation. We also found a transient population reminiscent of nascent amnion and then a spontaneous branch of differentiation trajectory leading to syncytiotrophoblast-like syncytial cells. These results provide insights into the possible extraembryonic differentiation pathway that is unique in primate embryogenesis and is relevant to the trophoblast competence of human primed pluripotent stem cells.

category: developmental biology


title: MicroRNA-202 prevents precocious spermatogonial differentiation and meiotic initiation during mouse spermatogenesis

abstract:
Spermatogonial differentiation and meiotic initiation during spermatogenesis are tightly regulated by a number of genes including those coding enzymes for miRNA biogenesis. However, whether and how single miRNAs regulate these processes remain unclear. Here, we report that miR-202, a member of the let-7 family, prevents precocious spermatogonial differentiation and meiotic initiation in spermatogenesis by regulating the timely expression of many genes including those for other key regulators. In miR-202 knockout (KO) mice, the undifferentiated spermatogonial pool is reduced, ultimately causing agametic seminiferous tubules. SYCP3, STRA8 and DMRT6 are expressed earlier in KO mice than in wild-type (WT) littermates, and Dmrt6 mRNA is a direct target of miR-202-5p. Moreover, the precocious spermatogonial differentiation and meiotic initiation were also observed in KO spermatogonial stem cells when cultured and induced in vitro, and could be rescued by the knockdown of Dmrt6. Therefore, we have not only shown that miR-202 is a novel regulator of meiotic initiation but also added a new module to the underlying regulatory network.

Summary statementA single miRNA, miR-202, prevents precocious differentiation and meiotic initiation during spermatogenesis. miR-202, DMRT6 and STRA8 act together as a novel module in the regulatory network of meiotic initiation.


title: PreDarwinian evolution, prebiotic change: a principle of least selection

abstract: Evolution prior to its Darwinian era occurred in an age of variation. Events allowed by starting materials, chemistry and physics were realized, directed by intrinsic probabilities. In this landscape, a principle of least selection defines the most probable evolutionary path. For selection from a population where x varies, the rate of x evolution dx/dt = {Delta}.  is the population mean of the selected property x,  a proportionality constant. Explicit equations for selected change {Delta} are presented. Simple results exist for common cases; for example, selecting for x [≥] a threshold. Maximizing {Delta} minimizes need for selection. Least selections for x demand maximal dependence on x, maximal x standard deviation{sigma} , and maximal lower x threshold (consistent with the need for a survivor). Least selection is free of the cost of natural selection, thus can support rapid evolution. Least selection also unites previous seemingly independent optima. As examples, evolution via  starting bloc selection and  chance utility (where selection spans a minimal change) and  near-ideal reactions (where variable reactant supplies recurrently create easy reactions) occur via transitory paths of least selection.  Bayesian convergence is guaranteed by least selections extremum (which sharply defines evolutions path).  Distribution fitness (specific outcomes selected among multiple concurrent variations) is least selection of rare functional minorities. Inheritance of encoded chemical capabilities first evolves because templating and catalysis are related, potentially joined in a single molecular complex. Least selection defines directions, biostructures define destinations for likely pathways of preDarwinian evolution.


title: Evolutionary graph theory beyond pairwise interactions: higher-order network motifs shape times to fixation in structured populations

abstract: To design population topologies that can accelerate rates of solution discovery in directed evolution problems or in evolutionary optimization applications, we must first systematically understand how population structure shapes evolutionary outcome. Using the mathematical formalism of evolutionary graph theory, recent studies have shown how to topologically build networks of population interaction that increase probabilities of fixation of beneficial mutations, at the expense, however, of longer fixation times, which can slow down rates of evolution under elevated mutation rate. Here we find that moving beyond dyadic interactions is fundamental to explain the trade-offs between probability and time to fixation. We show that higher-order motifs, and in particular three-node structures, allow tuning of times to fixation, without changes in probabilities of fixation. This gives a near-continuous control over achieving solutions that allow for a wide range of times to fixation. We apply our algorithms and analytic results to two evolutionary optimization problems and show that the rate at which evolving agents learn to navigate their environment can be tuned near continuously by adjusting the higher-order topology of the agent population. We show that the effects of population structure on the rate of evolution critically depend on the optimization landscape and find that decelerators, with longer times to fixation of new mutants, are able to reach the optimal solutions faster than accelerators in complex solution spaces. Our results highlight that no one population topology fits all optimization applications, and we provide analytic and computational tools that allow for the design of networks suitable for each specific task.


title: Oct4 primarily controls enhancer activity rather than accessibility

abstract: The transcription factor Oct4 is essential for maintaining stem cell pluripotency and for efficient cell reprogramming, but its functional roles are far from being understood. Here, we investigate the functions of Oct4 by rapidly depleting Oct4 from mouse embryonic stem cells and conducting a time-resolved multiomics analysis. Oct4 depletion leads to an immediate loss of its binding to putative enhancers that are accessible in chromatin. Loss of Oct4 is accompanied by a concomitant decrease in mRNA synthesis from putative target genes that are part of the transcriptional network that maintains pluripotency. Oct4 binding to enhancers does not correlate with chromatin accessibility, whereas Sox2 can apparently retain accessibility after Oct4 depletion even in the absence of eRNA synthesis. These results are consistent with the model that Sox2 primarily acts as a pioneer factor that renders enhancers accessible, whereas Oct4 acts primarily as a transcriptional activator that stimulates transcription of pluripotency enhancers and their target genes.


title: Neural ADMIXTURE: rapid population clustering with autoencoders

abstract: Characterizing the genetic substructure of large cohorts has become increasingly important as genetic association and prediction studies are extended to massive, increasingly diverse, biobanks. ADMIXTURE and STRUCTURE are widely used unsupervised clustering algorithms for characterizing such ancestral genetic structure. These methods decompose individual genomes into fractional cluster assignments with each cluster representing a vector of DNA marker frequencies. The assignments, and clusters, provide an interpretable representation for geneticists to describe population substructure at the sample level. However, with the rapidly increasing size of population biobanks and the growing numbers of variants genotyped (or sequenced) per sample, such traditional methods become computationally intractable. Furthermore, multiple runs with different hyperparameters are required to properly depict the population clustering using these traditional methods, increasing the computational burden. This can lead to days of compute. In this work we present Neural ADMIXTURE, a neural network autoencoder that follows the same modeling assumptions as ADMIXTURE, providing similar (or better) clustering, while reducing the compute time by orders of magnitude. Indeed, the equivalent of one month of continuous compute can be reduced to hours. In addition, Neural ADMIXTURE can include multiple outputs, providing the equivalent results as running the original ADMIXTURE algorithm many times with different numbers of clusters. Our models can also be stored, allowing later cluster assignment to be performed with a linear computational time. The software implementation of Neural ADMIXTURE can be found at https://github.com/ai-sandbox/neural-admixture.


title: Multiple reproductive females in family groups of smooth-coated otters

abstract: Smooth-coated otters (Lutrogale perspicillata) are inhabitants of the waterways of India and Singapore. Otter families typically consist of a single mating pair with mature, nonbreeding siblings living in family groups, or "romps". We note here the presence of multiple reproductive female otters within some romps, as well as the possible existence of simultaneous litters by different mothers. This phenomenon has not been recorded among L. perspicillata before. Here we address possible influences leading to multiple reproductive females within romps of smooth-coated otters, including inclusive fitness, incomplete suppression of reproduction, and existing in an urban environment. The numerous, recurring observations of multiple reproductive females warrant further investigation; while uncommon, this phenomenon is not as rare as once thought.

category: animal behavior and cognition


title: Structure of the mini-RNA-guided endonuclease CRISPR-Cas{Phi}3

abstract: CRISPR-Cas{Phi} is a novel family of miniaturized RNA-guided endonucleases from phages 1,2. These novel ribonucleoproteins (RNPs) provide a compact scaffold gathering all key activities of a genome editing tool2. Here, we provide the first structural insight into Cas{Phi} singular DNA targeting and cleavage mechanism by determining the cryoEM structure of Cas{Phi}3 with the triple strand R-loop generated after DNA cleavage. The structure reveals the unique machinery for target unwinding to form the crRNA-DNA hybrid and cleaving the target DNA. The protospacer adjacent motif (PAM) is recognised by the target strand (T-strand) and non-target strand (NT-strand) PAM interacting domains (TPID and NPID). Unwinding occurs after insertion of the conserved 1 helix disrupting the dsDNA, thus facilitating the crRNA-DNA hybrid formation. The NT-strand is funnelled towards the RuvC catalytic site, while a long helix of TPID separates the displaced NT-strand and the crRNA-DNA hybrid avoiding DNA re-annealing. The crRNA-DNA hybrid is directed to the stop (STP) domain that splits the hybrid guiding the T-strand towards the RuvC active site. The conserved RuvC insertion of the Cas{Phi} family is extended along the hybrid, interacting with the phosphate backbone of the crRNA. A cluster of hydrophobic residues anchors the RuvC insertion in a cavity of the STP domain. The assembly of the hybrid promotes the shortening of the RuvC insertion, thus pulling the STP towards the RuvC active site to activate catalysis. These findings illustrate why Cas{Phi} unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. Site-directed mutagenesis in key residues support Cas{Phi}3 target DNA and non-specific ssDNA cutting mechanism. Our analysis provides new avenues to redesign the compact CRISPR-Cas{Phi} nucleases for genome editing.


title: Injectable Nanoparticle-Based Hydrogels Enable the Safe and Effective Deployment of Immunostimulatory CD40 Agonist Antibodies

abstract: When properly deployed, the immune system can eliminate deadly pathogens, eradicate metastatic cancers, and provide long-lasting protection from diverse diseases. Unfortunately, realizing these remarkable capabilities is inherently risky as disruption to immune homeostasis can elicit dangerous complications or autoimmune disorders. While current research is continuously expanding the arsenal of potent immunotherapeutics, there is a technological gap when it comes to controlling when, where, and how long these drugs act on the body. Here, we explore the ability of a slow-releasing injectable hydrogel depot to reduce the problematic dose-limiting toxicities of immunostimulatory CD40 agonist (CD40a) while maintaining their potent anti-cancer efficacy. We leverage a previously described polymer-nanoparticle (PNP) hydrogel system that exhibits shear-thinning and yield-stress properties that we hypothesized would improve locoregional delivery of the CD40a immunotherapy. Using PET imaging, we demonstrate that prolonged hydrogel-based delivery redistributes CD40a exposure to the tumor and the tumor draining lymph node (TdLN), thereby reducing weight loss, hepatotoxicity, and cytokine storm associated with standard treatment. Moreover, CD40a-loaded hydrogels mediate improved local cytokine induction in the TdLN and improve treatment efficacy in the B16F10 melanoma model. PNP hydrogels, therefore, represent a facile, drug-agnostic method to ameliorate immune-related adverse effects and explore locoregional delivery of immunostimulatory drugs.


title: Robust integration of single-cell cytometry datasets

abstract: Combining single-cell cytometry datasets increases the analytical flexibility and the statistical power of data analyses. However, in many cases the full potential of co-analyses is not reached due to technical variance between data from different experimental batches. Here, we present cyCombine, a method to robustly integrate cytometry data from different batches, experiments, or even different experimental techniques, such as CITE-seq, flow cytometry, and mass cytometry. We demonstrate that cyCombine maintains the biological variance and the structure of the data, while minimizing the technical variance between datasets. cyCombine does not require technical replicates across datasets, and computation time scales linearly with the number of cells, allowing for integration of massive datasets. Robust, accurate, and scalable integration of cytometry data enables integration of multiple datasets for primary data analyses and the validation of results using public datasets.


title: On Improving an Already Competitive Segmentation Algorithm for the Cell Tracking Challenge - Lessons Learned

abstract: The virtually error-free segmentation and tracking of densely packed cells and cell nuclei is still a challenging task. Especially in low-resolution and low signal-to-noise-ratio microscopy images erroneously merged and missing cells are common segmentation errors making the subsequent cell tracking even more difficult. In 2020, we successfully participated as team KIT-Sch-GE (1) in the 5th edition of the ISBI Cell Tracking Challenge. With our deep learning-based distance map regression segmentation and our graph-based cell tracking, we achieved multiple top 3 rankings on the diverse data sets. In this manuscript, we show how our approach can be further improved by using another optimizer and by fine-tuning training data augmentation parameters, learning rate schedules, and the training data representation. The fine-tuned segmentation in combination with an improved tracking enabled to further improve our performance in the 6th edition of the Cell Tracking Challenge 2021 as team KIT-Sch-GE (2).


title: Pancreatic cancer risk predicted from disease trajectories using deep learning

abstract: Pancreatic cancer is an aggressive disease that typically presents late with poor patient outcomes. There is a pronounced medical need for early detection of pancreatic cancer, which can be addressed by identifying high-risk populations. Here we apply artificial intelligence (AI) methods to a dataset of more than 6 million patient records with 24,000 pancreatic cancer cases in the Danish National Patient Registry (Denmark) and, for comparison, a dataset of one million records with 4,000 pancreatic cancer cases in the Mass General Brigham Healthcare System (Boston, US). In contrast to existing methods that do not use temporal information, we explicitly train machine learning models on the time sequence of diseases in patient clinical histories and test the ability to predict cancer occurrence in time intervals of 3 to 60 months after risk assessment. We extract from the AI machine an estimate of the contribution to prediction of individual disease features. For cancer occurrence within 36 months, the performance of the best model (AUROC=0.88), trained and tested on disease trajectories in the Danish dataset, substantially exceeds that of a model without time information, even when disease events within a 3 month window before cancer diagnosis are excluded from training (AUROC[3m]=0.84). Independent training and testing on the Boston dataset reaches comparable performance (AUROC=0.87, AUROC[3m]=0.80), while cross-application of the Danish deep learning model on the Boston dataset has lower accuracy (AUROC=0.78, AUROC[3m]=0.70), indicating a requirement of independent training in health systems with different coding practices. These results raise the state-of-the-art level of performance of cancer risk prediction on real-world data sets and provide support for the design of future screening trials for high-risk patients, e.g., to serial imaging or blood-based biomarkers to facilitate earlier cancer detection. AI on real-world clinical records has the potential to shift focus from treatment of late-stage to early-stage cancer, benefiting patients by improving lifespan and quality of life.


title: Atomic reconstruction of biomolecular structures from AFM images and quantitative validation of experimental data using simulated AFM scanning

abstract: We provide the BioAFMviewer-Toolbox, an extension of our previously developed software platform for simulated AFM scanning of biomolecular structures and dynamics. The focus was on developing a toolbox of methods which employ simulated AFM scanning combined with quantitative analysis to facilitate the interpretation of resolution-limited AFM images. The key advancement is the automatized fitting of biomolecular structures to experimental AFM images, which allows to reconstruct 3D atomistic structures from AFM surface scans. Moreover, several methods for detailed analysis and comparison of surface topographies in simulated and experimental AFM images are provided. We demonstrate the applicability of the developed tools in the interpretation of high-speed AFM observations of proteins. The toolbox is implemented into the versatile interactive interface of the BioAFMviewer, which is a free software package available at www.bioafmviewer.com.


title: The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

abstract: Phosphatidylglycerol is a crucial phospholipid found ubiquitously in biological membranes of prokaryotic and eukaryotic cells. The phosphatidylglycerol phosphate (PGP) synthase (PgsA), a membrane-embedded enzyme, catalyzes the primary reaction of phosphatidylglycerol biosynthesis. Mutations in pgsA frequently correlate with daptomycin resistance in Staphylococcus aureus and other prevalent infectious pathogens. Here we report the structures of S. aureus PgsA (SaPgsA) captured at two distinct states of the catalytic process, with lipid substrate (cytidine diphosphate-diacylglycerol, CDP-DAG) or product (PGP) bound to the active site within a trifurcated amphipathic cavity. The hydrophilic head groups of CDP-DAG and PGP occupy two different pockets in the cavity, inducing local conformational changes. An elongated membrane-exposed surface groove accommodates the fatty acyl chains of CDP-DAG/PGP and opens a lateral portal for lipid entry/release. Remarkably, the daptomycin resistance-related mutations mostly cluster around the active site, causing reduction of enzymatic activity. Our results provide detailed mechanistic insights into the dynamic catalytic process of PgsA and structural frameworks beneficial for development of antimicrobial agents targeting PgsA from pathogenic bacteria.


title: Damaged goods? Evaluating the impact of X-ray damage on conformational heterogeneity in room temperature and cryo-cooled protein crystals

abstract:
X-ray crystallography is a cornerstone of biochemistry. Traditional freezing of protein crystals to cryo-temperatures mitigates X-ray damage and facilitates crystal handling but provides an incomplete window into the ensemble of conformations at the heart of protein function and energetics. Room temperature (RT) X-ray crystallography provides more extensive ensemble information, and recent developments allow conformational heterogeneity, the experimental manifestation of ensembles, to be extracted from single crystal data. However, high sensitivity to X-ray damage at RT raises concerns about data reliability. To systematically address this critical question, we obtained increasingly X-ray-damaged high-resolution datasets (1.02-1.52 [A]) from single thaumatin, proteinase K, and lysozyme crystals. Heterogeneity analyses indicated a modest increase in conformational disorder with X-ray damage. Nevertheless, these effects do not alter overall conclusions and can be minimized by limiting the extent of X-ray damage or eliminated by extrapolation to obtain heterogeneity information free from X-ray damage effects. To compare these effects to damage at cryo temperature and to learn more about damage and heterogeneity in cryo-cooled crystals, we carried out an analogous analysis of increasingly damaged proteinase K cryo datasets (0.9-1.16 [A]). We found X-ray damage-associated heterogeneity changes that were not observed at RT. This observation and the scarcity of reported X-ray doses and damage extent render it difficult to distinguish real from artifactual conformations, including those occurring as a function of temperature. The ability to aquire reliable heterogeneity information from single crystals at RT provides strong motivation for further development and routine implementation of RT X-ray crystallography to obtain conformational ensemble information.

SignificanceX-ray crystallography has allowed biologists to visualize the proteins that carry out complex biological processes and has provided powerful insights into how these molecules function. Our next level of understanding requires information about the ensemble of conformations that is at the heart of protein function and energetics. Prior results have shown that room temperature (RT) X-ray crystallography provides extensive ensemble information, but are subject to extenstive X-ray damage. We found that ensemble information with little or no effects from X-ray damage can be collected at RT. We also found that damage effects may be more prevalent than recognized in structures obtained under current standard cryogenic conditions. RT X-ray crystallography can be routinely implemented to obtain needed information about conformational ensembles.


title: Aerobiology in High Latitudes: Evidence of Bacteria Acting as Tracer of Warm Air Mass Advection reaching Northern Antarctic Peninsula

abstract: Despite the extent use of geochemical tracers to track warm air mass origin reaching the Antarctic continent, we present here evidences that microorganisms being transported by the atmosphere and deposited in fresh snow layers of Antarctic ice sheets do act as tracers of air mass advection from the Southern Patagonia region to Northern Antarctic Peninsula. We combined atmospheric circulation data with microorganism content in snow/firn samples collected in two sites of the Antarctic Peninsula (King George Island/Wanda glacier and Detroit Plateau) by using flow cytometer quantification. In addition, we cultivated, isolated and submitted samples to molecular sequencing to precise species classification. Viable gram-positive bacteria were found and recovered in different snow/firn layers samples, among dead and living cells, their number concentration was compared to northern wind component, stable isotopes of oxygen, {delta}18O, and the concentration of crustal elements (Fe, Ti and Ca). Use of satellite images combined with air mass back-trajectory analysis obtained from the NOAA/ HYSPLIT model corroborated the results.


title: Markovian Weighted Ensemble Milestoning (M-WEM): Long-time Kinetics from Short Trajectories

abstract: We introduce a rare-event sampling scheme, named Markovian Weighted Ensemble Milestoning (M-WEM), which inlays a weighted ensemble framework within a Markovian milestoning theory to efficiently calculate thermodynamic and kinetic properties of long-timescale biomolecular processes from short atomistic molecular dynamics simulations. M-WEM is tested on the Muller-Brown potential model, the conformational switching in alanine dipeptide, and the millisecond timescale protein-ligand unbinding in a trypsin-benzamidine complex. Not only can M-WEM predict the kinetics of these processes with quantitative accuracy, but it also allows for a scheme to reconstruct a multidimensional free energy landscape along additional degrees of freedom which are not part of the milestoning progress coordinate. For the ligand-receptor system, the experimental residence time, association and dissociation kinetics, and binding free energy could be reproduced using M-WEM within a simulation time of a few hundreds of nanoseconds, which is a fraction of the computational cost of other currently available methods, and close to four orders of magnitude less than the experimental residence time. Due to the high accuracy and low computational cost, the M-WEM approach can find potential application in kinetics and free-energy based computational drug design.


title: Origins and timing of emerging lesions in advanced renal cell carcinoma

abstract:
PurposeRenal cell carcinoma (RCC) with venous tumor thrombus (VTT) arising from the primary tumor occurs in 4-10% of cases and is associated with advanced disease. RCC with VTT and distant metastasis represents a unique clinical entity, and provides opportunities to examine the origins and relative timing of tumor lesion emergence and to identify molecular correlates with disease state.

Experimental DesignWe performed genomic and evolutionary analyses on 16 RCC patients with VTT, with eight also having metastases, using multi-region exome and RNA sequencing.

ResultsNo genomic alterations were specifically associated with the VTT or metastasis lesions; each tumor had multiple hallmark driver alterations, consistent with advanced disease state. We found that 21% (3/14) of clear-cell RCC cases could be assigned a previously defined "evolutionary subtype". Somatic mutation signatures were largely consistent with previously established RCC signatures, and showed low heterogeneity across regions of each tumor. Mismatch repair and homologous recombination ("BRCA-ness") deficiency signatures consistently co-occurred across most tumors, suggesting a pervasive role for intracellular DNA damage in RCC and the potential for related treatment strategies. Phylogenetic timing analysis of metastatic cases suggested that in most tumors, metastases branched from the primary tumor prior to formation of VTT and in some cases before diversification of the primary tumor. Both VTT and the earliest metastases were predicted to emerge many years prior to diagnosis. Transcriptional landscape analysis identified key differences distinguishing each lesion type from primary tumor: VTT upregulated TNF signaling and associated inflammatory pathways, whereas metastases upregulated MTOR signaling.

ConclusionsOur results provide a map of how RCC tumors can evolve, with metastatic clones typically emerging early in RCC development and taking hold via MTOR signaling, and later formation of VTT via local inflammatory processes.

Statement of Translational RelevanceRenal cell carcinoma (RCC) is a deadly and relatively common malignancy, which often presents as or progresses to metastatic disease. We used multi-region sequencing of RCC patients with venous tumor thrombus (VTT) and metastasis to ask how and when new lesions arise from the primary tumor, and what genomic factors contribute to their spread. Phylogenetic analysis of patients with VTT and co-presenting metastases suggested that in most cases, the VTT and metastases derive from distinct tumor clones. Moreover, metastatic clones often appear many years prior to diagnosis. We found that local TNF inflammation may contribute to VTT formation, whereas MTOR signaling is associated with metastases. Our study sheds light on the relationship of VTT and metastases, suggests therapeutic and biomarker strategies for RCC, and points to the need for early detection studies in RCC to better understand when metastases emerge and to identify at-risk patients.

category: cancer biology


title: DAB2IP inhibits p53 ubiquitin-mediated degradation by competitively binding to GRP75 and suppresses tumor malignancy in colon cancer

abstract: Increasing evidence has shown that DAB2IP acts as a tumor suppressor and plays an inhibition role in many tumors. However, the underlying mechanism is still uncertain. Our study shows that DAB2IP is positively associated with a better prognosis in colon cancer patients with wild-type TP53 expression. In vitro assay shows that DAB2IP elicits potent tumor-suppressive effects on inhibiting cell invasiveness, colony formation and promoting cell apoptosis in wild-type TP53 colon cancer cell lines. Subsequently, DAB2IP is demonstrated to up-regulate the stability of wild-type TP53 by inhibiting its degradation in a ubiquitin-proteasome-dependent manner. Using mass spectrometry profiling, we unveil that DAB2IP and p53 could both interact with the ubiquitin ligase-related protein, GRP75. Mechanistically, DAB2IP could competitively bind with GRP75, thus reducing GRP75-mediated p53 ubiquitination and degradation. Finally, animal experiments also reveal that DAB2IP inhibits the tumor progression in vivo. In conclusion, our study presents a novel function of DAB2IP in GRP75-driven wild-type p53 degradation, which provides a new insight in DAB2IP-induced tumor suppression and provides a novel molecular aspect of the p53 pathway.


title: Inhibition of cancer cells in culture. The effects of treatment with ketone bodies and/or rapamycin treatment

abstract:
BackgroundThe potential for ketogenic diets or administration of exogenous ketone bodies to treat or prevent to cancer remains encouraging. Of particular interest is the possibility that, whatever the effect of a nutritional intervention alone, the diet might enhance the effect of existing cancer drugs, thereby requiring lower doses and a reduction in toxicity and side effects.

MethodsSW480, a human cell line derived from colon, was treated with ketone bodies (sodium 3-hydroxy butyrate (common name, {square}-hydroxy butyrate) or with sodium acetoacetate in the presence or absence of rapamycin. Cells were incubated for 96 hours in DMEM with 10 mM glucose medium. HSF2617, a human epithelial fibroblast line served as control and cells were subjected to similar treatment as the SW480 cells. Cell proliferation and glucose consumption were determined with standard reagents.

ResultsThe ketone bodies inhibited proliferation of SW480 cells in culture. Rapamycin also inhibited proliferation and its action was enhanced by the ketone bodies although there was little synergistic effect under these conditions. Human fibroblast controls were not inhibited by the ketone bodies. Both SW480 and control lines showed consumption of glucose during a 96 hour incubation period, suggesting that normal controls can switch to ketogenic metabolism while the cancer cells, which proliferate poorly, cannot. Results are consistent with recent reports of a mouse model showing the synergy of rapamycin and a ketogenic diet (Zou Y, et al. (2020) PLoS ONE 15 (5)) as well as earlier publications describing additive or synergistic effects of ketogenic diets with other modalities of cancer treatment.

ConclusionsThe results show that the growth of a cancer cell line in culture can be inhibited by the addition of ketone bodies or rapamycin to the growth medium. The combination of treatments was found to be additive, consistent with results from a previously published mouse model. The data demonstrate the potential for a strategy whereby doses of anti-cancer agents that have detrimental or toxic side-effects can be reduced if coupled to an appropriate source of ketone bodies.


title: Release of endogenous dynorphin opioids in the prefrontal cortex disrupts cognition

abstract: Following repeated opioid use, some dependent individuals experience persistent cognitive deficits that contribute to relapse of drug-taking behaviors, and one component of this response may be mediated by the endogenous dynorphin/kappa opioid system in neocortex. In mice, we find that acute morphine withdrawal evokes dynorphin release in the medial prefrontal cortex (PFC) and disrupts cognitive function by activation of local kappa opioid receptors (KORs). Immunohistochemical analyses using a phospho-KOR antibody confirmed that both withdrawal-induced and optically evoked dynorphin release activated KOR in PFC. Using a genetically encoded sensor based on inert KOR (kLight1.2a), we revealed the in vivo dynamics of endogenous dynorphin release in the PFC. Local activation of KOR in PFC produced multi-phasic disruptions of memory processing in an operant delayed alternation behavioral task, which manifest as reductions in response number and accuracy during early and late phases of an operant session. Local pretreatment in PFC with the selective KOR antagonist norbinaltorphimine (norBNI) blocked the disruptive effect of systemic KOR activation during both early and late phases of the session. The early, but not late phase disruption was blocked by viral excision of PFC KORs, suggesting an anatomically dissociable contribution of pre- and postsynaptic KORs. Naloxone-precipitated withdrawal in morphine-dependent mice or optical stimulation of pdynCre neurons using Channelrhodopsin-2 (ChR2) disrupted delayed alternation performance, and the dynorphin-induced effect was blocked by local norBNI. Our findings describe a mechanism for control of cortical function during opioid dependence and suggest that KOR antagonism could promote abstinence.


title: Nanoscopical analysis reveals an orderly arrangement of the presynaptic scaffold protein Bassoon at the Golgi-apparatus

abstract:
1.Bassoon is a large, 440 kDa, scaffold protein of the specialized sites mediating neurotransmitter release from presynaptic nerve terminals, called active zones. At active zones of the brain, Bassoon is arranged with its C-terminus facing the plasma membrane. In young, cultured neurons Bassoon is associated with the Golgi-apparatus, where it may assemble active zone precursors structures, but whether or not Bassoon is an extended protein at the Golgi-apparatus and whether it is arranged in an orderly fashion is unknown. Understanding the topology of this large scaffold protein is important for models of active zone biogenesis.

Here, we generated recombinant Bassoon constructs for expression in neurons, with tags positioned to allow for the specific detection of N- and C-terminal regions of Bassoon by single-domain antibodies, called nanobodies. Using stimulated emission depletion (STED) nanoscopy in cultured hippocampal neurons, we confirmed that recombinant Bassoon is oriented with its C-terminus towards the active zone plasma membrane at synapses. Focusing on the soma, we found that an intramolecular tag located immediately downstream of amino acid 97 of Bassoon, showed prominent colocalization with markers of the trans Golgi network, including TGN38 and syntaxin-6. In contrast, tags located immediately downstream of the C-terminal amino acid 3938 of Bassoon showed significantly less colocalization with these Golgi-markers. The intramolecular N-terminal tag was located between 48 and 69 nm away from TGN38, while C-terminal tags were located between 100 and 115 nm away from TGN38. Sequences within the first 95 amino acids of Bassoon, but not its N-myristoylation sequence, were required for this arrangement.

Our results indicate that at the Golgi-apparatus Bassoon is oriented with its N-terminus towards and its C-terminus away from the trans Golgi network membrane. Moreover, they suggest that Bassoon is an extended molecule at the trans Golgi network with the distance between amino acids 97 and 3938 estimated to be between 46 and 52 nm. Our data are consistent with a model, in which the N-terminus of Bassoon binds to the membranes of the trans-Golgi network, while the C-terminus associates with active zone components, thus reflecting the topographic arrangement characteristic of synapses also at the Golgi-apparatus.


title: Contraction-dependent intracortical inhibition supports gravity-efficient motor control

abstract: Efficient control of voluntary movements along the gravity axis requires adapted shifts in muscular contraction modes. In daily life, rising the arm up involves shortening (i.e., concentric) contractions of arm flexors, while the reverse movement can rely on lengthening (i.e., eccentric) contractions of the same muscles with the help of gravity force. Although this muscular-control mode is universal, the neuromuscular mechanisms that subserve the control of such gravity-oriented movements remain unknown. In this study, we designed two neurophysiological experiments that allowed tracking modulations of cortical, spinal, and muscular outputs of arm flexors while healthy humans carried out vertical pointing movements. In conditions where upward and downward movements revealed kinematic features reminiscent of optimal motor commands (i.e., directional asymmetries), we report fine contraction-dependent modulations of the corticospinal output. The overall corticospinal excitability dropped during lengthening contractions (downward movements) compared with shortening contractions (upward movements). Specifically, we did not observe any change in spinal motoneuron responsiveness from cervicomedullary stimulations but a specific increase in intracortical inhibition during lengthening vs. shortening contractions. We discuss these fine contraction-dependent modulations of the supraspinal motor output in the light of feedforward mechanisms that may support gravity-tuned motor control. Generally, these results shed a new perspective on the neural policy that optimize movement control along the gravity axis.


title: The role of temporal coherence and temporal stability in the build-up of auditory grouping

abstract: The cochlea decomposes sounds into separate frequency channels, from which the auditory brain must reconstruct the auditory scene. To do this the auditory system must make decisions about which frequency information should be grouped together, and which should remain distinct. Two key cues for grouping are temporal coherence, resulting from coherent changes in power across frequency, and temporal predictability, resulting from regular or predictable changes over time. To test how these cues contribute to the construction of a sound scene we present listeners with a range of precursor sounds, which act to prime the auditory system by providing information about each sounds structure, followed by a fixed masker in which participants were required to detect the presence of an embedded tone. By manipulating temporal coherence and/or temporal predictability in the precursor we assess how prior sound exposure influences subsequent auditory grouping. In Experiment 1, we measure the contribution of temporal predictability by presenting temporally regular or jittered precursors, and temporal coherence by using either narrow or broadband sounds, demonstrating that both independently contribute to masking/unmasking. In Experiment 2, we measure the relative impact of temporal coherence and temporal predictability and ask whether the influence of each in the precursor signifies an enhancement or disruption of unmasking. We observed that interfering precursors produced the largest changes to thresholds.


title: Chronic adolescent stress increases exploratory behavior but does not change the acute stress response in adult male C57BL/6 mice

abstract: Chronic stress exposure in adolescence can lead to a lasting change in stress responsiveness later in life and is associated with increased mental health issues in adulthood. Here we investigate whether the Chronic Social Instability (CSI) paradigm in mice influences the behavioural and molecular responses to novel acute stressors, and whether it alters physiological responses influenced by the noradrenergic system. Using large cohorts of mice, we show that CSI mice display a persistent increase in exploratory behaviors in the open field test alongside small but widespread transcriptional changes in the ventral hippocampus. However, both the transcriptomic and behavioural responses to novel acute stressors are indistinguishable between groups. In addition, the pupillometric response to a tail shock, known to be mediated by the noradrenergic system, remains unaltered in CSI mice. Ultra-high performance liquid chromatography analysis of monoaminergic neurotransmitter levels in the ventral hippocampus also shows no differences between control or CSI mice at baseline or in response to acute stress. We conclude that CSI exposure during adolescence leads to persistent changes in exploratory behavior and gene expression in the hippocampus, but it does not alter the response to acute stress challenges in adulthood and is unlikely to alter the function of the noradrenergic system.


title: Carbohydrate sulfation as a mechanism for fine-tuning Siglec ligands

abstract: The immunomodulatory family of Siglecs recognize sialic acid-containing glycans as  self, which is exploited in cancer for immune-evasion. The biochemical nature of Siglec ligands remains incompletely understood with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs (CHST1, CHST2, or CHST4) significantly enhances the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, pharmacological blockade of N- and O-glycan maturation reveals a cell type-specific pattern of importance for either class of glycan. Production of a highly homogenous CD33 (Siglec-3) fragment enabled a mass spectrometry-based binding assay to determine 10-fold and 3-fold enhanced affinity for Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4GlcNAc and Neu5Ac2-3Gal{beta}1-4(6-O- sulfo)GlcNAc, respectively, over Neu5Ac2-3Gal{beta}1-4GlcNAc. CD33 showed significant additivity in affinity (36-fold) for the disulfated ligand, Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4(6-O-sulfo)GlcNAc. Moreover, overexpression of both CHST1 and CHST2 in cells greatly enhanced the binding of several Siglecs, including CD33. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a modification that is a general mechanism for tuning Siglec ligands on cells, including in cancer.

category: immunology