title: Flexible utilization of spatial- and motor-based codes for the storage of visuo-spatial information

abstract: Working memory provides flexible storage of information in service of upcoming behavioral goals. Some models propose specific fixed loci and mechanisms for the storage of visual information in working memory, such as sustained spiking in parietal and prefrontal cortex during working memory maintenance. An alternative view is that information can be remembered in a flexible format that best suits current behavioral goals. For example, remembered visual information might be stored in sensory areas for easier comparison to future sensory inputs, or might be re-coded into a more abstract action-oriented format and stored in motor areas. Here, we tested this hypothesis using a visuo-spatial working memory task where the required behavioral response was either known or unknown during the memory delay period. Using fMRI and multivariate decoding, we found that there was less information about remembered spatial position in early visual and parietal regions when the required response was known versus unknown. Further, a representation of the planned motor action emerged in primary somatosensory, primary motor, and premotor cortex during the same task condition where spatial information was reduced in early visual cortex. These results suggest that the neural networks supporting working memory can be strategically reconfigured depending on specific behavioral requirements during a canonical visual working memory paradigm.


title: Standardizing workflows in imaging transcriptomics with the abagen toolbox

abstract: Gene expression fundamentally shapes the structural and functional architecture of the human brain. Open-access transcriptomic datasets like the Allen Human Brain Atlas provide an unprecedented ability to examine these mechanisms in vivo; however, a lack of standardization across research groups has given rise to myriad processing pipelines for using these data. Here, we develop the abagen toolbox, an open-access software package for working with transcriptomic data, and use it to examine how methodological variability influences the outcomes of research using the Allen Human Brain Atlas. Applying three prototypical analyses to the outputs of 750,000 unique processing pipelines, we find that choice of pipeline has a large impact on research findings, with parameters commonly varied in the literature influencing correlations between derived gene expression and other imaging phenotypes by as much as{rho} [≥] 1.0. Our results further reveal an ordering of parameter importance, with processing steps that influence gene normalization yielding the greatest impact on downstream statistical inferences and conclusions. The presented work and the development of the abagen toolbox lay the foundation for more standardized and systematic research in imaging transcriptomics, and will help to advance future understanding of the influence of gene expression in the human brain.

category: neuroscience


title: Improved prediction of behavioral and neural similarity spaces using pruned DNNs

abstract: Deep Neural Networks (DNNs) have become an important tool for modeling brain and behaviour. One key area of interest has been to apply these networks to model human similarity judgements. Several previous works have used the embeddings from the penultimate layer of vision DNNs and showed that a reweighting of these features improves the fit between human similarity judgments and DNNs. These studies underline the idea that these embeddings form a good basis set but lack the correct level of salience. Here we re-examined the grounds for this idea and on the contrary, we hypothesized that these embeddings, beyond forming a good basis set, also have the correct level of salience to account for similarity judgments. It is just that the huge dimensional embedding needs to be pruned to select those features relevant for the considered domain for which a similarity space is modeled. In Study 1 we supervised DNN pruning based on a subset of human similarity judgments. We found that pruning: i) improved out-of-sample prediction of human similarity judgments from DNN embeddings, ii) produced better alignment with WordNet hierarchy, and iii) retained much higher classification accuracy than reweighting. Study 2 showed that pruning by neurobiological data is highly effective in improving out-of-sample prediction of brain-derived representational dissimilarity matrices from DNN embeddings, at times fleshing out isomorphisms not otherwise observable. Pruning supervised by human brain/behavior therefore effectively identifies alignable dimensions of semantic knowledge between DNNs and humans and constitutes an effective method for understanding the organization of knowledge in neural networks.


title: Knock-in tagging in zebrafish facilitated by insertion into non-coding regions

abstract:
Zebrafish provide an excellent model for in vivo cell biology studies due to their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers due to inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N- or C termini with fluorescent markers by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes critical for epithelial biology and organ development including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, and the ECM receptor {beta}1 integrin. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies.

Summary statementGeneration of endogenously tagged stable zebrafish knock-in lines is simplified by the integration of fluorescent protein cassettes with mRNA splicing elements into non-coding regions of genes.

category: developmental biology


title: NGS comparison of Downy and Powdery mildew resistance genomic regions between 'Regent' and 'Red Globe'

abstract:
Powdery and downy mildew have a large negative impact on grape production worldwide. Quantitative trait loci (QTL) mapping projects have identified several loci for the genetic factors responsible for resistance to these pathogens. Several of these studies have focused on the cultivar  Regent, which carries the resistance loci to downy mildew on chromosome 18 (Rpv3), as well powdery mildew on chromosome 15 (Ren3, Ren9). Several other minor resistance loci have also been identified on other chromosomes.

Here we report on the re-sequencing of the  Regent and  Red Globe (susceptible) genomes using next generation sequencing. While the genome of  Regent has more SNP variants than  Red Globe, the distribution of these variants across the two genomes is not the same, nor is it uniform. The variation per gene shows that some genes have higher SNP density than others and that the number of SNPs for a given gene is not always the same for the two cultivars. In this study, we investigate the effectiveness of studying the variation of non-synonymous to synonymous SNP ratios between resistant and susceptible cultivars in the target QTL regions as a strategy to narrow down the number of likely candidate genes for Rpv3, Ren3 and Ren9.

category: genetics


title: The Ubiquitous Vigilance Signal in fMRI Time Series Data

abstract: Vigilance and wakefulness modulate estimates of functional connectivity, and, if unaccounted for, they can become a substantial confound in resting-state fMRI. Unfortunately, wakefulness is rarely monitored due to the need for additional concurrent recordings (e.g., eye tracking, EEG). Recent work has shown that strong fluctuations around 0.05Hz, hypothesized to be CSF inflow, appear in the fourth ventricle (FV) when subjects fall asleep. The analysis of these fluctuations could provide an easy way to evaluate wakefulness in fMRI-only data. Here we evaluate this possibility using the 7T resting-state sample from the Human Connectome Project. Our results confirm the presence of those fluctuations in the HCP sample despite this data having relatively small inflow weighting. Moreover, we show that fluctuations of a similar frequency appear in large portions of grey matter with different temporal delays, and that they can substantially influence estimates of functional connectivity. Finally, we demonstrate that the temporal evolution of this signal cannot only help us reproduce previously reported overall sleep patterns in resting-state data, but also predict individual periods of eye closure with 70% accuracy, matching predictions attainable using the amplitude of the global signal (a common fMRI marker of arousal). In summary, our results demonstrate the ubiquitous presence of this signal in a large, publicly available, fMRI sample, its value as a marker of arousal in absence of a better metric, its relationship to the global signal, and its potential nuisance effects on functional connectivity estimates when ignored.


title: A specific relationship between musical sophistication and auditory working memory

abstract:
Musical engagement may be associated with better listening skills, such as the perception of and working memory for notes, in addition to the appreciation of musical rules. The nature and extent of this association is controversial. In this study we assessed the relationship between musical engagement and both sound perception and working memory.

We developed a task to measure auditory perception and working memory for sound using a behavioural measure for both, precision. We measured the correlation between these tasks and musical sophistication based on a validated measure (the Goldsmiths Musical Sophistication Index) that can be applied to populations of both musicians and non-musicians. The data show that musical sophistication accounts for 21% of the variance in the precision of working memory for frequency in an analysis that accounts for age and non-verbal intelligence. Musical sophistication was not significantly associated with the precision of working memory for amplitude modulation rate or with the precision of perception of either acoustic feature.

The work supports a specific association between musical sophistication and working memory for sound frequency.


title: Time-resolved structure-function coupling in brain networks

abstract: The relationship between structural and functional connectivity in the brain is a key question in systems neuroscience. Modern accounts assume a single global structure-function relationship that persists over time. Here we show that structure-function coupling is dynamic and regionally heterogeneous. We use a temporal unwrapping procedure to identify moment-to-moment co-fluctuations in neural activity, and reconstruct time-resolved structure-function coupling patterns. We find that patterns of dynamic structure-function coupling are highly organized across the cortex. These patterns reflect cortical hierarchies, with stable coupling in unimodal and transmodal cortex, and dynamic coupling in intermediate regions, particularly in insular cortex (salience network) and frontal eye fields (dorsal attention network). Finally, we show that the variability of structure-function coupling is shaped by the distribution of connection lengths. The time-varying coupling of structural and functional connectivity points towards an informative feature of the brain that may reflect how cognitive functions are flexibly deployed and implemented.


title: Cell-type specific transcriptional adaptations of nucleus accumbens interneurons to amphetamine

abstract: Parvalbumin-expressing (PV+) interneurons of the nucleus accumbens (NAc) play an essential role in the addictive-like behaviors induced by psychostimulant exposure. To identify molecular mechanisms of PV+ neuron plasticity, we isolated interneuron nuclei from the NAc of male and female mice following acute or repeated exposure to amphetamine (AMPH) and sequenced for cell type-specific RNA expression and chromatin accessibility. AMPH regulated the transcription of hundreds of genes in PV+ interneurons, and this program was largely distinct from that regulated in other NAc GABAergic neurons. Chromatin accessibility at enhancers predicted cell-type specific gene regulation, identifying transcriptional mechanisms of differential AMPH responses. Finally, we observed dysregulation of multiple PV-specific, AMPH-regulated genes in an Mecp2 mutant mouse strain that shows heightened behavioral sensitivity to psychostimulants, suggesting the functional importance of this transcriptional program. Together these data provide novel insight into the cell-type specific programs of transcriptional plasticity in NAc neurons that underlie addictive-like behaviors.


title: Instrumental aversion coding in the basolateral amygdala and its reversion by a benzodiazepine

abstract: Punishment involves learning the relationship between actions and their adverse consequences. Both the acquisition and expression of punishment learning depend on the basolateral amygdala (BLA), but how BLA supports punishment remains poorly understood. To address this, we measured calcium (Ca2+) transients in BLA principal neurons during punishment. Male rats were trained to press two individually presented levers for food; when one of these levers also yielded aversive footshock, responding on this punished lever decreased relative to the other, unpunished lever. In rats with the Ca2+indicator GCaMP6f targeted to BLA principal neurons, we observed excitatory activity transients to the footshock punisher and inhibitory transients to lever-presses. Critically, as rats learned punishment, activity around the punished response transformed from inhibitory to excitatory and similarity analyses showed that these punished lever-press transients resembled BLA transients to the punisher itself. Systemically administered benzodiazepine (midazolam) selectively alleviated punishment. Moreover, the degree to which midazolam alleviated punishment was associated with how much punished response-related BLA transients reverted to their pre-punishment state. Together, these findings show that punishment learning is supported by aversion-coding of instrumental responses in the BLA and that the anti-punishment effects of benzodiazepines are associated with a reversion of this aversion coding.


title: Investigating Regional Specificity of Alzheimers Disease Pathology in the 5xFAD Mouse Model

abstract: Alzheimers disease (AD) is a neurodegenerative disease that affects cognition and memory. Mouse models such as 5xFAD, incorporate mutations found in familial or early-onset (EOAD) AD; these mutations increase the production of the toxic AB species that contributes to plaque formation in the brain. However, not all brain regions in the 5xAD animals accumulate plaques to the same degree. For example, the cerebellum appears to be resistant to AB plaque formation, while other regions such as the cortex and subiculum develop copious plaques. The mechanism(s) underlying this regional specificity remains unclear. Thus, we used a fluorescent antibody to APP/AB to quantify the amount of these proteins in several brain regions of interest, including subiculum, cortex, and cerebellum. We found that the cerebellum had less APP/AB than the other regions quantified, which demonstrates a correlation between the levels of APP/AB and plaque formation. Taken together, this suggests that the regional specificity of AD pathology may be a result of different levels of protein expression.


title: Microbiome divergence of marine gastropod species separated by the Isthmus of Panama

abstract: The rise of the Isthmus of Panama ~3.5 mya separated populations of many marine organisms, which then diverged into new geminate sister species currently living in the Eastern Pacific Ocean and Caribbean Sea. However, we know very little about how such evolutionary divergences of host species have shaped their microbiomes. Here, we compared the microbiomes of whole-body and shell-surface samples of geminate species of marine gastropods in the genera Cerithium and Cerithideopsis to those of congeneric outgroups. Our results show that the effects of the Isthmus on microbiome composition varied among host genera and between sample types within the same hosts. In the whole-body samples, microbiome compositions of geminate species pairs in the focal genera tended to be similar, likely due to host filtering, although the strength of this relationship varied among the two groups and across similarity metrics. Shell-surface communities showed contrasting patterns, with co-divergence between the host taxa and a small number of microbial clades evident in Cerithideopsis, but not Cerithium. These results suggest that (i) the rise of the Isthmus of Panama affected microbiomes of geminate hosts in a complex and clade-specific manner and (ii) host-associated microbial taxa respond differently to vicariance events than the hosts themselves.

category: ecology


title: Phylotranscriptomics reveals discordance in the phylogeny of Hawaiian Drosophila and Scaptomyza (Diptera: Drosophilidae)

abstract: Island radiations present natural laboratories for studying the evolutionary process. The Hawaiian Drosophil-idae are one such radiation, with nearly 600 described species and substantial morphological and ecological diversification. These species are largely divided into a few major clades, but the relationship between these clades remains uncertain. Here we present 12 new assembled transcriptomes from across these clades, and use these transcriptomes to resolve the base of the evolutionary radiation. We recover a new hypothesis for the relationship between clades, and demonstrate its support over previously published hypotheses. We then use the evolutionary radiation to explore dynamics of concordance in phylogenetic support, by analyzing the gene and site concordance factors for every possible topological combination of major groups. We show that high bootstrap values mask low evolutionary concordance, and we demonstrate that the most likely topology is distinct from the topology with the highest support across gene trees and from the topology with highest support across sites. We then combine all previously published genetic data for the group to estimate a time-calibrated tree for over 300 species of drosophilids. Finally, we digitize dozens of published Hawaiian Drosophilidae descriptions, and use this to pinpoint probable evolutionary shifts in reproductive ecology as well as body, wing, and egg size. We show that by examining the entire landscape of tree and trait space, we can gain a more complete understanding of how evolutionary dynamics play out across an island radiation.

category: evolutionary biology


title: Multi-morph eco-evolutionary dynamics in structured populations

abstract: Our understanding of the evolution of quantitative traits in nature is still limited by the challenge of including realistic trait distributions in the context of frequency-dependent selection and ecological feedbacks. We extend to class-structured populations a recently introduced "oligomorphic approximation" which bridges the gap between adaptive dynamics and quantitative genetics approaches and allows for the joint description of the dynamics of ecological variables and of the moments of multimodal trait distributions. Our theoretical framework allows us to analyse the dynamics of populations composed of several morphs and structured into distinct classes (e.g. age, size, habitats, infection status, species...). We also introduce a new approximation to simplify the eco-evolutionary dynamics using reproductive values. We illustrate the effectiveness of this approach by applying it to the important conceptual case of two-habitat migration-selection models. In particular, we show that our approach allows us to predict both the long-term evolutionary endpoints and the short-term transient dynamics of the eco-evolutionary process, including fast evolution regimes. We discuss the theoretical and practical implications of our results and sketch perspectives for future work.


title: Dimensionality reduction and data integration for scRNA-seq data based on integrative hierarchical Poisson factorisation

abstract: Single-cell RNA sequencing (scRNA-seq) data sets consist of high-dimensional, sparse and noisy feature vectors, and pose a challenge for classic methods for dimensionality reduction. We show that application of Hierarchical Poisson Factorisation (HPF) to scRNA-seq data produces robust factors, and outperforms other popular methods. To account for batch variability in composite data sets, we introduce Integrative Hierarchical Poisson Factorisation (IHPF), an extension of HPF that makes use of a noise ratio hyper-parameter to tune the variability attributed to technical (batches) vs. biological (cell phenotypes) sources. We exemplify the advantageous application of IHPF under data integration scenarios with varying alignments of technical noise and cell diversity, and show that IHPF produces latent factors with a dual block structure in both cell and gene spaces for enhanced biological interpretability.

category: bioinformatics


title: Hepatoprotective effects of Aureobasidium pullulans derived Beta 1,3-1,6 biological response modifier glucans in a STAM- animal model of non-alcoholic steatohepatitis

abstract:
BackgroundNon-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are highly prevalent conditions characterized by inflammation and fibrosis of the liver which can progress to cirrhosis and hepatocellular carcinoma if left untreated. Lifestyle disorders such as obesity, diabetes and dyslipidaemia predispose to and are associated with the disease progression. Conventional modalities are mainly symptomatic, with no definite solution. Beta glucan-based biological response modifiers are a potential strategy in lieu of their beneficial metabolic effects. Aureobasidium pullulans strains AFO-202 and N-163 beta glucans were evaluated for anti-fibrotic and anti-inflammatory hepatoprotective potentials in a NASH animal model in this study.

MethodsIn the STAM murine model of NASH, five groups were studied for eight weeks-- (1) vehicle (RO water), (2) AFO-202 beta glucan; (3) N-163 beta glucan, (4) AFO-202+N-163 beta glucan, and (5) telmisartan (standard pharmacological intervention). Evaluation of biochemical parameters in plasma and hepatic histology including Sirius red staining and F4/80 immunostaining were performed.

ResultsAFO-202 beta glucan significantly decreased inflammation-associated hepatic cell ballooning and steatosis. N-163 beta glucan decreased fibrosis and inflammation significantly (p value<0.05). The combination of AFO-202 with N-163 significantly decreased the NAFLD Activity Score (NAS) compared with other groups.

ConclusionThis preclinical study supports the potential of N-163 and AFO-202 beta glucans alone or in combination as potential preventive and therapeutic agent(s), for NASH.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: pharmacology and toxicology


title: The mitochondrial Na+/Ca2+ exchanger, NCLX, mediates PDE2 dependent neuronal survival and learning

abstract: Impaired phosphodiesterase (PDE) function and mitochondrial Ca2+ - [Ca2+]m signaling leads to cardiac failure, ischemic damage and dysfunctional learning and memory. Yet, a causative link between these pathways is unknown. Here, we fluorescently monitored [Ca2+]m transients in hippocampal neurons evoked by caffeine followed by depolarization. [Ca2+]m efflux was apparent in WT but diminished in neurons deficient in the mitochondrial Na+/Ca2+ exchanger NCLX. Surprisingly, neuronal depolarization-induced Ca2+ transients alone failed to evoke strong [Ca2+]m efflux in WT neurons. Caffeine is also a PDE inhibitor. Pretreatment with the PDE2 inhibitor Bay 60-7550 rescued [Ca2+]m efflux triggered by neuronal depolarization. Inhibition of PDE2 acted by diminishing the Ca2+ dependent reduction of mitochondrial cAMP, thereby promoting NCLX phosphorylation. Selective PDE2 inhibition also enhanced [Ca2+]m efflux triggered by neuromodulators. We found that protection of neurons against excitotoxic insults, conferred by PDE2 inhibition, was diminished in NCLX KO neurons, thus is NCLX dependent. Finally, administration of Bay 60-7550 enhanced new object recognition learning in WT but not in NCLX KO mice. Our results identify a long-sought link between PDE and [Ca2+]m signaling thereby providing new therapeutic targets.

category: physiology


title: Dysregulated myogenesis and autophagy in genetically induced pulmonary emphysema

abstract: Patients with chronic obstructive pulmonary disease (COPD)-pulmonary emphysema often develop locomotor muscle dysfunction, which is independently associated with disability and higher mortality in that population. Muscle dysfunction entails reduced muscle mass and force-generation capacity, which are influenced by fibers integrity. Myogenesis, which is muscle turnover driven by progenitor cells such as satellite cells, contributes to the maintenance of muscle integrity in the context of organ development and injury-repair cycles. Injurious events crucially occur in COPD patients skeletal muscles in the setting of exacerbations and infections which lead to acute decompensations for limited periods of time after which, patients typically fail to recover the baseline status they had before the acute event. Autophagy, which is dysregulated in muscles from COPD patients, is a key regulator of satellite cells activation and myogenesis, yet very little research has so far investigated the mechanistic role of autophagy dysregulation in COPD muscles. Using a genetically inducible murine model of COPD-driven muscle dysfunction and confirmed with a second genetic animal model, we found a significant myogenic dysfunction associated with a reduced proliferative capacity of freshly isolated satellite cells. Transplantation experiments followed by lineage tracing suggest that an intrinsic defect in satellite cells, and not in the COPD environment, plays a dominant role in the observed myogenic dysfunction. RNA sequencing analysis of freshly isolated satellite cells suggests dysregulation of transcripts associated with control of cell cycle and autophagy, which is confirmed by a direct observation of COPD mice satellite cells fluorescent-tracked autophagosome formation. Moreover, spermidine-induced autophagy stimulation leads to improved satellite cells autophagosome turnover, replication rate and myogenesis. Our data suggests that pulmonary emphysema causes a disrupted myogenesis, which could be improved with stimulation of autophagy and satellite cells activation, leading to an attenuated muscle dysfunction in this context.


title: Persistent autism-relevant phenotype produced by in utero and lactational exposure of female mice to the commercial PBDE mixture, DE-71

abstract: Polybrominated diphenyl ethers (PBDEs) are ubiquitous persistent organic pollutants (POPs) that are known neuroendocrine disrupting chemicals with adverse neurodevelopmental effects. PBDEs may act as risk factors for autism spectrum disorders (ASD), characterized by abnormal psychosocial functioning, although direct evidence is currently lacking. Using a translational exposure model, we tested the hypothesis that maternal transfer of a commercial mixture of PBDEs, DE-71, produces ASD-relevant behavioral and neurochemical deficits in female offspring. C57Bl6/N mouse dams (F0) were exposed to DE-71 via oral administration of 0 (VEH/CON), 0.1 (L-DE-71) or 0.4 (H-DE-71) mg/kg bw/d from 3 wk prior to gestation through lactation. Mass spectrometry analysis indicated in utero and lactational transfer of PBDEs (ppb) to F1 female offspring brain tissue at postnatal day (PND) 15 which was reduced by PND 110. Neurobehavioral testing of social novelty preference (SNP) and social recognition memory (SRM) revealed that adult L-DE-71 F1 offspring display altered short- and long-term SRM, in the absence of reduced sociability, and increased repetitive behavior. These effects were concomitant with reduced olfactory discrimination of social odors. Additionally, L-DE-71 exposure also altered short-term novel object recognition memory but not anxiety or depressive-like behavior. Moreover, F1 L-DE-71 displayed downregulated mRNA transcripts for oxytocin (Oxt) in the bed nucleus of the stria terminalis (BNST) and supraoptic nucleus, vasopressin (Avp) in the BNST and upregulated Avp1ar in BNST, and Oxtr in the paraventricular nucleus. Our work demonstrates that developmental PBDE exposure produces ASD-relevant neurochemical, olfactory processing and behavioral phenotypes that may result from early neurodevelopmental reprogramming within central social and memory networks.


title: Impact of temperature on the affinity of SARS-CoV-2 Spike for ACE2

abstract: The seasonal nature in the outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. The current COVID-19 pandemic makes no exception, and temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2. The receptor binding domain (RBD) of the Spike glycoprotein binds to the angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Studying the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike to ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide, bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

category: microbiology


title: Reduced neutralization of SARS-CoV-2 B.1.617 variant by inactivated and RBD-subunit vaccine

abstract: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The Spike protein that mediates coronavirus entry into host cells is a major target for COVID-19 vaccines and antibody therapeutics. However, multiple variants of SARS-CoV-2 have emerged, which may potentially compromise vaccine effectiveness. Using a pseudovirus-based assay, we evaluated SARS-CoV-2 cell entry mediated by the viral Spike B.1.617 and B.1.1.7 variants. We also compared the neutralization ability of monoclonal antibodies from convalescent sera and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine) and ZF2001 (RBD-subunit vaccine) against B.1.617 and B.1.1.7 variants. Our results showed that, compared to D614G and B.1.1.7 variants, B.1.617 shows enhanced viral entry and membrane fusion, as well as more resistant to antibody neutralization. These findings have important implications for understanding viral infectivity and for immunization policy against SARS-CoV-2 variants.


title: Therapeutic efficacy of CT-P59 against P.1 variant of SARS-CoV-2

abstract:
P.1. or gamma variant also known as the Brazil variant, is one of the variants of concern (VOC) which appears to have high transmissibility and mortality. To explore the potency of the CT-P59 monoclonal antibody against P.1 variant, we tried to conduct binding affinity, in vitro neutralization, and in vivo animal tests. In in vitro assays revealed that CT-P59 is able to neutralize P.1 variant in spite of reduction in its binding affinity against a RBD (receptor binding domain) mutant protein including K417T/E484K/N501Y and neutralizing activity against P.1 pseudoviruses and live viruses. In contrast, in vivo hACE2 (human angiotensin-converting enzyme 2)-expressing TG (transgenic) mouse challenge experiment demonstrated that a clinically relevant or lower dosages of CT-P59 is capable of lowering viral loads in the respiratory tract and alleviates symptoms such as body weight losses and survival rates. Therefore, a clinical dosage of CT-P59 could compensate for reduced in vitro antiviral activity in P.1-infected mice, implying that CT-P59 has therapeutic potency for COVID-19 patients infected with P.1 variant.

HighlightsO_LICT-P59 could bind to and neutralize P.1 variant, but CT-P59 showed reduced susceptibility in in vitro tests.
C_LIO_LIThe clinical dosage of CT-P59 demonstrated in vivo therapeutic potency against P.1 variants in hACE2-expressing mice challenge study.
C_LIO_LICT-P59 ameliorates their body weight loss and prevents the lethality in P.1 variant-infected mice.
C_LI


title: A photoactivable natural product with broad antiviral activity against enveloped viruses including highly pathogenic coronaviruses

abstract: The SARS-CoV-2 outbreak has highlighted the need for broad-spectrum antivirals against coronaviruses (CoVs). Here, pheophorbide a (Pba) was identified as a highly active antiviral molecule against HCoV-229E after bioguided fractionation of plant extracts. The antiviral activity of Pba was subsequently shown for SARS-CoV-2 and MERS-CoV, and its mechanism of action was further assessed, showing that Pba is an inhibitor of coronavirus entry by directly targeting the viral particle. Interestingly, the antiviral activity of Pba depends on light exposure, and Pba was shown to inhibit virus-cell fusion by stiffening the viral membrane as demonstrated by cryo-electron microscopy. Moreover, Pba was shown to be broadly active against several other enveloped viruses, and reduced SARS-CoV-2 and MERS-CoV replication in primary human bronchial epithelial cells. Pba is the first described natural antiviral against SARS-CoV-2 with direct photosensitive virucidal activity that holds potential for COVID-19 therapy or disinfection of SARS-CoV-2 contaminated surfaces.


title: Critical genetic program for Drosophila imaginal disc regeneration revealed by single-cell analysis

abstract: Whether regeneration is primarily accomplished by re-activating gene regulatory networks used previously during development or by activating novel regeneration-specific transcriptional programs remains a longstanding question. Currently, most genes implicated in regeneration also function during development. Using single-cell transcriptomics in regenerating Drosophila wing discs, we identified two regeneration-specific cell populations within the blastema. They are each composed of cells that upregulate multiple genes encoding secreted proteins that promote regeneration. In this regenerative secretory zone, the transcription factor Ets21C controls the expression of multiple regenerationpromoting genes. While eliminating Ets21C function has no discernible effect on development, it severely compromises regeneration. This Ets21C-dependent gene regulatory network is also activated in blastema-like cells in tumorous discs, suggesting that pro-regenerative mechanisms can be co-opted by tumors to promote aberrant growth.


title: Unraveling patterns of disrupted gene expression across a complex tissue

abstract: Whole tissue RNASeq is the standard approach for studying gene expression divergence in evolutionary biology and provides a snapshot of the comprehensive transcriptome for a given tissue. However, whole tissues consist of diverse cell types differing in expression profiles, and the cellular composition of these tissues can evolve across species. Here, we investigate the effects of different cellular composition on whole tissue expression profiles. We compared gene expression from whole testes and enriched spermatogenesis populations in two species of house mice, Mus musculus musculus and M. m. domesticus, and their sterile and fertile F1 hybrids, which differ in both cellular composition and regulatory dynamics. We found that cellular composition differences skewed expression profiles and differential gene expression in whole testes samples. Importantly, both approaches were able to detect large-scale patterns such as disrupted X chromosome expression although whole testes sampling resulted in decreased power to detect differentially expressed genes. We encourage researchers to account for histology in RNASeq and consider methods that reduce sample complexity whenever feasible. Ultimately, we show that differences in cellular composition between tissues can modify expression profiles, potentially altering inferred gene ontological processes, insights into gene network evolution, and processes governing gene expression evolution.


title: Multifactorial chromatin regulatory landscapes at single cell resolution

abstract: Chromatin profiling at locus resolution uncovers gene regulatory features that define cell types and developmental trajectories, but it remains challenging to map and compare distinct chromatin-associated proteins within the same sample. Here we describe a scalable antibody barcoding approach for profiling multiple chromatin features simultaneously in the same individual cells, Multiple Target Identification by Tagmentation (MulTI-Tag). MulTI-Tag is optimized to retain high sensitivity and specificity of enrichment for multiple chromatin targets in the same assay. We use MulTI-Tag to resolve distinct cell types using multiple chromatin features on a commercial single-cell platform, and to distinguish unique, coordinated patterns of active and repressive element regulatory usage in the same individual cells. Multifactorial profiling allows us to detect novel associations between histone marks in single cells and holds promise for comprehensively characterizing cell-specific gene regulatory landscapes in development and disease.

category: genomics


title: An Empirical Bayes Approach to Estimating Dynamic Models of Co-regulated Gene Expression

abstract: Time-course gene expression datasets provide insight into the dynamics of complex biological processes, such as immune response and organ development. It is of interest to identify genes with similar temporal expression patterns because such genes are often biologically related. However, this task is challenging due to the high dimensionality of these datasets and the nonlinearity of gene expression time dynamics. We propose an empirical Bayes approach to estimating ordinary differential equation (ODE) models of gene expression, from which we derive a similarity metric between genes called the Bayesian lead-lag R2 (LLR2). Importantly, the calculation of the LLR2 leverages biological databases that document known interactions amongst genes; this information is automatically used to define informative prior distributions on the ODE models parameters. As a result, the LLR2 is a biologically-informed metric that can be used to identify clusters or networks of functionally-related genes with co-moving or time-delayed expression patterns. We then derive data-driven shrinkage parameters from Steins unbiased risk estimate that optimally balance the ODE models fit to both data and external biological information. Using real gene expression data, we demonstrate that our methodology allows us to recover interpretable gene clusters and sparse networks. These results reveal new insights about the dynamics of biological systems.


title: Photoredox-catalyzed decarboxylative C-terminal differentiation for bulk and single molecule proteomics

abstract: Methods for the selective labeling of biogenic functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. To achieve successful labeling with any one method requires that the peptide fragments contain the functional group for which the labeling chemistry is designed. In practice, only two functional groups are present on every peptide fragment regardless of the protein cleavage site, namely, an N-terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the N- and/or C-terminus of peptides. In this work, we showcase the first successful application of photocatalyzed C-terminal decarboxylative-alkylation for peptide mass-spectrometry and single molecule protein sequencing, that can be broadly applied to any proteome. We demonstrate that peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts, can be site-specifically labeled at their C-terminal residue with a Michael acceptor. Using two distinct analytical approaches, we characterize C-terminal labeling efficiencies of greater than 50% across complete proteomes and document the proclivity of various C-terminal amino acid residues for decarboxylative-labeling, showing histidine and tryptophan to be the most disfavored. Finally, we combine C-terminal decarboxylative labeling with an orthogonal carboxylic acid labeling technology in tandem, to establish a new platform for fluorosequencing.

category: biochemistry


title: CALIPER: A software for blood-free parametric Patlak mapping using PET/MRI input function.

abstract:
Routine clinical use of absolute PET quantification techniques is limited by the need for serial arterial blood sampling for input function and more importantly by the lack of automated pharmacokinetic analysis tools that can be readily implemented in clinic with minimal effort. PET/MRI provides the ability for absolute quantification of PET probes without the need for serial arterial blood sampling using image-derived input functions (IDIFs). Here we introduce caliPER, a modular and scalable software for simplified pharmacokinetic modelling of PET probes with irreversible uptake or binding based on PET/MR IDIFs and Patlak Plot analysis. caliPER generates regional values or parametric maps of net influx rate (Ki) using reconstructed dynamic PET images and anatomical MRI aligned to PET for IDIF vessel delineation. We evaluated the performance of caliPER for blood-free region-based and pixel-wise Patlak analyses of [18F] FDG by comparing caliPER IDIF to serial arterial blood input functions and its application in imaging brain glucose hypometabolism in Frontotemporal dementia. IDIFs corrected for partial volume errors including spill-out and spill-in effects were similar to arterial blood input functions with a general bias of around 6-8%, even for arteries <5 mm. The Ki and cerebral metabolic rate of glucose estimated using caliPER IDIF were similar to estimates using arterial blood sampling (<2%) and within limits of whole brain values reported in literature. Overall, caliPER is a promising tool for irreversible PET tracer quantification and can simplify the ability to perform parametric analysis in clinical settings without the need for blood sampling.

HighlightsO_LIcaliPER is an adaptable image processing software for extracting image-derived input functions and generating parametric maps of irreversible PET tracer uptake.
C_LIO_LIAnatomical (T1-weighted/T2-weighted/time-of-flight angiography) MRI carefully aligned to PET provides a robust approach for delineation of vessels on PET, eliminating the need for serial blood sampling for input functions.
C_LIO_LIApplication of caliPER in modelling glucose uptake in patients with Frontotemporal dementia, demonstrates the feasibility of absolute quantification of cerebral metabolic rate of glucose in clinical populations.
C_LI


title: caliPER: A software for blood-free parametric Patlak mapping using PET/MRI input function.


title: Obtaining Spatially Resolved Tumor Purity Maps Using Deep Multiple Instance Learning In A Pan-cancer Study

abstract: Tumor purity is the proportion of cancer cells in the tumor tissue. An accurate tumor purity estimation is crucial for accurate pathologic evaluation and for sample selection to minimize normal cell contamination in high throughput genomic analysis. We developed a novel deep multiple instance learning model predicting tumor purity from H&E stained digital histopathology slides. Our model successfully predicted tumor purity from slides of fresh-frozen sections in eight different TCGA cohorts and formalin-fixed paraffin-embedded sections in a local Singapore cohort. The predictions were highly consistent with genomic tumor purity values, which were inferred from genomic data and accepted as the golden standard. Besides, we obtained spatially resolved tumor purity maps and showed that tumor purity varies spatially within a sample. Our analyses on tumor purity maps also suggested that pathologists might have chosen high tumor content regions inside the slides during tumor purity estimation in the TCGA cohorts, which resulted in higher values than genomic tumor purity values. In short, our model can be utilized for high throughput sample selection for genomic analysis, which will help reduce pathologists workload and decrease inter-observer variability. Moreover, spatial tumor purity maps can help better understand the tumor microenvironment as a key determinant in tumor formation and therapeutic response.

category: cancer biology


title: Your head is there to move you around: Goal-driven models of the primate dorsal pathway

abstract: Neurons in the dorsal visual pathway of the mammalian brain are selective for motion stimuli, with the complexity of stimulus representations increasing along the hierarchy. This progression is similar to that of the ventral visual pathway, which is well characterized by artificial neural networks (ANNs) optimized for object recognition. In contrast, there are no image-computable models of the dorsal stream with comparable explanatory power. We hypothesized that the properties of dorsal stream neurons could be explained by a simple learning objective: the need for an organism to orient itself during self-motion. To test this hypothesis, we trained a 3D ResNet to predict an agents self-motion parameters from visual stimuli in a simulated environment. We found that the responses in this network accounted well for the selectivity of neurons in a large database of single-neuron recordings from the dorsal visual stream of non-human primates. In contrast, ANNs trained on an action recognition dataset through supervised or self-supervised learning could not explain responses in the dorsal stream, despite also being trained on naturalistic videos with moving objects. These results demonstrate that an ecologically relevant cost function can account for dorsal stream properties in the primate brain.


title: Curcumin micronization by supercritical fluid: in vitro and in vivo biological relevance

abstract: Curcumin, a polyphenol extracted from the rhizome of Curcuma longa L. (Zingiberaceae), is shown to have antioxidant, anti-inflammatory, neuroprotective, anxiolytic, and antidepressant properties in both preclinical and clinical studies. However, its low bioavailability is a limitation for its potential adoption as a therapeutic agent. The process of micronization can overcome this barrier by reducing the particle size and increasing the dissolution rate, potentially improving the bioavailability of the compounds of interest. In this study, we compared the in vitro antioxidant effects of curcumin (CUR) and micronized curcumin (MC) and studied their effects on behavioral and neurochemical parameters in zebrafish submitted to unpredictable chronic stress (UCS). MC (1 g/L) presented higher antioxidant activity in vitro as compared to CUR, as measured by iron-reducing antioxidant power (FRAP), 1,1-diphenyl-2-2-picyryl-hydrazyl radical removal (DPPH), and deoxyribose tests. UCS increased total distance traveled in the social interaction test (SI), while decreased crossings, time, and entries to the top area in the novel tank test (NTT). No effects of UCS were observed in the open tank test (OTT). The behavioral effects induced by UCS were not blocked by any curcumin preparation. UCS also decreased non-protein thiols (NPSH) levels, while increased glutathione reductase (GR) activity and thiobarbituric acid reactive substances (TBARS) levels on zebrafish brain. MC presented superior antioxidant properties than CUR in vivo, blocking the stress-induced neurochemical effects. Although this study did not measure the concentration of curcumin on the zebrafish brain, our results suggest that micronization increases the bioavailability of curcumin, potentiating its antioxidant activity both in vitro and in vivo. Our study also demonstrates that counteracting the oxidative imbalance induced by UCS is not sufficient to block its behavioral effects.


title: Luminance and contrast of images in the THINGS database

abstract: The THINGS database is a freely available stimulus set that has the potential to facilitate the generation of theory that bridges multiple areas within cognitive neuroscience. The database consists of 26,107 high quality digital photos that are sorted into 1,854 concepts. While a valuable resource, relatively few technical details relevant to the design of studies in cognitive neuroscience have been described. We present an analysis of two key low-level properties of THINGS images, luminance and luminance contrast. These image statistics are known to influence common physiological and neural correlates of perceptual and cognitive processes. In general, we found that the distributions of luminance and contrast are in close agreement with the statistics of natural images reported previously. However, we found that image concepts are separable in their luminance and contrast: we show that luminance and contrast alone are sufficient to classify images into their concepts with above chance accuracy. We describe how these factors may confound studies using the THINGS images, and suggest simple controls that can be implemented a priori or post-hoc. We discuss the importance of using such natural images as stimuli in psychological research.


title: Investigating brain mechanisms underlying natural reading by co-registering eye tracking with combined EEG and MEG

abstract: Linking brain and behavior is one of the great challenges in cognitive neuroscience. Ultimately, we want to understand how the brain processes information to guide every-day behavior. However, most neuroscientific studies employ very simplistic experimental paradigms whose ecological validity is doubtful. Reading is a case in point, since most neuroscientific studies to date have used unnatural word-by-word stimulus presentation and have often focused on single word processing. Previous research has therefore actively avoided factors that are important for natural reading, such as rapid self-paced voluntary saccadic eye movements. Recent methodological developments have made it possible to deal with associated problems such as eye movement artefacts and the overlap of brain responses to successive stimuli, using a combination of eye-tracking and neuroimaging. A growing number of electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) are successfully using this methodology. Here, we provide a proof-of-concept that this methodology can be applied to combined EEG and magnetoencephalography (MEG) data. Our participants naturally read 4-word sentences that could end in a plausible or implausible word while eye-tracking, EEG and MEG were being simultaneously recorded. Eye-movement artefacts were removed using independent-component analysis. Fixation-related potentials and fields for sentence-final words were subjected to minimum-norm source estimation. We detected an N400-type brain response in our EEG data starting around 200 ms after fixation of the sentence-final word. The brain sources of this effect, estimated from combined EEG and MEG data, were mostly located in left temporal lobe areas. We discuss the possible use of this method for future neuroscientific research on language and cognition.


title: Organization of cortical and thalamic input to inhibitory neurons in mouse motor cortex

abstract: Intracortical inhibition in motor cortex (M1) regulates movement and motor learning. If inhibitory cell types and cortical laminae targeted by cortical and thalamic afferents differ, then these afferents play different roles in regulating M1 output. We quantified input to two classes of M1 interneurons, parvalbumin+ (PV) fast-spiking cells and somatostatin+ (SOM) low-threshold-spiking cells, using monosynaptic rabies tracing. We then compared anatomical connectivity and functional connectivity based on synaptic strength from sensory cortex and thalamus. Functionally, each input innervated M1 interneurons with a unique laminar profile. Different interneuron types were excited in a distinct, complementary manner, suggesting feedforward inhibition proceeds selectively via distinct circuits. Specifically, somatosensory cortex (S1) inputs primarily targeted PV+ neurons in upper layers (L2/3) but SOM+ neurons in middle layers (L5). Somatosensory thalamus (PO) inputs targeted PV+ neurons in middle layers (L5). In contrast to sensory cortical areas, thalamic input to SOM+ neurons was equivalent to PV+ neurons. Thus, long-range excitatory inputs target inhibitory neurons in an area and cell type-specific manner which contrasts with input to neighboring pyramidal cells. In contrast to feedforward inhibition providing generic inhibitory tone in cortex, circuits are selectively organized to recruit inhibition matched to incoming excitatory circuits.


title: Behaviorally emergent hippocampal place maps remain stable during memory recall

abstract: The hippocampus is critical for the formation and recall of episodic memories1, 2 which store past experience of events ( what) occurring at particular locations ( where) in time ( when). Hippocampal place cells, pyramidal neurons which show location-specific modulation of firing rates during navigation3, 4, together form a spatial representation of the environment. It has long been hypothesized that place cells serve as the neural substrate for long-term episodic memory of space5, 6. However, recent studies call to question this tenet of the field by demonstrating unexpected levels of representational drift in hippocampal place cells with respect to the duration of episodic memories in mice7, 8. In the present study, we examined behaviorally driven long-term organization of the place map, to resolve the relationship between memory and place cells. Leveraging the stability of two-photon calcium imaging, we tracked activity of the same set of CA1 pyramidal neurons during learning and memory recall in an operant, head-fixed, odorcued spatial navigation task. We found that place cells are rapidly recruited into task-dependent spatial maps, resulting in emergence of orthogonal as well as overlapping representations of space. Further, task-selective place cells used a diverse set of remapping strategies to represent changing task demands that accompany learning. We found behavioral performance dependent divergence of spatial maps between trial types occurs during learning. Finally, imaging during remote recall spanning up to 30 days revealed increased stabilization of learnt place cell maps following memory consolidation. Our findings suggest that a subset of place cells is recruited by rule based spatial learning, actively reconfigured to represent task-relevant spatial relationships, and stabilized following successful learning and consolidation.


title: Lipopolysaccharide-induced neuroinflammation disrupts functional connectivity and community structure in primary cortical microtissues

abstract: Three-dimensional (3D) neural microtissues are a powerful in vitro paradigm for studying brain development and disease under controlled conditions, while maintaining many key attributes of the in vivo environment. Here, we used primary cortical microtissues to study the effects of neuroinflammation on neural microcircuits. We demonstrated the use of a genetically encoded calcium indicator combined with a novel live-imaging platform to record spontaneous calcium transients in microtissues from day 14-34 in vitro. We implemented graph theory analysis of calcium activity to characterize underlying functional connectivity and community structure of microcircuits, which are capable of capturing subtle changes in network dynamics during early diseases states. We found that microtissues cultured for 34 days displayed functional remodeling of microcircuits and that community structure strengthened over time. Lipopolysaccharide, a neuroinflammatory agent, significantly increased functional connectivity and disrupted community structure 5-9 days after exposure. These microcircuit-level changes have broad implications for the role of neuroinflammation in functional dysregulation of neural networks.


title: In silico proof of principle of machine learning-based antibody design at unconstrained scale

abstract:
Generative machine learning (ML) has been postulated to be a major driver in the computational design of antigen-specific monoclonal antibodies (mAb). However, efforts to confirm this hypothesis have been hindered by the infeasibility of testing arbitrarily large numbers of antibody sequences for their most critical design parameters: paratope, epitope, affinity, and developability. To address this challenge, we leveraged a lattice-based antibody-antigen binding simulation framework, which incorporates a wide range of physiological antibody binding parameters. The simulation framework enables both the computation of antibody-antigen 3D-structures as well as functions as an oracle for unrestricted prospective evaluation of the antigen specificity of ML-generated antibody sequences. We found that a deep generative model, trained exclusively on antibody sequence (1D) data can be used to design native-like conformational (3D) epitope-specific antibodies, matching or exceeding the training dataset in affinity and developability variety. Furthermore, we show that transfer learning enables the generation of high-affinity antibody sequences from low-N training data. Finally, we validated that the antibody design insight gained from simulated antibody-antigen binding data is applicable to experimental real-world data. Our work establishes a priori feasibility and the theoretical foundation of high-throughput ML-based mAb design.

HighlightsO_LIA large-scale dataset of 70M [3 orders of magnitude larger than the current state of the art] synthetic antibody-antigen complexes, that reflect biological complexity, allows the prospective evaluation of antibody generative deep learning
C_LIO_LICombination of generative learning, synthetic antibody-antigen binding data, and prospective evaluation shows that deep learning driven antibody design and discovery at an unconstrained level is feasible
C_LIO_LITransfer learning (low-N learning) coupled to generative learning shows that antibody-binding rules may be transferred across unrelated antibody-antigen complexes
C_LIO_LIExperimental validation of antibody-design conclusions drawn from deep learning on synthetic antibody-antigen binding data
C_LI

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/451480v1_ufig1.gif" ALT="Figure 1">
View larger version (64K):
org.highwire.dtl.DTLVardef@1d947bcorg.highwire.dtl.DTLVardef@1f4c51corg.highwire.dtl.DTLVardef@b8df67org.highwire.dtl.DTLVardef@1530ea5_HPS_FORMAT_FIGEXP  M_FIG We leverage large synthetic ground-truth data to demonstrate the (A,B) unconstrained deep generative learning-based generation of native-like antibody sequences, (C) the prospective evaluation of conformational (3D) affinity, paratope-epitope pairs, and developability. (D) Finally, we show increased generation quality of low-N-based machine learning models via transfer learning.

C_FIG

category: immunology


title: Using Domain-Knowledge to Assist Lead Discovery in Early-Stage Drug Design

abstract: We are interested in generating new small molecules which could act as inhibitors of a biological target, when there is limited prior information on target-specific inhibitors. This form of drug-design is assuming increasing importance with the advent of new disease threats for which known chemicals only provide limited information about target inhibition. In this paper, we propose the combined use of deep neural networks and Inductive Logic Programming (ILP) that allows the use of symbolic domain-knowledge (B) to explore the large space of possible molecules. Assuming molecules and their activities to be instances of random variables X and Y, the problem is to draw instances from the conditional distribution of X, given Y, B (DX|Y,B). We decompose this into the constituent parts of obtaining the distributions DX|B and DY|X,B, and describe the design and implementation of models to approximate the distributions. The design consists of generators (to approximate DX|B and DX|Y,B) and a discriminator (to approximate DY|X,B). We investigate our approach using the well-studied problem of inhibitors for the Janus kinase (JAK) class of proteins. We assume first that if no data on inhibitors are available for a target protein (JAK2), but a small numbers of inhibitors are known for homologous proteins (JAK1, JAK3 and TYK2). We show that the inclusion of relational domain-knowledge results in a potentially more effective generator of inhibitors than simple random sampling from the space of molecules or a generator without access to symbolic relations. The results suggest a way of combining symbolic domain-knowledge and deep generative models to constrain the exploration of the chemical space of molecules, when there is limited information on target-inhibitors. We also show how samples from the conditional generator can be used to identify potentially novel target inhibitors.

category: molecular biology


title: Global patterns of subgenome evolution in organelle-targeted genes of sixallotetraploid angiosperms

abstract:
Whole-genome duplications (WGDs), in which the number of nuclear genome copies is elevated as a result of autopolyploidy or allopolyploidy, are a prominent process of diversification in eukaryotes. The genetic and evolutionary forces that WGD imposes upon cytoplasmic genomes are not well understood, despite the central role that cytonuclear interactions play in eukaryotic function and fitness. Cellular respiration and photosynthesis depend upon successful interaction between the 3000+ nuclear-encoded proteins destined for the mitochondria or plastids and the gene products of cytoplasmic genomes in multi-subunit complexes such as OXPHOS, organellar ribosomes, Photosystems I and II, and Rubisco. Allopolyploids are thus faced with the critical task of coordinating interactions between nuclear and cytoplasmic genes that were inherited from different species. Because cytoplasmic genomes share a more recent history of common descent with the maternal nuclear subgenome than the paternal subgenome, evolutionary "mismatches" between the paternal subgenome and the cytoplasmic genomes in allopolyploids might lead to accelerated rates of evolution in the paternal homoeologs of allopolyploids, either through relaxed purifying selection or strong directional selection to rectify these mismatches. We tested this hypothesis in maternal vs. paternal copies of organelle-targeted genes in six allotetraploids: Brachypodium hybridum, Chenopodium quinoa, Coffea arabica, Gossypium hirsutum, Nicotiana tabacum, and Triticum dicoccoides. We report evidence that allopolyploid subgenomes exhibit unequal rates of protein-sequence evolution, but we did not observe global effects of cytonuclear incompatibilities on paternal homoeologs of organelle-targeted genes. Analyses of gene content revealed mixed evidence for whether organelle-targeted genes re-diploidize more rapidly than non-organelle-targeted genes. Together, these global analyses provide insights into the complex evolutionary dynamics of allopolyploids, showing that allopolyploid subgenomes have separate evolutionary trajectories despite sharing the same nucleus, generation time, and ecological context.

AUTHOR SUMMARY

Whole genome duplication, in which the size and content of the nuclear genome is instantly doubled, represents one of the most profound forms of mutational change. The consequences of duplication events are equally monumental, especially considering that almost all eukaryotes have undergone whole genome duplications during their evolutionary history. While myriad genetic, cellular, organismal, and ecological effects of whole genome duplications have been extensively documented, relatively little attention has been paid to the diminutive but essential "other" genomes present inside the cell, those of chloroplasts and mitochondria. In this study, we compared the evolutionary patterns of >340,000 genes from 23 species to test whether whole genome duplications are associated with genetic mismatches between the nuclear, mitochondrial, and chloroplast genomes. We discovered tremendous differences between duplicated copies of nuclear genomes; however, mitochondria-nuclear and chloroplast-nuclear mismatches do not appear to be common following whole genome duplications. Together these genomic data represent the most extensive analysis yet performed on how polyploids maintain the delicate and finely tuned balance between the nuclear, mitochondrial, and chloroplast genomes.


title: Neural encoding of phrases and sentences in spoken language comprehension

abstract: Speech stands out in the natural world as a biological signal that communicates formally-specifiable complex meanings. However, the acoustic and physical dynamics of speech do not injectively mark the linguistic structure and meaning that we perceive. Linguistic structure must therefore be inferred through the human brains endogenous mechanisms, which remain poorly understood. Using electroencephalography, we investigated the neural response to synthesized spoken phrases and sentences that were closely physically-matched but differed in syntactic structure, under either linguistic or non-linguistic task conditions. Differences in syntactic structure were well-captured in theta band ([~] 2 to 7 Hz) phase coherence, phase connectivity degree at low frequencies (< [~] 2 Hz), and in both intensity and degree of power connectivity of induced neural response in the alpha band ([~] 7.5 to 13.5 Hz). Theta-gamma phase-amplitude coupling was found when participants listened to speech, but it did not discriminate between syntactic structures. Spectral-temporal response function modelling suggested different encoding states in both temporal and spectral dimensions as a function of the amount and type of linguistic structure perceived, over and above the acoustically-driven neural response. Our findings provide a comprehensive description of how the brain separates linguistic structures in the dynamics of neural responses, and imply that phase synchronization and strength of connectivity can be used as readouts for constituent structure, providing a novel basis for future neurophysiological research on linguistic structure in the brain.


title: Modulation in Alpha Band Activity Reflects Syntax Composition: An MEG Study of Minimal Syntactic Binding

abstract: Successful sentence comprehension requires the binding, or composition, of multiple words into larger structures to establish meaning. Using magnetoencephalography, we investigated the neural mechanisms involved in binding at the syntax level, in a task where contributions from semantics were minimized. Participants were auditorily presented with minimal sentences that required binding (pronoun and pseudo-verb with the corresponding morphological inflection; "she grushes") and pseudo-verb wordlists that did not require binding ("cugged grushes"). Relative to no binding, we found that syntactic binding was associated with a modulation in alpha band (8-12 Hz) activity in left-lateralized language regions. First, we observed a significantly smaller increase in alpha power around the presentation of the target word ("grushes") that required binding (-0.05s to 0.1s), which we suggest reflects an expectation of binding to occur. Second, during binding of the target word (0.15s to 0.25s), we observed significantly decreased alpha phase-locking between the left inferior frontal gyrus and the left middle/inferior temporal cortex, which we suggest reflects alpha-driven cortical disinhibition serving to strengthen communication within the syntax composition neural network. Together, our findings highlight the critical role of rapid spatial-temporal alpha band activity in controlling the allocation, transfer and coordination of the brains resources during syntax composition.


title: Structural covariance of amygdala subregions is associated with trait aggression and endogenous testosterone in healthy individuals

abstract: Many studies point toward volume reductions in the amygdala as a potential neurostructural marker for trait aggression. However, most of these findings stem from clinical samples, rendering unclear whether the findings generalize to non-clinical populations. Furthermore, the notion of neural networks suggests that interregional correlations in grey matter volume (i.e., structural covariance) can explain individual differences in aggressive behavior beyond local univariate associations. Here, we tested whether structural covariance between amygdala subregions and the rest of the brain is associated with self-reported aggression in a large sample of healthy young students (n=263; 51% women). Salivary testosterone concentrations were measured for a subset of n=76 participants (45% women), allowing us to investigate the influence of endogenous testosterone on structural covariance. Aggressive individuals showed enhanced covariance between superficial amygdala (SFA) and dorsal anterior insula (dAI), but lower covariance between laterobasal amygdala (LBA) and dorsolateral prefrontal cortex (dlPFC). These structural patterns overlap with functional networks involved in the genesis and regulation of aggressive behavior, respectively. With increasing endogenous testosterone, we observed stronger structural covariance between centromedial amygdala (CMA) and medial prefrontal cortex in men and between CMA and orbitofrontal cortex in women. These results speak for structural covariance of amygdala subregions as a robust correlate of trait aggression in healthy individuals. Moreover, regions that showed structural covariance with the amygdala modulated by either testosterone or aggression did not overlap, speaking for a more complex role of testosterone in human social behavior rather than the simple assumption that testosterone only increases aggressiveness.


title: Experimental evolution of Bacillus subtilis on Arabidopsis thaliana roots reveals fast adaptation and improved root colonization in the presence of soil microbes

abstract: The soil ubiquitous Bacillus subtilis is known to promote plant growth and protect plants against disease. These characteristics make B. subtilis highly relevant in an agricultural perspective, fueling the interest in studying B. subtilis-plant interactions. Here, we employ an experimental evolution approach to explore adaptation of B. subtilis to Arabidopsis thaliana roots. We found that B. subtilis rapidly adapted to the plant root environment, as evidenced by improved root colonizers observed already after 12 consecutive transfers between seedlings in a hydroponic setup. In addition, two selected evolved isolates from independent populations from transfer 30 outcompeted the ancestor during root colonization. Re-sequencing of single evolved isolates and endpoint populations revealed mutations in genes related to different bacterial traits. Further, phenotypic characterization of evolved isolates from transfer 30 showed that increased root colonization was associated with robust biofilm formation in response to the plant polysaccharide xylan. Additionally, several evolved isolates across independent populations were impaired in motility, a redundant trait in the selective environment. Interestingly, two evolved isolates suffered a fitness disadvantage in a non-selective environment, demonstrating an evolutionary cost of adaptation to the plant root. Finally, increased root colonization by a selected evolved isolate was also demonstrated in the presence of resident soil microbes. Our findings provide novel insights into how a well-known plant growth-promoting rhizobacterium rapidly adapts to an ecologically relevant environment and reveal evolutionary consequences that are fundamental to consider when evolving strains for biocontrol purposes.


title: Independent host- and bacterium-based determinants protect a model symbiosis from phage predation

abstract: Summary/AbstractBacteriophages (phages) are diverse and abundant constituents of microbial communities worldwide, and are capable of modulating bacterial populations in diverse ways. Here we describe a novel phage, {phi}HNL01, which infects the marine bacterium Vibrio fischeri. We use culture-based approaches to demonstrate that mutations in the exopolysaccharide locus of V. fischeri render this bacterium resistant to infection by {phi}HNL01, highlighting the extracellular matrix as a key determinant of phage tropism in this interaction. Additionally, using the natural symbiosis between V. fischeri and the squid Euprymna scolopes, we show that during colonization, V. fischeri is protected from phage present in the ambient seawater. Taken together, these findings shed light on independent yet synergistic host- and bacterium-based strategies for resisting symbiosis-disrupting phage predation, and present important implications for understanding these strategies in the context of host-associated microbial ecosystems.


title: Co-cultivation of the anaerobic fungus Caecomyces churrovis with Methanobacterium bryantii enhances transcription of carbohydrate binding modules

abstract: Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid forming anaerobic fungus. C. churrovis possess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal-methanogen physical associations and fungal cell wall development and remodeling.


title: SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells

abstract: Coactivator complexes regulate chromatin accessibility and transcription. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved coactivator complex. The core module scaffolds the entire SAGA complex and adopts a histone octamer-like structure, which consists of six histone fold domain (HFD)-containing proteins forming three histone fold (HF) pairs, to which the double HFD-containing SUPT3H adds an HF pair. Spt3, the yeast ortholog of SUPT3H, interacts genetically and biochemically with the TATA binding protein (TBP) and contributes to global RNA polymerase II (Pol II) transcription. Here we demonstrate that i) SAGA purified from human U2OS or mouse embryonic stem cells (mESC) can assemble without SUPT3H; ii) SUPT3H is not essential for mESC survival, iii) SUPT3H is required for mESC growth and self-renewal, and iv) the loss of SUPT3H from mammalian cells affects the transcription of only a specific subset of genes. Accordingly, in the absence of SUPT3H no major change in TBP accumulation at gene promoters was observed. Thus, SUPT3H is not required for the assembly of SAGA, TBP recruitment, or overall Pol II transcription, but plays a role in mESC growth and self-renewal. Our data further suggest that yeast and mammalian SAGA complexes contribute to transcription regulation by distinct mechanisms.


title: The 3D enhancer network of the developing T cell genome is controlled by SATB1

abstract: Mechanisms of tissue-specific gene expression regulation via spatial coordination of gene promoters and distal regulatory elements are still poorly understood. We investigated the 3D genome organization of developing murine T cells and identified SATB1, a tissue-specific genome organizer, enriched at the anchors of promoter-enhancer chromatin loops. We assessed the function of SATB1 in T cell chromatin organization and compared it to the conventional genome organizer CTCF. SATB1 builds a more refined layer of genome organization upon a CTCF scaffold. To understand the regulatory implications of SATB1 loopscape structure, we generated Satb1fl/flCd4-Cre+ (Satb1 cKO) conditional knockout animals which suffered from autoimmunity. We aimed to identify molecular mechanisms responsible for the deregulation of the immune system in Satb1 cKO animals. H3K27ac HiChIP and Hi-C experiments indicated that SATB1 primarily mediates promoter-enhancer loops affecting master regulator genes (such as Bcl6), the T cell receptor locus and adhesion molecule genes, collectively being critical for cell lineage specification and immune system homeostasis. Our findings unravel the function of a tissue-specific factor that controls transcription programs, via spatial chromatin arrangements complementary to the chromatin structure imposed by ubiquitously expressed genome organizers.


title: Structural and mechanistic insight into CRISPR-Cas9 inhibition by anti-CRISPR protein AcrIIC4

abstract: Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPRs have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal struture of AcrIIC4 and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4 likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.


title: End-to-end computational approach to the design of RNA biosensors for miRNA biomarkers of cervical cancer

abstract: Cervical cancer is a global public health subject as it affects women in the reproductive ages, and accounts for the second largest burden among cancer patients worldwide with an unforgiving 50% mortality rate. Poor awareness and access to effective diagnosis have led to this enormous disease burden, calling for point-of-care, minimally invasive diagnosis methods. Here, an end-to-end quantitative approach for a new kind of diagnosis has been developed, comprising identification of optimal biomarkers, design of the sensor, and simulation of the diagnostic circuit. Using miRNA expression data in the public domain, we identified circulating miRNA biomarkers specific to cervical cancer using multi-tier screening. Synthetic riboregulators called toehold switches specific for the biomarker panel were then designed. To predict the dynamic range of toehold switches for use in genetic circuits as biosensors, we developed a generic grammar of these switches, and built a multivariate linear regression model using thermodynamic features derived from RNA secondary structure and interaction. The model yielded predictions of toehold efficacy with an adjusted R2 = 0.59. Reaction kinetics modelling was performed to predict the sensitivity of the second-generation toehold switches to the miRNA biomarkers. Simulations showed a linear response between 10nM and 100nM before saturation. Our study demonstrates an end-to-end workflow for the efficient design of genetic circuits geared towards the effective detection of unique genomic signatures that would be increasingly important in todays world. The approach has the potential to direct experimental efforts and minimise costs. All resources including the machine learning toolkit, reaction kinetics simulation, designed toehold sequences, genetic circuits, data and sbml files for replicating and utilizing our study are provided open-source with the iGEM Foundation (https://github.com/igem2019) under GNU GPLv3 licence.

category: synthetic biology


title: Serotonergic neural network related to behavioral inhibition system

abstract: Rationale The tendency to avoid punishment, called behavioral inhibition system, is an essential aspect of motivational behavior. Behavioral inhibition system is related to negative affect, such as anxiety, depression and pain, but its neural basis has not yet been clarified. Objectives To clarify the association between individual variations in behavioral inhibition system and brain 5-HT2A receptor availability and specify which brain networks were involved in healthy male subjects, using [18F]altanserin positron emission tomography and resting-state functional magnetic resonance imaging. Results Behavioral inhibition system score negatively correlated with 5-HT2A receptor availability in anterior cingulate cortex. A statistical model indicated that the behavioral inhibition system score was associated with 5-HT2A receptor availability, which was mediated by the functional connectivity between anterior cingulate cortex and left middle frontal gyrus, both of which involved in the cognitive control of negative information processing. Conclusions Individuals with high behavioral inhibition system displays low 5-HT2A receptor availability in anterior cingulate cortex and this cognitive control network links with prefrontal-cingulate integrity. These findings have implications for underlying the serotonergic basis of physiologies in aversion.


title: SAXS analysis of the intrinsic tenase complex bound to lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

abstract: The intrinsic tenase (Xase) complex, formed by factors (f)VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI) and small angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.


title: Identification of general acid catalyst for the ATPase activity of Lynch syndrome-related MutL homologs

abstract: Mutations of mismatch repair MutL homologs are causative of a hereditary cancer, Lynch syndrome. Investigation of MutL facilitates genetic diagnoses essential for cancer risk managements and therapies. We characterized MutL homologs from human and a hyperthermophile, Aquifex aeolicus, (aqMutL) to reveal the catalytic mechanism for the ATPase activity. Although existence of a general acid catalyst had not been conceived in the mechanism, analysis of the pH dependence of the aqMutL ATPase activity revealed that the reaction is accelerated by general acid-base catalysis. Analyses of mutant aqMutLs showed that Lys79 is the general acid, and the corresponding residues were confirmed to be critical for activities of human homologs, on the basis of which a catalytic mechanism for MutL ATPase is proposed. These and other results described here would contribute to evaluating the pathogenicity of Lynch syndrome-associated missense mutations.


title: Thioredoxin reductase controls the capacity of peroxiredoxins to limit mitochondrial H2O2 release

abstract:
H2O2 performs central roles in signaling at physiological levels, while at elevated levels it causes molecular damage. Mitochondria are major producers of H2O2, which has been implied in regulating diverse processes inside and outside the organelle. However, it still remains unclear whether and how mitochondria in intact cells release H2O2. Here we employed the genetically encoded high-affinity H2O2 sensor HyPer7 in mammalian tissue culture cells to investigate different modes of mitochondrial H2O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells, and observed H2O2 released from mitochondria directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus nor on the plasma membrane, pointing to steep gradients emanating from mitochondria. These gradients are controlled by cytosolic peroxiredoxins that act redundantly and are present with a substantial reserve capacity. Furthermore, dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2O2 handling and explains previously observed cell-to-cell differences. Thus, our data indicate that H2O2-mediated signaling likely occurs close to mitochondria during specific metabolic conditions.

HIGHLIGHTSO_LIMitochondrial H2O2 can be detected in the cytosol in intact human cells
C_LIO_LIMitochondrial H2O2 gradients are steep and controlled by peroxiredoxins 1 and 2
C_LIO_LIPeroxiredoxins 1 and 2 complement for each other
C_LIO_LIPeroxiredoxins 1 and 2 are present with a substantial reserve capacity
C_LIO_LIMetabolism-induced changes of reducing processes control peroxiredoxin activity
C_LI


title: Multi-environment QTL analysis delineates a major locus associated with homoeologous exchanges for water-use efficiency and seed yield in allopolyploid Brassica napus

abstract:
O_LICanola varieties exhibit discernible variation in drought avoidance and drought escape traits, suggesting its adaptation to water-deficit environments. However, the underlying mechanisms are poorly understood.
C_LIO_LIA doubled haploid (DH) population was analysed to identify QTL associated with water use efficiency (WUE) related traits. Based on the resequenced parental genome data, we developed sequence-capture based markers for fine mapping. mRNA-Seq was performed to determine the expression of candidate genes underlying QTL for carbon isotope discrimination ({Delta}13C).
C_LIO_LIQTL contributing to main and QTL x Environment interaction effects for {Delta}13C and for agronomic WUE were identified. One multi-trait QTL for {Delta}13C, days to flower, plant height and seed yield was identified on chromosome A09, in the vicinity of ERECTA. Interestingly, this QTL region was overlapped with a homoeologous exchange event (HE), suggesting its association with the major QTL. Transcriptome analysis revealed several differentially expressed genes between parental lines, including in HE regions.
C_LIO_LIThis study provides insights into the complexity of WUE related genes in the context of canola adaptation to water-deficit conditions. Our results suggest that alleles for high {Delta}13C contribute positively to canola yield. Genetic and genomic resources developed herein could be utilised to make genetic gains for improving canola WUE.
C_LI

category: plant biology


title: Double nerve transfer to a single target muscle: experimental model in the upper extremity

abstract: Surgical nerve transfers are used to efficiently treat peripheral nerve injuries, neuromas, phantom limb pain or improve bionic prosthetic control. Commonly, one donor nerve is transferred to one target muscle. However, the transfer of multiple nerves onto a single target muscle may increase the number of muscle signals for myoelectric prosthetic control and facilitate the treatment of multiple neuromas. Currently, no experimental models are available for multiple nerve transfers to a common target muscle in the upper extremity. This study describes a novel experimental model to investigate the neurophysiological effects of peripheral double nerve transfers. For this purpose, we developed a forelimb model to enable tension-free transfer of one or two donor nerves in the upper extremity. Anatomic dissections were performed to design the double nerve transfer model (n=8). In 62 male Sprague-Dawley rats the ulnar nerve of the antebrachium alone (n=30) or together with the anterior interosseus nerve (n=32) was transferred to reinnervate the long head of the biceps brachii. Before neurotization, the motor branch to the biceps long head was transected at the motor entry point and resected up to its original branch to prevent auto-reinnervation. In all animals, coaptation of both nerves to the motor entry point could be performed tension-free. Mean duration of the procedure was 49 {+/-} 13 min for the single nerve transfer and 78 {+/-} 20 min for the double nerve transfer. Twelve weeks after surgery, muscle response to neurotomy, behavioral testing, retrograde labeling and structural analyses were performed to assess reinnervation. These analyses indicated that all nerves successfully reinnervated the target muscle. No aberrant reinnervation was observed by the originally innervating nerve. Our observations suggest a minimal burden for the animal with no signs of functional deficit in daily activities or auto-mutilation in both procedures. Furthermore, standard neurophysiological analyses for nerve and muscle regeneration were applicable. This newly developed nerve transfer model allows for the reliable and standardized investigation of neural and functional changes following the transfer of multiple donor nerves to one target muscle.


title: Loss of TREM2 reduces hyperactivation of progranulin deficient microglia but not lysosomal pathology

abstract: GRN haploinsufficiency causes frontotemporal lobar degeneration and results in microglial hyperactivation, lysosomal dysfunction and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology we evaluated genetic and pharmacological approaches suppressing TREM2 dependent transition of microglia from a homeostatic to a disease associated state. Trem2 deficiency in Grn KO mice led to a reduction of microglia activation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies allowed a complete rescue of elevated levels of TREM2 together with increased shedding and reduction of TREM2 signaling. Furthermore, antibody-treated PGRN deficient hiMGL showed dampened microglial hyperactivation, reduced TREM2 signaling and phagocytic activity, however, lack of rescue of lysosomal dysfunction. Similarly, lysosomal dysfunction, lipid dysregulation and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Furthermore, NfL, a biomarker for neurodegeneration, was elevated in the Grn/Trem2 KO. These findings suggest that microglia hyperactivation is not necessarily contributing to neurotoxicity, and instead demonstrates that TREM2 exhibits neuroprotective potential in this model.


title: De novo synthesized polyunsaturated fatty acids operate as both host immunomodulators and nutrients for Mycobacterium tuberculosis

abstract: Successful control of Mycobacterium tuberculosis (Mtb) infection by macrophages relies on immunometabolic reprogramming, where the role of fatty acids (FAs) remains poorly understood. Recent studies unraveled Mtbs capacity to acquire saturated and monounsaturated FAs via the Mce1 importer. However, upon activation macrophages produce polyunsaturated FAs (PUFAs), mammal-specific FAs mediating the generation of key immunomodulatory eicosanoids. Here, we asked whether de novo synthesis of PUFAs is modulated in Mtb-infected macrophages and benefits host or pathogen. Quantitative lipidomics revealed that Mtb infection activates the early PUFA biosynthetic pathway for production of eicosanoids. While PUFA synthesis blockade significantly impaired the inflammatory and antimicrobial responses of infected macrophages, it had no effect on Mtb growth in vivo. Using a click-chemistry approach, we found that Mtb efficiently imports PUFAs of the {omega}6 subset via Mce1 in axenic culture, including the eicosanoid precursor arachidonic acid (AA). Notably, Mtb preferentially internalized AA over all other FAs within infected macrophages, but here Mtbs import of AA was largely Mce1-independent and correlated with elevated AA uptake by host cells. Together, these findings reveal AA as a major FA substrate for intracellular Mtb. They suggest that Mtbs hijacking of host-derived AA may counteract its stimulatory effect on anti-mycobacterial immune responses.


title: Visualizing the pH in Escherichia coli colonies via the sensor protein mCherryEA allows high-throughput screening of mutant libraries

abstract:
Cytoplasmic pH is tightly regulated by diverse active mechanisms and interconnected regulatory processes in bacteria. Many processes and regulators underlying pH-homeostasis have been identified via phenotypic screening of strain libraries towards non-growth at low or high pH values. Direct screens with respect to changes of the internal pH in mutant strain collections are limited by laborious methods including fluorescent dyes or radioactive probes. Genetically encoded biosensors equip single organisms or strain libraries with an internal sensor molecule already during the generation of the strain. In this study, we used the pH-sensitive mCherry variant mCherryEA as ratiometric pH biosensor. We visualized the internal pH of E. coli colonies on agar plates by the use of a Gel-Doc imaging system. Combining this imaging technology with robot-assisted colony picking and spotting allowed us to screen and select mutants with altered internal pH values from a small transposon mutagenesis derived E. coli library. Identification of the TN- insertion sites in strains with altered internal pH levels revealed that the transposon was inserted into trkH (encoding a transmembrane protein of the potassium uptake system) or the rssB gene (encoding the anti-adaptor protein RssB which mediates the proteolytic degradation of the general stress response regulator RpoS), two genes known to be associated with pH-homeostasis and pH stress adaptation. This successful screening approach demonstrates that the pH- sensor based analysis of arrayed colonies on agar plates is a sensitive approach for the fast identification of genes involved in pH-homeostasis or pH stress adaptation in E. coli.

ImportancePhenotypic screening of strain libraries on agar plates has become a versatile tool to understand gene functions and to optimize biotechnological platform organisms. Screening is supported by genetically encoded biosensors that allow to easily measure intracellular processes. For this purpose, transcription-factor-based biosensors have emerged as the sensor-type of choice. Here, the target stimulus initiates the activation of a response gene (e.g. a fluorescent protein) followed by transcription, translation and maturation. Due to this mechanistic principle, biosensor readouts are delayed and cannot report the actual intracellular state of the cell in real-time. To capture fast intracellular processes adequately, fluorescent reporter proteins are extensively applied. But these sensor-types are not utilized for phenotypic screenings so far. To take advantage of their properties, we here established an imaging method, which allows to apply a fast ratiometric sensor protein for assessing the internal pH of colonies in a high-thoughput manner.