title: Evolution of the quorum sensing systems in Pseudomonas aeruginosa can involve both loss of regulon function and network modulation

abstract:
Pseudomonas aeruginosa populations evolving in cystic fibrosis (CF) lungs, animal infection models, natural environments or in vitro undergo extensive genetic adaption and diversification. A common mutational target is the quorum sensing (QS) regulon, a three-unit regulatory system that controls the expression of a suite of virulence factors and secreted public goods. Three scenarios have been advocated to explain selection for QS mutants, which include (I) disuse of the regulon, (II) cheating on public goods, or (III) modulation of the network. Here, we test these scenarios by examining a set of 61 QS mutants from an experimental evolution study. We observed non-synonymous mutations in all three QS systems - Las, Rhl and PQS. Most Las mutants carried large deletions, resulting in loss of QS function, and the inability to produce QS-regulated traits (scenario I or II). Conversely, phenotypic and gene expression analyses of Rhl mutants support network modulation (scenario III), as these mutants overexpressed the Las and Rhl regulators and showed an altered QS-regulated trait production portfolio. PQS mutants also showed patterns of network modulation (scenario III), spurring strain diversification and phenotypic trade-offs, where the upregulation of certain QS traits is associated with the downregulation of others. Overall, our results indicate that mutations in different QS systems lead to diverging effects on the social portfolio of bacterial populations. These mutations might not only affect the plasticity and diversity of evolved populations but could also impact bacterial fitness and virulence in infections.

ImportancePseudomonas aeruginosa uses quorum sensing (QS), a three-unit multi-layered network, to coordinate expression of traits for growth and virulence in the context of infections. Despite its importance for bacterial fitness, the QS regulon appears to be a common mutational target during long-term adaptation of P. aeruginosa in the host, natural environments and experimental evolutions. This raises the questions why such an important regulatory system is under selection and how mutations change the portfolio of QS-regulated traits. Here, we examine a set of 61 naturally evolved mutants to address these questions. We found that mutations involving the master regulator, LasR, resulted in an almost complete breakdown of QS, while mutations in RhlR and PqsR resulted in modulations of the QS regulon, where both the QS regulon structure and the QS-regulated trait portfolio changed. Our work reveals that natural selection drives diversification in QS activity patterns in evolving populations.


title: Stem cell heterogeneity and reiteration of developmental signaling underlie melanocyte regeneration in zebrafish

abstract: Tissue-resident stem cells are present in many adult organs, where they are important for organ homeostasis and repair in response to injury. However, the signals that activate these cells and the mechanisms governing how these cells self-renew or differentiate are highly context-dependent and incompletely understood, particularly in non-hematopoietic tissues. In the skin, melanocyte stem cells (McSCs) are responsible for replenishing mature pigmented melanocytes. In mammals, these cells reside in the hair follicle bulge and bulb niches where they are activated during homeostatic hair follicle turnover and following melanocyte destruction, as occurs in vitiligo and other skin hypopigmentation disorders. Recently, we identified adult McSCs in the zebrafish. To elucidate mechanisms governing McSC self-renewal and differentiation we analyzed individual transcriptomes from thousands of melanocyte lineage cells during the regeneration process. We identified transcriptional signatures for McSCs, deciphered transcriptional changes and intermediate cell states during regeneration, and analyzed cell-cell signaling changes to discover mechanisms governing melanocyte regeneration. We identified KIT signaling via the RAS/MAPK pathway as a regulator of McSC direct differentiation. Analysis of the scRNAseq dataset also revealed a population of mitfa/aox5 co-expressing cells that divides following melanocyte destruction, likely corresponding to cells that undergo self-renewal. Our findings show how activation of different subpopulations of mitfa-positive cells underlies self-renewal and differentiation to properly reconstitute the melanocyte pigmentary system following injury.

category: cell biology


title: Low mutation load in a supergene underpinning alternative male mating strategies in ruff

abstract: Ruffs are shorebirds with an elaborate lekking behavior involving three male morphs with different mating strategies: Independents, Satellites, and Faeders1,2. The latter two are heterozygous for different versions of a supergene maintained by an inversion that were estimated to have occurred about 4 million years ago3. Faeders carry an intact inversion while the Satellite allele is recombinant, both of which are expected to accumulate high mutational load because they are recessive lethals. Here we have constructed a highly contiguous genome assembly of the inversion region for both the Independent and Satellite haplotypes. The recombination event(s) between an inverted and non-inverted chromosome creating the Satellite allele must have occurred recently (within the last 100,000 years) based on the minute sequence divergence between the Satellite and Independent alleles in the recombinant regions. Contrary to expectations4,5, we find no expansion of repeats and only a very modest mutation load on the Satellite allele in the nonrecombinant region despite high sequence divergence (1.46%). The essential centromere protein CENPN gene is disrupted by the inversion, and surprisingly is as well conserved on the inversion haplotypes as on the noninversion haplotype. The results suggest that the inversion may be much younger than previously thought. The lack of mutation load despite recessive lethality can be explained by the introgression of the inversion from a now extinct lineage.

category: evolutionary biology


title: UniTVelo: temporally unified RNA velocity reinforces single-cell trajectory inference

abstract: The recent breakthrough of single-cell RNA velocity methods brings attractive promises to reveal directed trajectory on cell differentiation, states transition and response to perturbations. However, the existing RNA velocity methods are often found to return erroneous results, partly due to model violation or lack of temporal regularization. Here, we present UniTVelo, a statistical framework of RNA velocity that models the dynamics of spliced and unspliced RNAs via flexible transcription activities. Uniquely, it also supports the inference of a unified latent time across the transcriptome. With ten datasets, we demonstrate that UniTVelo returns the expected trajectory in different biological systems, including hematopoietic differentiation and those even with weak kinetics or complex branches.

category: bioinformatics


title: FASTAptameR 2.0: A Web Tool for Combinatorial Sequence Selections

abstract: Combinatorial selections are powerful strategies for identifying biopolymers with specific biological, biomedical, or chemical characteristics. Unfortunately, most available software tools for high-throughput sequencing analysis have high entrance barriers for many users because they require extensive programming expertise. FASTAptameR 2.0 is an R-based reimplementation of FASTAptamer designed to minimize this barrier while maintaining the ability to answer complex sequence-level and population-level questions. This opensource toolkit features a user-friendly web tool, interactive graphics, up to 100x faster clustering, an expanded module set, and an extensive user guide. FASTAptameR 2.0 accepts diverse input polymer types and can be applied to any sequence-encoded selection.


title: High transposase abundance in the deep ocean is linked to a particle-associated lifestyle

abstract: Transposases are mobile genetic elements (MGEs) that move within and between genomes, enabling genomic plasticity within microbes. Transposases often carry functional genes, termed cassette sequences, when they migrate within and between genomes. In marine microbial communities, transposase abundance increases with depth, but the reasons for this trend are unclear. Through analysis of metagenomes from the Tara Oceans and Malaspina Expeditions, we identify the particle-associated lifestyle as a main covariate for the high occurrence of transposases in the deep ocean. The abundance of biofilm-associated genes correlates with transposases in a strong and depth-independent manner, suggesting that MGEs readily propagate among microbial communities in crowded biofilms. Genome size positively correlates with particle-associated populations and transposase abundance. This relationship may result from selection for larger genomes, in which essential genes are more sparse and less likely to be disrupted by the recombination activities of MGEs. Genes related to defense mechanisms are enriched in cassette sequences and upregulated in the deep sea. Further, defense mechanism genes in cassette sequences experience stronger selection than other cassette genes. Thus, while transposons can spread at the expense of their microbial hosts, they introduce novel genes and potentially benefit the hosts in accessing heavily contested resources. Together, our results reveal an emerging picture of deep ocean particles as gene sharing highways that result in expanded and defensively oriented microbial genomes.


title: Perfusion Quantification in the Human Brain Using DSC MRI - Simulations and Validations at 3T

abstract: Gadolinium (Gd) and deoxyhemoglobin (dOHb) are paramagnetic contrast agents capable of inducing changes in T2*-weighted MRI signal, utilized in dynamic susceptibility contrast (DSC) MRI. With multiple contrast agents and analysis choices, there are a variety of questions as to its capability to accurately quantify perfusion values. To address these questions, we developed a novel signal model for DSC MRI that incorporates signal contributions from intravascular and extravascular water proton spins at 3T for arterial, venous, and cerebral tissue voxels. This framework allowed us to model the MRI signal in response to changes in Gd and dOHb concentrations, and the effects that various experimental and tissue parameters have on perfusion quantification. We compared the predictions of the numerical simulations with those obtained from experimental data at 3T on six healthy human subjects using Gd and dOHb boluses as contrast agents. Using standard DSC analysis, we identified perfusion quantification dependencies in the experimental results that were in close agreement with the simulations. We found that a reduced baseline oxygen saturation (base-SaO2), greater susceptibility of applied contrast agent (Gd vs dOHb), and larger magnitude of the hypoxic drop ({Delta}SaO2) reduces overestimation of the cerebral blood volume (rCBV) and flow (rCBF). Furthermore, shortening the bolus duration increases the accuracy and reduces the calculated values of mean transit time (MTT). This study demonstrates that changes in Gd and dOHb can be described by the same unifying theoretical framework, as validated by the experimental results. Based on our work, we suggest practices in DSC MRI that increase accuracy and reduce inter- and intra-subject variability. In uncovering a wide array of quantification dependencies, we argue that caution must be exercised when comparing perfusion values obtained from a standard DSC MRI analysis when employing different experimental paradigms.

category: biophysics


title: Quality Control Framework of TCM Preparations based on Multi-type Fingerprints using a Source Proportion Estimation Model

abstract: Traditional Chinese Medicine (TCM) preparations have been widely used in clinical practice for the treatment of various diseases. The quality of TCM preparations is related to clinical efficacy and safety and is highly valued by researchers. The authenticity of TCM preparation can be guaranteed objectively by accurate quality control according to the composition. Here, we proposed a quality control framework of TCM preparations, which is based on multi-type fingerprints using the source proportion estimation model (SPEM). The high-performance liquid chromatography (HPLC) analysis and the high-throughput sequencing analysis are employed to acquire the chemical and taxonomic fingerprints of samples, respectively. The quality of TCM preparations among different manufacturers or batches is evaluated by using SPEM, which is an unsupervised method for source identification of TCM samples. Results showed the good performance of the quality control framework, for example, SPEM achieved a mean accuracy of 0.778 based on the ITS2 taxonomic fingerprint when differentiating manufacturer of BazhenYimu Wan pill. Applications of the quality control framework revealed the batch effect in TCM samples, and environmental factors, such as geography have a profound impact on the consistency of TCM preparations. In summary, this study is an exploration in the field of digital development of TCM preparations and provide a new insight to quantify the batch effect among different batches of TCM samples.

category: pharmacology and toxicology


title: Resource competition drives an invasion-replacement event among shrew species on an island

abstract:
O_LIInvasive mammals are responsible for the majority of native species extinctions on islands. While most of these extinction events will be due to novel interactions between species (e.g. exotic predators and naive prey), it is more unusual to find incidences where a newly invasive species causes the decline/extinction of a native species on an island when they normally coexist elsewhere in their overlapping mainland ranges.
C_LIO_LIWe investigated if resource competition between two insectivorous small mammals was playing a significant role in the rapid replacement of the native pygmy shrew (Sorex minutus) in the presence of the recently invading greater white-toothed shrew (Crocidura russula) on the island of Ireland.
C_LIO_LIWe used DNA metabarcoding of gut contents from >300 individuals of both species to determine each species diet and measured the size of individuals (weight and length) during different stages of the invasion in Ireland (before, during and after the species come into contact with one another) and on a French island where both species have long coexisted (acting as a natural  control site). Dietary composition, niche width and overlap and size were compared in these different stages.
C_LIO_LIThe size of the invasive C. russula and composition of its diet changes between when it first invades an area and after it becomes established. Individuals are larger and they consume larger invertebrates at the invasion front, before switching towards the smaller prey taxa that are more essential for the survival of the native species after establishment. As a result, the level of interspecific dietary overlap increases from between 11-14% when they first come into contact with each other to between 39-46% after the invasion.
C_LIO_LIHere we show that an invasive species can quickly alter its dietary niche in a new environment, leading to negative impacts that were not previously predicted based on the coexistence of these species in other parts of their mainland ranges. As well as causing the replacement of a native small mammal, the invasive shrew may be rapidly exhausting local resources of larger invertebrate species. These subsequent changes in terrestrial invertebrate communities could have severe impacts further downstream on ecosystem functioning and services.
C_LI

category: ecology


title: Quantitative environmental DNA metabarcoding reflects quantitative capture data of fish community obtained by electrical shocker

abstract: Quantitative assessment of biodiversity provides invaluable information for ecosystem conservation and the sustainable use of biological resources. Environmental DNA (eDNA) metabarcoding has been used as a powerful tool for identifying community composition, but it does not necessarily provide quantitative information due to several methodological limitations. Thus, the quantification of eDNA through metabarcoding is an important frontier of eDNA-based biomonitoring. Particularly, the qMiSeq approach has recently been developed as a quantitative metabarcoding method and has attracted much attention due to its usefulness. The aim here was to evaluate the performance of the qMiSeq approach as a quantitative monitoring tool for fish communities by comparing the quantified eDNA concentrations with the results of fish capture surveys. The eDNA water sampling and the capture surveys using the electrical shocker were conducted at a total of 21 sites in four river systems in Japan. As a result, we found significant positive relationships between eDNA concentrations of each species quantified by qMiSeq and both the abundance and biomass of each captured taxon at each site. Furthermore, for seven out of eleven taxa, a significant positive relationship was observed between quantified DNA concentrations by sample and both the abundance and biomass. In total, our results demonstrated that eDNA metabarcoding with the qMiSeq approach is a suitable and useful tool for quantitative monitoring of fish communities. Due to the simplicity of the eDNA analysis, the eDNA metabarcoding with qMiSeq approach would promote further growth of quantitative monitoring of biodiversity.


title: Floral humidity as a signal--not a cue--in a nocturnal pollination system

abstract: Although visual and olfactory floral signals attract pollinators from a distance, at the flowers threshold, pollinators can use floral humidity as an index cue for nectar presence. We evaluate the role of floral humidity in the Datura wrightii-Manduca sexta nocturnal pollination system. In addition to our finding that M. sexta shows strong innate attraction toward humid flowers, we identify the hygrosensing sensillum on their antennae, demonstrate its extreme sensitivity to minute changes in RH, and observe the elimination of moths behavioral preference towards humid flowers following experimental occlusion of the sensilla. Despite Manducas attraction toward humid flowers, we find that floral humidity is not a reliable cue for nectar presence in this system. While Datura floral headspace sustains an enormous humidity gradient, it is not a consequence of nectar evaporation, but an outcome of gas exchange through floral stomata and is decoupled from nectar presence. Using interdisciplinary tools, we demonstrate the function of floral humidity as an attractive signal, not a cue, in this pollination system, thus showcasing an underappreciated modality by which flowers may manipulate their visitors.


title: Temperature-dependent gene regulatory divergence underlies local adaptation with gene flow in the Atlantic silverside

abstract: Gene regulatory divergence is thought to play an important role in adaptation, yet its extent and underlying mechanisms remain largely elusive under scenarios of local adaptation with gene flow. Local adaptation is widespread in marine species despite generally high connectivity and often associated with tightly-linked genomic architectures, such as chromosomal inversions. To investigate gene regulatory evolution under gene flow and the role of discrete genomic regions associated with local adaptation to a steep thermal gradient, we generated RNA-seq data from Atlantic silversides (Menidia menidia) from two locally adapted populations and their F1 hybrids, reared under two different temperatures. We found substantial divergence in gene expression and thermal plasticity, with up to 31% of genes being differentially expressed, and primarily trans-rather than cis-regulatory divergence between populations, despite ongoing gene flow. Substantially reduced thermal plasticity, temperature-dependent gene misexpression and the disruption of co-expression networks in hybrids point toward a role of regulatory incompatibilities in maintaining local adaptation, particularly under colder temperatures, which appear more challenging for this species. Adaptive chromosomal inversions seem to play an important role in gene regulatory divergence through the accumulation of regulatory incompatibilities but are not consistently enriched for divergently regulated genes. Together, these results highlight that gene regulation can diverge substantially among populations connected by strong gene flow in marine environments, partly due to the accumulation of temperature-dependent regulatory incompatibilities within inversions.


title: Leverage drug perturbation to reveal genetic regulators of hepatic gene expression in African Americans

abstract:
BackgroundExpression quantitative loci (eQTL) studies have paved the way in identifying genetic variation impacting gene expression levels. African Americans (AAs) are disproportionately underrepresented in eQTL studies, resulting in a lack of power to identify population-specific regulatory variations especially related to drug response. Specific drugs are known to affect the biosynthesis of drug metabolism enzymes as well as other genes. We used drug perturbation in cultured primary hepatocytes derived from AAs to determine the effect of drug treatment on eQTL mapping and to identify the drug response eQTLs (reQTLs) that show altered effect size following drug treatment.

MethodsWhole-genome genotyping (Illumina MEGA array) and RNA-sequencing were performed on 60 primary hepatocyte cultures after treatment with 6 drugs (Rifampin, Phenytoin, Carbamazepine, Dexamethasone, Phenobarbital, and Omeprazole) and at baseline (no treatment). eQTLs were mapped by treatment and jointly using Meta Tissue.

ResultsWe found varying transcriptional changes across different drug treatments and identified Nrf2 as a potential general transcriptional regulator. We jointly mapped eQTL with gene expression data for across all drug treatments and baseline which increased our power to detect eQTLs by 2.7-fold. We also identified 2,988 reQTLs (eQTLs with altered effect size after drug treatment), which were more likely to overlap transcription factor binding sites and uncovered a novel reQTL, rs61017966 that increases CYP3A5 gene expression, a major drug metabolizing enzyme responsible for both drug response and adverse events across several drug classes.

ConclusionsOur results provide novel insights into the genetic regulation of gene expression in hepatocytes through drug perturbation and provide insight into SNPs that effect the livers ability to respond to transcription upregulation.

category: genomics


title: Predicting mechanisms of action at genetic loci associated with discordant effects on type 2 diabetes and abdominal fat accumulation

abstract: Obesity is a major risk factor for cardiovascular disease, stroke and type 2 diabetes (T2D). One risk factor that couples the adipose tissue accumulation characteristic of obesity to increased T2D risk is abdominal fat accumulation, measured by calculating the ratio of waist-to-hip circumference adjusted for the body-mass index (WHRadjBMI). Body fat distribution is estimated to have 40-80% heritability, which suggests a strong genetic component. Genetic loci associated with WHRadjBMI identified in genome-wide association studies (GWAS) are predicted to act through adipose tissues, but the molecular mechanisms regulating the accumulation of adipose tissue in various fat depots and their consequences for T2D risk are poorly understood. By integrating multi-omic data with colocalization analysis and Tissue of Action score analysis, we predicted colocalization of 79 genetic loci associated with both T2D adjusted for BMI (T2DadjBMI) and WHRadjBMI. Six of these loci were significantly associated with discordant effects on T2DadjBMI and WHRadjBMI, meaning that the alleles of the lead genetic variants in these loci that were protective for T2D were associated with increased abdominal obesity. Genes in these loci could explain the metabolically healthy obesity and lean insulin resistance (MHO/LIR) phenotype; therefore, we predicted functional genes and tissues of action at the discordant loci, and investigated how expression of these genes was linked to metabolic phenotypes. Through this rigorous consensus analysis, we provide evidence for the hypothesis that the genes encoding Thyroid Adenoma Associated Protein Antisense RNA (THADA-AS), Gypsy Retrotransposon Integrase 1 (GIN1), and Peptidylglycine Alpha-Amidating Monooxygenase (PAM) are involved in molecular mechanisms that uncouple abdominal fat accumulation from risk for T2D.


title: TopoGAN: Unsupervised manifold alignment of single-cell data

abstract:
MotivationSingle-cell technologies allow deep characterization of different molecular aspects of cells. Integrating these modalities provides a comprehensive view of cellular identity. Current integration methods rely on overlapping features or cells to link datasets measuring different modalities, limiting their application to experiments where different molecular layers are profiled in different subsets of cells.

ResultsWe present TopoGAN, a method for unsupervised manifold alignment of single-cell datasets with non-overlapping cells or features. We use topological autoencoders to obtain latent representations of each modality separately. A topology-guided Generative Adversarial Network then aligns these latent representations into a common space. We show that TopoGAN outperforms state-of-the-art manifold alignment methods in complete unsupervised settings. Interestingly, the topological autoencoder for individual modalities also showed better performance in preserving the original structure of the data in the low-dimensional representations when compared to using UMAP or a variational autoencoder. Taken together, we show that the concept of topology preservation might be a powerful tool to align multiple single modality datasets, unleashing the potential of multi-omic interpretations of cells.

Availability and implementationImplementation available on GitHub (https://github.com/AkashCiel/TopoGAN). All datasets used in this study are publicly available.

Contactt.r.m.abdelaal@lumc.nl


title: Focus-ISM for Sharp and Gentle Super-Resolved Microscopy

abstract: Super-resolution microscopy is routinely used for fixed and thin samples, while its feasibility for imaging live and thick samples is still limited. In the case of stimulated emission depletion (STED) microscopy, the high-intensity illumination required to achieve effective sub-diffraction resolution can introduce photo-damage, thus reducing the compatibility of the technique with live-cell imaging. Moreover, the out-of-focus fluorescence background may overcome the often faint signal stemming from the focal point, thus constraining imaging to thin samples. Here, we combined STED microscopy with image-scanning microscopy (ISM) to mitigate these limitations without any practical disadvantages. We first enhanced a laser scanning microscope (LSM) by introducing a detector array, hence providing access to a set of additional spatial information that is not available with a typical single-element detector. Then, we exploited this extended dataset to implement focus-ISM, a novel method that relaxes the high-intensity requirement of STED microscopy and removes the out-of-focus background. Additionally, we generalized the focus-ISM method to conventional LSM, namely without a STED beam. The proposed approach requires minimal architectural changes compared with conventional STED microscopes but provides substantial advantages for live and thick sample imaging while maintaining all compatibility with all recent advances in STED and confocal microscopy. As such, focus-ISM represents an essential step towards a universal super-resolved LSM technique for subcellular imaging.


title: Volitional generation of reproducible energy-efficient temporal patterns

abstract: One of the extraordinary characteristics of the biological brain is its low energy expense to implement a variety of biological functions and intelligence compared to the modern artificial intelligence (AI). Spike-based energy-efficient temporal codes have long been suggested as the contributor for the brain to run with a low energy expense. Despite this code having been largely reported in the sensory cortex, whether this code can be implemented in other brain areas to serve broader functions and how the brain learns to generate it have remained unaddressed. In this study, we designed a novel brain-machine interface (BMI) paradigm, by learning which two macaques could volitionally generate reproducible energy-efficient temporal patterns in the primary motor cortex (M1). Moreover, most neurons that were not directly assigned for controlling the BMI did not boost their excitability, demonstrating an overall energy-efficiency manner in performing the task. Over the course of learning, we found that the firing rates and temporal precision of selected neurons co-evolved to generate the energy-efficient temporal patterns, suggesting a cohesive rather than dissociable processing underlie the refinement of energy-efficient temporal patterns.

category: neuroscience


title: Sleep spindles in primates: modelling the effects of distinct laminar thalamocortical connectivity in core, matrix, and reticular thalamic circuits

abstract: Sleep spindles are associated with the beginning of deep sleep and memory consolidation and are disrupted in schizophrenia and autism. In primates, distinct core and matrix thalamocortical (TC) circuits regulate sleep-spindle activity, through communications that are filtered by the inhibitory thalamic reticular nucleus (TRN) however, little is known about typical TC network interactions and the mechanisms that are disrupted in brain disorders. We developed a primate-specific, circuit-based TC computational model with distinct core and matrix loops that can simulate sleep spindles. We implemented novel multilevel cortical and thalamic mixing, and included local thalamic inhibitory interneurons, and direct layer 5 projections of variable density to TRN and thalamus to investigate the functional consequences of different ratios of core and matrix node connectivity contribution to spindle dynamics. Our simulations showed that spindle power in primates can be modulated based on the level of cortical feedback, thalamic inhibition, and engagement of model core vs. matrix, with the latter having a greater role in spindle dynamics. The study of the distinct spatial and temporal dynamics of core-, matrix-, and mix-generated sleep spindles establishes a framework to study disruption of TC circuit balance underlying deficits in sleep and attentional gating seen in autism and schizophrenia.


title: The lateral habenula mediates the association of a conditioned stimulus with the absence of an appetitive unconditioned stimulus

abstract: The lateral habenula (LHb) has been implicated in conditioned inhibition. Here, the modulating effects of LHb activity on the association of a conditioned stimulus (CS) with the nonoccurrence of an unconditioned stimulus (US) were assessed in vivo using chemogenetic methods. Animals initially received explicitly unpaired CS light and US food presentations. Animals subjected to a retardation-of-acquisition task subsequently underwent light and food pairings, whereas those subjected to a summation task underwent compound light-tone and food pairings. The inhibitory light strength was assessed based on retardation of the acquisition of food-cup conditioned responses (CRs) in light-food pairings and comparisons of food-cup CRs to each stimulus in a CS-alone test following compound training. Neurotoxic LHb lesions and chemogenetic LHb inhibition throughout unpaired training attenuated the inhibitory light strength. Furthermore, chemogenetic LHb activation accelerated the decline in CR induced by repeated light-alone presentations following light-food pairings. Therefore, the LHb critically contributes to conditioned inhibition.


title: A translational MRI approach to validate acute axonal damage detection in multiple sclerosis

abstract:
Axonal degeneration is a central pathological feature of multiple sclerosis and is closely associated with irreversible clinical disability. Current noninvasive methods to detect axonal damage in vivo are limited in their specificity, clinical applicability and lack of proper validation. We aimed to validate an MRI framework based on multicompartment modeling of the diffusion signal (AxCaliber) in rats in the presence of axonal pathology, achieved through injection of a neurotoxin damaging the neuronal terminal of axons. We then applied the same MRI protocol to map axonal integrity in the whole brain of multiple sclerosis relapsing-remitting patients and age-matched healthy controls.

AxCaliber is sensitive to microstructural axonal damage in rats, as demonstrated by a significant increase in the mean axonal caliber along the target tract, which correlated with the neurotoxin neurofilament staining. In humans, we uncovered a diffuse increase in mean axonal caliber in demyelinating lesions and, importantly, in most areas of the normal-appearing white matter. Our results demonstrate that axonal diameter mapping is a sensitive and specific imaging biomarker able to link noninvasive imaging contrasts with the underlying biological substrate, supporting the key role of generalized axonal damage in multiple sclerosis.


title: Global brain health modulates the impact of lesion damage on post-stroke sensorimotor outcomes

abstract:
Sensorimotor performance after stroke is strongly related to focal injury measures such as corticospinal tract lesion load. However, the role of global brain health is less clear. Here, we examined the impact of brain age, a measure of neurobiological aging derived from whole brain structural neuroimaging, on sensorimotor outcomes. We hypothesized that stroke lesion damage would result in older brain age, which would in turn be associated with poorer sensorimotor outcomes. We also expected that brain age would mediate the impact of lesion damage on sensorimotor outcomes and that these relationships would be driven by post-stroke secondary atrophy (e.g., strongest in the ipsilesional hemisphere in chronic stroke). We further hypothesized that structural brain resilience, which we define in the context of stroke as the brains ability to maintain its global integrity despite focal lesion damage, would differentiate people with better versus worse outcomes.

We analyzed cross-sectional high-resolution brain MRI and outcomes data from 963 people with stroke from 38 cohorts worldwide using robust linear mixed-effects regressions to examine the relationship between sensorimotor behavior, lesion damage, and brain age. We used a mediation analysis to examine whether brain age mediates the impact of lesion damage on stroke outcomes and if associations are driven by ipsilesional measures in chronic ([≥]180 days) stroke. We assessed the impact of brain resilience on sensorimotor outcome using logistic regression with propensity score matching on lesion damage.

Stroke lesion damage was associated with older brain age, which in turn was associated with poorer sensorimotor outcomes. Brain age mediated the impact of corticospinal tract lesion load on sensorimotor outcomes most strongly in the ipsilesional hemisphere in chronic stroke. Greater brain resilience, as indexed by younger brain age, explained why people have better versus worse sensorimotor outcomes when lesion damage was fixed.

We present novel evidence that global brain health is associated with superior post-stroke sensorimotor outcomes and modifies the impact of focal damage. This relationship appears to be due to post-stroke secondary degeneration. Brain resilience provides insight into why some people have better outcomes after stroke, despite similar amounts of focal injury. Inclusion of imaging-based assessments of global brain health may improve prediction of post-stroke sensorimotor outcomes compared to focal injury measures alone. This investigation is important because it introduces the potential to apply novel therapeutic interventions to prevent or slow brain aging from other fields (e.g., Alzheimers disease) to stroke.


title: Normative Decision Rules in Changing Environments

abstract: Models based on normative principles have played a major role in our understanding of how the brain forms decisions. However, these models have typically been derived for simple, stable environments, and their relevance to decisions under more naturalistic, dynamic conditions is unclear. We previously derived a normative decision model in which evidence accumulation is adapted to environmental dynamics (Glaze et al., 2015), but the evolution of commitment rules (e.g., thresholds on the accumulated evidence) under such dynamic conditions is not fully understood. Here we derive a normative model for decisions based on changing evidence or reward. In these cases, performance (reward rate) is maximized using adaptive decision thresholds that best account for diverse environmental changes, in contrast to predictions of many previous decision models. These adaptive thresholds exhibit several distinct temporal motifs that depend on the specific, predicted and experienced changes in task conditions. These adaptive decision strategies perform robustly even when implemented imperfectly (noisily) and can account for observed response times on a task with time-varying evidence better than commonly used constant-threshold or urgency-gating models. These results further link normative and neural decision-making while expanding our view of both as dynamic, adaptive processes that update and use expectations to govern both deliberation and commitment.


title: Chemical screen of Arabidopsis zygote and proteomics in tobacco BY-2 cells identify general plant cell division inhibitors

abstract: Cell division is essential for growth and development and involves events such as spindle assembly, chromosome separation, and cell plate formation. In plants, the tools used to control these events at the desired time are still poor because the genetic approach is ineffective owing to a high redundancy and lethality, as well as harmful side effects. Accordingly, we screened cell division-affecting compounds, with a focus on Arabidopsis thaliana zygotes, which individually develop in maternal ovules; the cell division was reliably traceable without time-lapse observations. We then identified the target events of the identified compounds using tobacco BY-2 cells for live-cell imaging and proteomics. As a result, we isolated two compounds, PD-180970 and PP2. PD-180970 disrupts microtubule (MT) organization and, thus, nuclear separation, presumably by inhibiting MT-associated proteins (MAP70). PP2 affected class II Kinesin-12 localization at the phragmoplast emerging site and impaired cytokinesis. Moreover, neither chemical caused irreversible damage to viability but they were effective in multiple plant species such as cucumber (Cucumis sativus) and moss (Physcomitrium patens). We propose that the combination of chemical screening based on Arabidopsis zygotes and target event specification focusing on tobacco BY-2 cells can be used to effectively identify novel tools and transiently control specific cell division events that are conserved in diverse plant species.

category: plant biology


title: Sweet taste receptor cells may participate in mucosal immune surveillance

abstract: The oral microbiome is second only to its intestinal counterpart in diversity and abundance, but its effects on taste cells remains largely unexplored. Using single cell RNASeq, we found that mouse taste receptor cells (STRCs) have a gene expression signature reminiscent of Microfold (M) cells, a central player in immune surveillance in the mucosa associated lymphoid tissue (MALT) such as those in the Peyers patch and tonsils. Administration of Tumor Necrosis Factor Ligand Superfamily Member 11 (TNFSF11, also known as RANKL), a growth factor required for differentiation of M cells dramatically increased M cell proliferation and marker gene expression in the taste papillae and in cultured taste organoids from wild type (WT) mice. Taste papillae and organoids from knockout mice lacking Spib (SpibKO), a RANKL-regulated transcription factor required for M cell development and regeneration on the other hand, failed to respond to RANKL. Taste papillae from SpibKO mice also showed reduced expression of NF-{kappa}B signaling pathway components and proinflammatory cytokines and attracted fewer immune cells. However, lipopolysaccharide-induced expression of cytokines was strongly upregulated in SpibKO mice compared to their WT counterparts. Like M cells, STRCs from WT but not SpibKO mice readily took up fluorescently labeled microbeads, a proxy for microbial transcytosis. The proportion of STRCs and other taste cell subtypes are unaltered in SpibKO mice; however, they displayed increased attraction to sweet and umami taste stimuli. We propose that STRCs are involved in immune surveillance at the taste papillae and tune their taste responses to microbial signaling and infection.


title: Distributed and gradual microstructure changes track the emergence of behavioural benefit from memory reactivation

abstract: Memory traces develop gradually and link to neural plasticity. Memory reactivation during sleep is crucial for consolidation, but its precise impact on plasticity and contribution to long-term memory storage remains unclear. We used multimodal diffusion-weighted imaging to track the location and timescale of microstructural changes following Targeted Memory Reactivation (TMR) of a motor task. This showed continuous microstructure plasticity in precuneus across 10 days post-TMR, paralleling the gradual development of behavioural benefit. Both early (0 - 24 h post-TMR) and late (24 h - 10 days post-TMR) microstructural changes in striatum and sensorimotor cortex were associated with the emergence of behavioural effects of TMR at day 20. Furthermore, the baseline microstructural architecture of sensorimotor cortex predicted TMR susceptibility. These findings demonstrate that repeated reactivation of memory traces during sleep engenders microstructural plasticity which continues days after the stimulation night and is associated with the emergence of memory benefits at the behavioural level.


title: Analysis of allelic cross-reactivity of monoclonal IgG antibodies by a multiplexed reverse FluoroSpot assay

abstract: The issue of antibody cross-reactivity is of central importance in immunology, and not least in protective immunity to Plasmodium falciparum malaria, where key antigens show substantial allelic variation (polymorphism). However, serological analysis often does not allow the distinction between true cross-reactivity (one antibody recognizing multiple antigen variants) and apparent cross-reactivity (presence of multiple variant-specific antibodies), as it requires analysis at the single B-cell/monoclonal antibody level. ELISpot is an assay that enables that, and a recently developed multiplexed variant of ELISpot (FluoroSpot) facilitates simultaneous assessment of B-cell/antibody reactivity to several different antigens. In this study, we present a further enhancement of this assay that makes direct analysis of monoclonal antibody-level cross-reactivity with allelic variants feasible. Using VAR2CSA-type PfEMP1 - a notoriously polymorphic antigen involved in the pathogenesis of placental malaria - as a model, we demonstrate the robustness of the assay and its applicability to analysis of true cross-reactivity of monoclonal VAR2CSA-specific antibodies in naturally exposed individuals. The assay is adaptable to the analysis of other polymorphic antigens, rendering it a powerful tool in studies of immunity to malaria and many other diseases.

category: immunology


title: Multi-scale phase separation by explosive percolation with single chromatin loop resolution

abstract: The 2m-long human DNA is tightly intertwined into the cell nucleus of the size of 10m. The DNA packing is explained by folding of chromatin fiber. This folding leads to the formation of such hierarchical structures as: chromosomal territories, compartments; densely packed genomic regions known as Chromatin Contact Domains (CCDs), and loops. We propose models of dynamical genome folding into hierarchical components in human lymphoblastoid, stem cell, and fibroblast cell lines. Our models are based on explosive percolation theory. The chromosomes are modeled as graphs where CTCF chromatin loops are represented as edges. The folding trajectory is simulated by gradually introducing loops to the graph following various edge addition strategies that are based on topological network properties, chromatin loop frequencies, compartmentalization, or epigenomic features. Finally, we propose the genome folding model - a biophysical pseudo-time process guided by a single scalar order parameter. The parameter is calculated by Linear Discriminant Analysis. We simulate the loop formation by using Loop Extrusion Model (LEM) while adding them to the system. The chromatin phase separation, where fiber folds into topological domains and compartments, is observed when the critical number of contacts is reached. We also observe that 80% of the loops are needed for chromatin fiber to condense in 3D space, and this is constant through various cell lines. Overall, our in-silico model integrates the high-throughput 3D genome interaction experimental data with the novel theoretical concept of phase separation, which allows us to model event-based time dynamics of chromatin loop formation and folding trajectories.


title: Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery

abstract: The genome can be divided into two spatially segregated compartments, A and B,1,2 which broadly partition active and inactive chromatin states, respectively. Constitutive heterochromatin is predominantly located within the B compartment and comprises chromatin that is in close contact with the nuclear lamina.3-5 By contrast, facultative heterochromatin marked by H3K27me3 can span both compartments.2-5 How epigenetic modifications, A/B compartmentalization, and lamina association collectively maintain heterochromatin architecture and function remains unclear.6,7 Here we developed an approach termed Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C) that jointly measures chromosome conformation, DNA methylation, and nuclear lamina positioning. Through this approach, we identified topologically distinct A/B sub-compartments characterized by high levels of H3K27me3 and differing degrees of lamina association. To study the regulation of these sub-compartments, we inhibited Polycomb repressive complex 2 (PRC2), revealing that H3K27me3 is an essential factor in sub-compartment segregation. Unexpectedly, PRC2 inhibition also elicited broad gains in lamina association and constitutive heterochromatin spreading into H3K27me3-marked B sub-compartment regions. Consistent with repositioning to the lamina, genes originally marked with H3K27me3 in the B compartment, but not in the A compartment, remained largely repressed, suggesting that constitutive heterochromatin spreading can compensate for loss of H3K27me3 at a transcriptional level. These findings demonstrate that Polycomb sub-compartments and their antagonism with nuclear lamina association are fundamental organizational features of genome structure. More broadly, by jointly measuring nuclear position and Hi-C contacts, our study demonstrates how dynamic changes in compartmentalization and nuclear lamina association represent distinct but interdependent modes of heterochromatin regulation.


title: A magnetic pulse does not affect free-flight navigation behaviour of a medium-distance songbird migrant in spring

abstract:
Current evidence suggests that migratory animals extract map information from the geomagnetic field for true navigation. The sensory basis underlying this feat is elusive, but presumably involves magnetic particles. A common experimental manipulation procedure consists of pre-treating animals with a magnetic pulse. This aims at re-magnetising particles to alter the internal representation of the external field prior to a navigation task. While pulsing provoked deflected bearings in laboratory experiments, analogous studies with free-flying songbirds yielded inconsistent results. Here, we pulsed European robins (Erithacus rubecula), being medium-distance migrants, at an offshore stopover site during spring migration and monitored their free-flight behaviour with a regional-scale tracking system. We found no pulse effect on departure probability, nocturnal departure timing, or departure direction, in agreement with results on a long-distance migrant released at the same site in autumn. This necessitates a reassessment of the importance of geomagnetic maps for migratory decisions for free-flying birds.

Summary statementMagnetic pulse pre-treatment disturbs geomagnetic map usage of birds in lab environments. However, our free-flying birds show no effect, suggesting geomagnetic map information is less important in the natural environment.

category: animal behavior and cognition


title: The Repeating, Modular Architecture of the HtrA Proteases

abstract: A conserved, 26 residue sequence [AA(X2)[A/G][G/L](X2)GDV[I/L](X2)[V/L]NGE(X1)V(X6)] and corresponding structure repeating module was identified within the HtrA protease family using a non-redundant set (N=20) of publically available structures. While the repeats themselves were far from sequence perfect they had notable conservation to a statistically significant level with three or more repetitions identified within one protein at a level that would be expected to randomly occur only once per 1031 residues. This sequence repeat was associated with a six stranded antiparallel {beta}-barrel module, two of which are present in the core of the structures of the PA clan of serine proteases, while a modified version of this module could be identified in the PDZ-like domains. Automated structural alignment methods had difficulties in superimposing these {beta}-barrels but use of a target human HtrA2 structure showed that these modules had an average RMSD across the set of structures of less than 2 [A] (mean and median). Our findings support Dayhoffs hypothesis that complex proteins arose through duplication of simpler peptide motifs and domains.


title: Human cerebral tissue growth is a critical process

abstract: We develop a Fokker-Planck theory of tissue growth with three types of cells (symmetrically dividing, asymmetrically dividing and non-dividing) as main agents to study the growth dynamics of human cerebral organoids. Fitting the theory to lineage tracing data obtained in next generation sequencing experiments, we show that the growth of cerebral organoids is a self organized critical (SOC) process. We derive analytical expressions describing the evolution of clonal lineage sizes and discuss possible organizational mechanisms behind the critical growth.


title: All-trans retinoic acid enhances the antitumor effects of fimaporfin-based photodynamic therapy

abstract: The vitamin A metabolite all-trans retinoic acid (ATRA; tretinoin) has anticancer potential; however, lack of clinical success has prevented its approval for solid tumors. Herein, we propose combining low-dose ATRA preconditioning with fimaporfin-based photodynamic therapy (ATRA+PDT) for the treatment of solid cancers. Compared to monotherapies, ATRA+PDT induced synergistic cytotoxic responses including promotion of apoptosis in colon and breast carcinoma cell lines. In human HT-29 colon cancer cells, the effect of ATRA+PDT on gene expression was evaluated by RNA sequencing (RNA-seq). The unfolded protein response (UPR) and retinoic acid-mediated apoptosis signaling were predicted as strongly activated canonical pathways after ATRA+PDT compared to PDT. During the first 20 days post-ATRA+PDT, we obtained significant anti-tumor responses in HT-29 xenografts, including complete responses in 2/5 mice. In conclusion, ATRA+PDT represent a novel combination therapy for solid tumors that should be further tested in immunocompetent preclinical models.

category: cancer biology


title: ABA-induced SA accumulation causes higher susceptibility in B. juncea as compared to tolerant genotypes against A. brassicae

abstract: Alternaria brassicae, a necrotrophic pathogen causes Alternaria blight in members of the Brassicaceae family. An extensive yield-oriented breeding strategy has rendered Indian mustard (Brassica juncea) susceptible to many fungal pathogens however, Alternaria blight is one of the most pressing challenges of all because it causes almost 40-60% yield loss. Variable degree of tolerance is reported in wild relatives of B. juncea, Sinapis alba (White mustard) and camelina sativa (False flax) have been reported to exhibit moderate and high tolerance respectively against A. brassicae. Phytohormones are the essential regulator of the intricate mechanism of plant defence response. The hormones salicylic acid (SA) and jasmonic acid (JA) have been widely studied and recognized as important regulators of plant immune response. In the last decade, research has pointed out that other hormones like abscisic acid (ABA) also participate equally in plant defence. However, the role of ABA in defence responses and its cross-talk with SA and JA has not been fully understood in terms of Brassica-A. brassicae system. In this investigation, three genotypes-B. juncea, S. alba and C. sativa were selected and their response to exogenous application of SA, JA and ABA and their combination with A. brassicae were studied. Disease assessment, gene expression analysis and quantitative estimation of phytohormones showed that the B. juncea exhibited a weak JA-mediated defence response against A. brassicae and synergy between SA-ABA shifted the signalling mechanism to SA-mediated response leading to susceptibility in B. juncea. Tolerant genotypes, S. alba and C. sativa exhibited a robust JA-mediated response against A. brassicae and JA-ABA was found antagonistic in Brassica-A. brassicae phyto-pathosystem.


title: Content-aware image restoration improves spatiotemporal resolution in luminescence microscopy.

abstract: Luminescence microscopy is a powerful tool in biomedical imaging applications due to its intrinsically high signal to noise ratio. However, luminescence signal detection requires longer exposure times than fluorescence imaging and is consequently less suited for applications requiring high temporal resolution or throughput. Here we demonstrate that content-aware image restoration can drastically reduce the exposure time requirements in luminescence imaging, thus overcoming one of the major limitations of the technique.


title: Molecular architecture of the autoinhibited kinesin-1 lambda particle

abstract:
Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the alpha-helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1, in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drive kinesin-1 activation.

Summary statementIntegration of computational structure prediction with electron microscopy reveals the coiled-coil architecture of the autoinhibited compact conformer of the microtubule motor, kinesin-1.


title: An Ensemble Learning and Slice Fusion Strategy for Three-Dimensional Nuclei Instance Segmentation

abstract: Automated microscopy image analysis is a fundamental step for digital pathology and computer aided diagnosis. Most existing deep learning methods typically require post-processing to achieve instance segmentation and are computationally expensive when directly used with 3D microscopy volumes. Supervised learning methods generally need large amounts of ground truth annotations for training whereas manually annotating ground truth masks is laborious especially for a 3D volume. To address these issues, we propose an ensemble learning and slice fusion strategy for 3D nuclei instance segmentation that we call Ensemble Mask R-CNN (EMR-CNN) which uses different object detectors to generate nuclei segmentation masks for each 2D slice of a volume and propose a 2D ensemble fusion and a 2D to 3D slice fusion to merge these 2D segmentation masks into a 3D segmentation mask. Our method does not need any ground truth annotations for training and can inference on any large size volumes. Our proposed method was tested on a variety of microscopy volumes collected from multiple regions of organ tissues. The execution time and robustness analyses show that our method is practical and effective.


title: Sugar transporter Slc37a2 regulates bone metabolism via a dynamic tubular lysosomal network in osteoclasts

abstract: Osteoclasts are giant bone-digesting cells that harbour specialized lysosome-related organelles termed secretory lysosomes (SLs). SLs store cathepsin K and serve as a membrane precursor to the ruffled border, the osteoclasts  resorptive apparatus. Yet, the molecular composition and spatiotemporal organization of SLs remains incompletely understood. Here, using organelle-resolution proteomics, we identify member a2 of the solute carrier 37 family (Slc37a2) as a SL sugar transporter. We demonstrate that Slc37a2 localizes to the SL limiting membrane and that these organelles adopt a hitherto unnoticed but dynamic tubular network in living osteoclasts that is required for bone digestion. Accordingly, mice lacking Slc37a2 accrue high bone mass owing to uncoupled bone metabolism and disturbances in SL export of monosaccharide sugars, a prerequisite for SL delivery to the ruffled border. Thus, Slc37a2 is a physiological component of the osteoclasts unique secretory organelle and a potential therapeutic target for metabolic bone diseases.


title: High resolution quantitative and functional MRI indicate lower myelination of thin and thick stripes in human secondary visual cortex

abstract: The characterization of cortical myelination is essential for the study of structure-function relationships in the human brain. However, knowledge about cortical myelination is largely based on post mortem histology, which shows conflicting results depending on the staining method used, and generally renders direct comparison to function impossible. The repeating pattern of pale-thin-pale-thick stripes of cytochrome oxidase (CO) activity in the primate secondary visual cortex (V2) is a prominent columnar system, where heavier myelination in both thin/thick and pale stripes were found, respectively. We used quantitative magnetic resonance imaging (qMRI) in conjunction with functional magnetic resonance imaging (fMRI) at ultra-high field strength (7 T) to localize and study myelination of stripes in several humans at sub-millimeter resolution in vivo. Thin and thick stripes were functionally localized by exploiting their sensitivity to color and binocular disparity, respectively. Resulting functional activation maps showed robust stripe patterns in V2 which enabled further comparison of quantitative relaxation parameters between stripe types. Thereby, we found lower longitudinal relaxation rates (R1) of thin and thick stripes compared to surrounding gray matter in the order of 1-2%, indicating heavier myelination of pale stripes. No differences for effective transverse relaxation rates [Formula] were found. The study demonstrates the feasibility to investigate structure-function relationships in living humans within one cortical area at the level of columnar systems using qMRI.


title: Face Familiarity Revealed by Fixational Eye Movements and Fixation-Related Potentials in Free Viewing

abstract: Event-related potentials (ERPs) and the oculomotor inhibition (OMI) in response to transient visual stimuli are known to be sensitive to different stimulus properties, including attention and expectation. In natural vision, transient stimulation of the visual cortex is generated primarily by saccades. Recent studies suggest that the core EEG components in free viewing, induced by saccades (fixation-related potentials, RPs), are similar to the ERP components with flashed stimuli. We have recently found that the OMI in response to flashed stimuli is sensitive to face familiarity. Here, we investigated whether fixation-related-potentials (FRPs) and microsaccade inhibition (OMI) in free viewing are sensitive to face familiarity. Observers (N=15) freely watched a slideshow of seven unfamiliar and one familiar world leaders facial images presented randomly for 4-seconds periods, with multiple images per identity. We measured the occipital fixation-related N1 relative to the P1 magnitude as well as the associated fixation-triggered OMI. We found that the average N1 was significantly smaller and the OMI was shorter for the familiar face, compared with any of the 7 unfamiliar faces. Moreover, the P1 was suppressed across saccades for the familiar but not for the unfamiliar faces. Overall, the results indicate that the occipital FRP and the OMI in free viewing are sensitive to face familiarity; this could perhaps be used as a novel physiological measure for studying hidden suppressed memories.


title: Deficiency in pericyte remodeling as a basis for impaired capillary flow and structure during brain aging

abstract: Deterioration of brain capillary flow and architecture is a hallmark of aging and dementia. It remains unclear how loss of brain pericytes in these conditions contributes to capillary dysfunction. We conducted cause-and-effect studies by optically ablating pericytes in adult and aged mice in vivo. Focal pericyte loss induced capillary dilation without blood-brain barrier disruption. These abnormal dilations were exacerbated in the aged brain, and resulted in increased flow heterogeneity in capillary networks. A subset of affected capillaries reduced in perfusion due to flow steal. Some capillaries stalled in flow and regressed, leading to loss of capillary connectivity. Remodeling of neighboring pericytes restored endothelial coverage and vascular tone within days. Pericyte remodeling was slower in the aged brain, resulting in regions of persistent capillary dilation. These findings link pericyte loss to disruption of capillary flow and structure. They also identify pericyte remodeling as a therapeutic target to preserve capillary flow dynamics.


title: Pre-T cell receptor Self-MHC Sampling Restricts Thymocyte Dedifferentiation

abstract: Summary paragraphProgramming T lymphocytes to distinguish self from non-self is a vital, multi-step process arising in the thymus1-4. Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pT chain and a clone-specific {beta} chain, constitutes a critical early checkpoint in thymocyte development within the {beta} T-cell lineage5, 6. Recent work demonstrates that preTCRs arrayed on double negative (DN) thymocytes, like {beta} TCRs appearing on double positive (DP) thymocytes, ligate peptides bound to MHC molecules (pMHC) on thymic stroma but via a different molecular docking strategy7-10. Here we show the consequences of those distinctive interactions for thymocyte progression, using synchronized fetal thymic progenitor cultures differing in the presence or absence of pMHC on support stroma, determining single cell transcriptomes at key thymocyte developmental transitions. Although MHC negative stroma fosters {beta} T lymphocyte differentiation, the absence of pMHC-preTCR interplay leads to deviant thymocyte transcriptional programming associated with de-differentiation. Highly proliferative DN and DP subsets with antecedent characteristics of T cell lymphoblastic and myeloid malignancies emerge. Thus, at least in vitro, beyond fostering {beta} chain repertoire broadening for subsequent {beta} TCR utilization, preTCR-pMHC interaction limits cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduces significant developmental vulnerabilities.


title: Plasma membrane topography governs the three-dimensional dynamic localization of IgM B cell receptor clusters

abstract: B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces and are thought to perform sensory functions in screening antigen-bearing surfaces. Here, we have studied the cell surface features of Ramos B cells and the spatiotemporal organization of the IgM-BCR using lattice light sheet microscopy in combination with tailored custom-built 4D image analysis. Ramos B cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A proportion of membrane-localized IgM-BCR was found in clusters, which were associated with the ridges and the microvilli. The dynamic ridge network organization and the IgM-BCR cluster mobility were linked and both were controlled by Arp2/3 complex activity. Our results suggest that topographical features of the cell surface govern the distribution and dynamic localization of IgM-BCR clusters to facilitate antigen screening.


title: California's forest carbon offsets buffer pool is severely undercapitalized

abstract: California operates a large forest carbon offsets program that credits carbon stored in forests across the continental United States and parts of coastal Alaska. These credits can be sold to buyers who wish to justify ongoing emissions, including in Californias cap-and-trade program. Although fossil CO2 emissions have effectively permanent atmospheric consequences, carbon stored in forests is inherently less durable because forests are subject to significant socioeconomic and physical risks that can cause temporarily stored carbon to be re-released into the atmosphere. To address these risks, Californias program is nominally designed to provide a 100-year guarantee on forest carbon claims based on a self-insurance program known as a buffer pool. Projects contribute credits to the buffer pool based on a suite of project-specific risk factors, with buffer pool credits retired as needed to cover carbon losses from events such as wildfire or drought. So long as the buffer pool remains solvent, the programs permanence claim remains intact. Here, we perform an actuarial analysis of the performance of Californias buffer pool. We document how wildfires have depleted nearly one-fifth of the total buffer pool in less than a decade, equivalent to at least 95% of the program-wide contribution intended to manage all fire risks for 100 years. We also show that potential carbon losses from a single forest disease, sudden oak death, could fully encumber all credits set aside for disease and insect risks. These findings indicate that Californias buffer pool is severely undercapitalized and therefore unlikely to be able to guarantee the environmental integrity of Californias forest offsets program for 100 years.


title: Developmental changes of opsin gene expression in ray-finned fishes (Actinopterygii)

abstract: Fish often change their habitat and trophic preferences during development. Dramatic functional differences between embryos, larvae, juveniles and adults also concern sensory systems, including vision. Here we focus on the photoreceptors (rod and cone cells) in the retina and their gene expression profiles during the development. Using comparative transcriptomics on 63 species, belonging to 23 actinopterygian orders, we report general developmental patterns of opsin expression, mostly suggesting an increased importance of the rod opsin (RH1) gene and the long-wavelength sensitive (LWS) cone opsin, and a decreasing importance of the shorter wavelength sensitive cone opsin throughout development. Furthermore, we investigate in detail ontogenetic changes in 14 selected species (from Polypteriformes, Acipenseriformes, Cypriniformes, Aulopiformes and Cichliformes), and we report examples of expanded cone opsin repertoires, cone opsin switches (mostly within RH2) and increasing rod:cone ratio as evidenced by the opsin and phototransduction cascade genes. Our findings provide molecular support for developmental stage-specific visual palettes of rayfinned fishes and shifts between, which most likely arose in response to ecological, behavioural and physiological factors.


title: Complete sequences of six Major Histocompatibility Complex haplotypes, including all the major MHC class II structures

abstract: Accurate and comprehensive immunogenetic reference panels are key to the successful implementation of population-scale immunogenomics. The 5Mbp Major Histocompatibility Complex (MHC) is the most polymorphic region of the human genome and associated with multiple immune-mediated diseases, transplant matching and therapy responses. Analysis of MHC genetic variation is severely complicated by complex patterns of sequence variation, linkage disequilibrium and a lack of fully resolved MHC reference haplotypes, increasing the risk of spurious findings on analyzing this medically important region. Integrating Illumina and ultra-long Nanopore sequencing as well as bespoke bioinformatics, we completed five of the alternative MHC reference haplotypes of the current (B38) build of the human reference genome and added one other. The six assembled MHC haplotypes encompass the DR1 and DR4 haplotype structures in addition to the previously completed DR2 and DR3, as well as six distinct classes of the structurally variable C4 region. Analysis of the assembled haplotypes showed that MHC class II sequence structures, including repeat element positions, are generally conserved within the DR haplotype supergroups, and that sequence diversity peaks in three regions around HLA-A, HLA-B+C, and the HLA class II genes. Demonstrating the potential for improved short-read analysis, the number of proper read pairs recruited to the MHC was found to be increased by 0.32% - 0.69% in a 1000 Genomes Project read re-mapping experiment with seven diverse samples. Furthermore, the assembled haplotypes can serve as references for the community and provide the basis of a structurally accurate genotyping graph of the complete MHC region.


title: A Novel Y-Shaped, S-O-N-O-S-Bridged Crosslink between Three Residues C22, C44, and K61 Is a Redox Switch of the SARS-CoV-2 Main Protease

abstract: As the COVID-19 pathogen, SARS-CoV-2 relies on its main protease (MPro) for pathogenesis and replication. During the crystallographic analyses of MPro crystals that were exposed to the air, a uniquely Y-shaped, S-O-N-O-S-bridged posttranslational crosslink that connects three residues C22, C44, and K61 at their side chains was frequently observed. As a novel posttranslational modification, this crosslink serves as a redox switch to regulate the catalytic activity of MPro, a demonstrated drug target of COVID-19. The formation of this linkage leads to a much more opened active site that can be potentially targeted for the development of novel SARS-CoV-2 antivirals. The inactivation of MPro by this crosslink indicates that small molecules that lock MPro in the crosslinked form can be potentially used with other active site-targeting molecules such as paxlovid for synergistic effects in inhibiting the SARS-CoV-2 viral replication. Therefore, this new finding reveals a unique aspect of the SARS-CoV-2 pathogenesis and is potentially paradigm-shifting in our current understanding of the function of MPro and the development of its inhibitors as COVID-19 antivirals.

category: biochemistry


title: Systematic analysis of alternative splicing in time course data using Spycone

abstract: During disease progression or organism development, alternative splicing (AS) may lead to isoform switches (IS) that demonstrate similar temporal patterns and reflect the AS co-regulation of such genes. Tools for dynamic process analysis usually neglect AS. Here we propose Spycone (https://github.com/yollct/spycone), a splicing-aware framework for time course data analysis. Spycone exploits a novel IS detection algorithm and offers downstream analysis such as network and gene set enrichment. We demonstrate the performance of Spycone using simulated and real-world data of SARS-CoV-2 infection.


title: Differentiated dynamic response in C. elegans chemosensory cilia

abstract: Cilia are membrane-enveloped organelles that protrude from the surface of most eurokaryotic cells and play crucial roles in sensing the external environment. For maintenance and function cilia are dependent on intraflagellar transport (IFT). Here we use a combination of microfluidics and fluorescence microscopy to study the response of phasmid chemosensory neurons, in live Caenorhabditis elegans, to chemical stimuli. We found that chemical stimulation resulted in unexpected changes in IFT and ciliary structure. Notably, stimulation with hyperosmotic solutions or chemical repellents resulted in different responses, not only in IFT, ciliary structure and cargo distribution, but also in neuronal activity. The response to chemical repellents results in habituation of the neuronal activity, suggesting that IFT plays a role in regulating the chemosensory response. Our findings show that cilia are able to sense and respond to different external cues in distinct ways, highlighting the flexible nature of cilia as sensing hubs.


title: Smoke exposed roots causes reduced whole-plant vascular sectoriality.

abstract:
While photosynthates are partitioned by the relative strength of young developing leaves and roots as sinks for carbon-based resources, many plants also show a close relationship between partitioning, phyllotaxy and vascular connectivity, yielding sectorial patterns of allocation. We examined whether smoke influences phloem vascular sectoriality in a model plant, sunflower (Helianthis annuus L.). Using radioactive 11CO2 administered to single photosynthetically active source leaves, we examined the transport behavior and allocation patterns of 11C-photosynthates using gamma counting and autoradiography. Soil treated with liquid smoke caused significant reductions in phloem sectoriality involving young sink leaves and roots. The resulting increase in vascular connectivity could benefit young plant performance by allowing a more uniform allocation of nutrients and/or stress signal molecules at a critical time of their growth.

One-Sentence SummarySmoke-exposed roots exhibit a significant reduction in phloem sectoriality involving carbon transport to young sink leaves and roots.

category: physiology


title: Age-related changes in neural synchronization and the sustained response to amplitude and frequency modulation of sound

abstract: Perception of speech requires sensitivity to features, such as amplitude and frequency modulations, which are often temporally regular. Previous work suggests age-related changes in neural responses to temporally regular features, but little work has focused on age differences for different types of modulations. We recorded magnetoencephalography in younger (21-33 years) and older adults (53-73 years) to investigate age differences in neural responses to slow (2-6 Hz sinusoidal and non-sinusoidal) modulations in amplitude, frequency, or combined amplitude and frequency. Neural responses to sound onset (independent of temporal modulations) were enhanced in older compared to younger adults, suggesting hyperresponsivity and a loss of inhibition in the aged auditory system. Analyses of neural activity to modulations revealed enhanced neural synchronization with amplitude, frequency, and combined amplitude-frequency modulations for older compared to younger adults. This enhancement generalized across different degrees of temporal regularity (sinusoidal and non-sinusoidal), although neural synchronization was generally lower for non-sinusoidal modulation. Despite enhanced synchronization, sustained neural activity was reduced in older compared to younger adults for sounds modulated both sinusoidally and non-sinusoidally in frequency. Our results suggest substantial age differences in the sensitivity of the auditory system to features present in speech and other natural sounds.


title: A model of the mouse cortex with attractor dynamics explains the structure and emergence of rsfMRI co-activation patterns

abstract: Neural network models have been instrumental in revealing the foundational principles of whole-brain dynamics. Here we describe a new whole-cortex model of mouse resting-state fMRI (rsfMRI) activity. Our model implements neural input-output nonlinearities and excitatory-inhibitory interactions within areas, as well as a directed connectome obtained with viral tracing to model interareal connections. Our model makes novel predictions about the dynamic organization of rsfMRI activity on a fast scale of seconds, and explains its relationship with the underlying axonal connectivity. Specifically, the simulated rsfMRI activity exhibits rich attractor dynamics, with multiple stationary and oscillatory attractors. Guided by these theoretical predictions, we find that empirical mouse rsfMRI time series exhibit analogous signatures of attractor dynamics, and that model attractors recapitulate the topographical organization and temporal structure of empirical rsfMRI co-activation patterns (CAPs). The richness and complexity of attractor dynamics, as well as its ability to explain CAPs, are lost when the directionality of underlying axonal connectivity is neglected. Finally, complexity of fast dynamics on the scale of seconds was maximal for the values of inter-hemispheric axonal connectivity strength and of inter-areal connectivity sparsity measured in real anatomical mouse data.


title: Microglial ferroptotic stress causes non-cell autonomous neuronal death

abstract:
BackgroundFerroptosis is a form of regulated cell death characterised by lipid peroxidation as the terminal endpoint and a requirement for iron. Although it protects against cancer and infection, ferroptosis is also implicated in causing neuronal death in degenerative diseases of the central nervous system (CNS). The precise role for ferroptosis in causing neuronal death is yet to be fully resolved.

MethodsTo elucidate the role of ferroptosis in neuronal death we utilised co-culture and conditioned medium transfer experiments involving microglia, astrocytes and neurones. We ratified clinical significance of our cell culture findings via assessment of human CNS tissue from cases of the fatal, paralysing neurodegenerative condition of amyotrophic lateral sclerosis (ALS). Finally, we utilised the SOD1G37R mouse model of ALS and a novel CNS-permeant ferroptosis inhibitor to verify pharmacological significance in vivo.

ResultsWe found that sublethal ferroptotic stress selectively affecting microglia triggers an inflammatory cascade that results in non-cell autonomous neuronal death. Central to this cascade is the conversion of astrocytes to a neurotoxic state. We show that spinal cord tissue from cases of ALS exhibits a signature of ferroptosis that encompasses atomic, molecular and biochemical features. Moreover, a molecular correlation between ferroptosis and neurotoxic astrocytes evident in ALS-affected spinal cord is recapitulated in the SOD1G37R mouse model where treatment with the novel, CNS-permeant ferroptosis inhibitor, CuII(atsm), ameliorated these markers and was neuroprotective.

ConclusionsBy showing that microglia responding to sublethal ferroptotic stress culminates in non-cell autonomous neuronal death, our results implicate microglial ferroptotic stress as a rectifiable cause of neuronal death in neurodegenerative disease. As ferroptosis is currently primarily regarded as an intrinsic cell death phenomenon, these results introduce an entirely new pathophysiological role for ferroptosis in disease.


title: Sex Differences in Central Amygdala Reactivity following Psychedelic Drug Exposure

abstract: Psilocybin, and its active metabolite psilocin, have been shown to elicit rapid and long-lasting symptom improvements in a variety of affective psychiatric illnesses. However, the specific mechanisms behind these therapeutic effects remain relatively unknown. The central amygdala (CeA) is a primary output region within the extended amygdala that is heavily dysregulated in affective psychiatric disorders. Here, we utilized fiber photometry to measure changes in CeA reactivity to an aversive air puff stimulus after psilocin administration, both acutely and at varying prolonged time points. We found that administration of psilocin increased reactivity acutely in females, but not males. Additionally, we show that one dose of psilocin produced decreases in reactivity in males, but not females, 28-days post administration, with decreases seen as early as 2-days post administration. We further report stimulus-specific changes in CeA reactivity after psilocin with differential responsivity to an aversive air puff stimulus and an auditory stimulus. We also measured changes in behavioral response to the air puff stimulus and report sex differences in threat responding but not exploratory behavior or general locomotion. This study provides evidence that a single dose of psilocin can elicit sex-specific, dynamic, and enduring changes in CeA reactivity to an aversive stimulus. These data are an important step towards dissecting circuit-based mechanisms underlying the effects of psychedelics.


title: A bacteriophage-based, highly efficacious, needle and adjuvant-free, mucosal COVID-19 vaccine

abstract: The authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected and effective in preventing COVID-19, but do not induce efficient mucosal immunity, or prevent viral transmission. We developed a bacteriophage T4-based, multicomponent, needle and adjuvant-free, mucosal vaccine by engineering spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of T4-COVID vaccine induced higher virus neutralization antibody titers against multiple variants, balanced Th1/Th2 antibody and cytokine responses, stronger CD4+ and CD8+ T cell immunity, and higher secretory IgA titers in sera and bronchoalveolar lavage with no effect on the gut microbiota, compared to vaccination of mice intramuscularly. The vaccine is stable at ambient temperature, induces apparent sterilizing immunity, and provides complete protection against original SARS-CoV-2 strain and its Delta variant with minimal lung histopathology. This mucosal vaccine is an excellent candidate for boosting immunity of immunized and/or as a second-generation vaccine for the unimmunized population.

category: microbiology


title: Touch receptor end-organ innervation and function requires sensory expression of the transcription factor Meis2.

abstract: Touch sensation is primarily encoded by mechanoreceptors, sometimes called Low-Threshold Mechanoreceptors (LTMRs), with their cell bodies in the Dorsal Root Ganglia (DRG). LTMRs make up no more that 20% of all sensory neurons and exhibit great diversity in terms of molecular signature, terminal ending morphology and electrophysiological properties, mirroring the complexity of tactile experience. LTMRs are an interesting model to study the molecular cues controlling neuronal diversification in terms of both molecular specification and target-field innervation. The morphological specialization of the sensory end-organ of LTMRs exhibits striking diversity between different mechanoreceptor types and whether it occurs in the glabrous or hairy skin. Much has been learnt about transcriptional codes that define different LTMR subtypes, but the identification of molecular players that participate in their late maturation has not been extensively addressed. Here we identified the TALE homeodomain transcription factor Meis2 as a key regulator of LTMR targetfield innervation. Meis2 is specifically expressed in cutaneous LTMRs and its expression depends on target-derived signals. Meis2 gene inactivation in mouse sensory neurons precursors or early postmitotic neurons allows normal survival and specification of LTMRs. However, LTMRs lacking Meis2 show pronounced defects in end-organ innervation which was accompanied by severely impaired receptor properties and behavioral responses. These results establish Meis2 as a major transcriptional regulator controlling the orderly formation of peripheral end-organs required for touch.

category: developmental biology


title: Long-term temporal trends in gastrointestinal parasite infection in wild Soay sheep

abstract:
Monitoring the prevalence and abundance of parasites over time is important for addressing their potential impact on host life-histories, immunological profiles, and their influence as a selective force. Only long-term ecological studies have the potential to shed light on both the temporal trends in infection prevalence and abundance and the drivers of such trends, because of their ability to dissect drivers that may be confounded over shorter time scales. Despite this, only a relatively small number of such studies exist. Here, we analysed changes in the prevalence and abundance of gastrointestinal parasites in the wild Soay sheep population of St Kilda across 31 years. The host population density has increased across the study, and population density is known to increase parasite transmission, but we found that density and year explained temporal variation in parasite prevalence and abundance independently. Prevalence of both strongyle nematodes and coccidian microparasites increased during the study, and this effect varied between lambs, yearlings and adults. Meanwhile, abundance of strongyles was more strongly linked to host density than to temporal (yearly) dynamics, while abundance of coccidia showed a strong temporal trend without any influence of density. Strikingly, coccidian abundance increased threefold across the course of the study in lambs, while increases in yearlings and adults were negligible. Our decades-long, intensive, individual-based study will enable the role of environmental change and selection pressures in driving these dynamics to be determined, potentially providing unparalleled insight into the drivers of temporal variation in parasite dynamics in the wild.

Key findingsO_LIWe studied temporal trends in gastrointestinal parasites of wild sheep over 31 years
C_LIO_LIYear and host population density explained temporal variation in parasites independently
C_LIO_LIPrevalence of both strongyle nematodes and coccidia varied across the study period
C_LIO_LIAbundance of strongyles was more closely linked to host density than year
C_LIO_LIAbundance of coccidia increased threefold over time in lambs, but did not vary in adults
C_LI


title: A Unifying Statistical Framework to Discover Disease Genes from GWAS

abstract: Genome-wide association studies (GWAS) identify genomic loci associated with complex traits, but it remains an open challenge to identify the genes underlying the association signals. Here, we extend the equations of statistical fine-mapping, to compute the probability that each gene in the human genome is targeted by a causal variant, given a particular trait. Our computations are enabled by several key innovations. First, we partition the genome into optimal linkage disequilibrium blocks, enabling genome-wide detection of trait-associated genes. Second, we unveil a comprehensive mapping that associates genetic variants to the target genes they affect. The combined performance of the map on high-throughput functional genomics and eQTL datasets supersedes the state of the art. Lastly, we describe an algorithm which learns, directly from GWAS data, how to incorporate prior knowledge into the statistical computations, significantly improving their accuracy. We validate each component of the statistical framework individually and in combination. Among methods to identify genes targeted by causal variants, this paradigm rediscovers an unprecedented proportion of known disease genes. Moreover, it establishes human genetics support for many genes previously implicated only by clinical or preclinical evidence, and it discovers an abundance of novel disease genes with compelling biological rationale.


title: Argon plasma-modified bacterial cellulose filters for protection against respiratory pathogens

abstract:
Due to the global spread of the SARS-CoV-2 virus and the resultant pandemic, there has been a major surge in the demand for surgical masks, respirators, and other air filtration devices. Unfortunately, the fact that these filters are made of petrochemical-derived, non-biodegradable polymers means that the surge in production has also led to a surge in plastic waste. In this work, we present novel, sustainable filters based on bacterial cellulose (BC) functionalized with low-pressure argon plasma (LPP-Ar). The "green" production process involved BC biosynthesis by Komagataeibacter xylinus, followed by simple purification, homogenization, lyophilization, and finally LPP-Ar treatment. The obtained LPP-Ar-functionalized BC-based material (LPP-Ar-BC-bM) showed excellent antimicrobial and antiviral properties, with no cytotoxicity versus murine fibroblasts in vitro. Further, filters consisting of three layers of LPP-Ar-BC-bM had >99% bacterial and viral filtration efficiency, while maintaining sufficiently low airflow resistance (6 mbar at an airflow of 95 L/min). Finally, as a proof-of-concept, we were able to prepare 80 masks with LPP-Ar-BC-bM filter and ~85% of volunteer medical staff assessed them as good or very good in terms of comfort. We conclude that our novel sustainable, biobased, biodegradable filters are suitable for respiratory personal protective equipment (PPE), such as surgical masks and respirators. Further, with scale-up, they may be adapted for indoor air handling filtration in hospitals or schools.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/489859v1_ufig1.gif" ALT="Figure 1">
View larger version (46K):
org.highwire.dtl.DTLVardef@99ae8dorg.highwire.dtl.DTLVardef@192c4eforg.highwire.dtl.DTLVardef@bf2acborg.highwire.dtl.DTLVardef@92907a_HPS_FORMAT_FIGEXP  M_FIG C_FIG

category: bioengineering


title: Benchmark of tools for in silico prediction of MHC class I and class II genotypes from NGS data

abstract: The HLA genes are a group of highly polymorphic genes that are located in the MHC region on chromosome 6. The HLA genotype affects the presentability of tumour antigens to the immune system. Therefore, methods to accurately type HLA alleles are critical to study differences in immune response between cancer patients. PCR-based methods are the current gold standard, but large-scale datasets with PCR-based HLA genotypes are rarely available. A variety of methods for in silico NGS-based HLA genotyping have been developed, bypassing the need to determine these genotypes with separate experiments. However, there is currently no consensus on the best performing tool. Here, we compiled a list of 13 HLA callers and evaluated their accuracy on three different datasets. Based on these results, best-practice guidelines were constructed, and consensus HLA allele predictions were made for DNA and RNA samples from The Cancer Genome Atlas (TCGA).


title: Data-driven Identification of Total RNA Expression Genes (TREGs) for Estimation of RNA Abundance in Heterogeneous Cell Types

abstract: Next-generation sequencing technologies have facilitated data-driven identification of gene sets with different features including genes with stable expression, cell-type specific expression, or spatially variable expression. Here, we aimed to define and identify a new class of "control" genes called Total RNA Expression Genes (TREGs), which correlate with total RNA abundance in heterogeneous cell types of different sizes and transcriptional activity. We provide a data-driven method to identify TREGs from single cell RNA-sequencing (RNA-seq) data, available as an R/Bioconductor package at https://bioconductor.org/packages/TREG. We demonstrated the utility of our method in the postmortem human brain using multiplex single molecule fluorescent in situ hybridization (smFISH) and compared candidate TREGs against classic housekeeping genes. We identified AKT3 as a top TREG across five brain regions, especially in the dorsolateral prefrontal cortex.


title: Mechanism of multimodal substrate translocation in P-glycoprotein

abstract:
P-glycoprotein (Pgp) is a highly dynamic ATP-binding cassette transporter that confers cancer multidrug resistance and mediates the bioavailability and pharmacokinetics of many drugs. Structural and biochemical data have provided insights into the binding of diverse compounds, but how they are translocated through the membrane has remained elusive. The biggest hurdle to address this fundamental question has been isolating and characterizing the short-lived transition states during transport. Here, we covalently attached a cyclopeptide substrate to discrete sites of Pgp. Cryo-EM analyses of inward- and outward-facing conformations enabled tracing of the substrate passage and revealed how a cascade of conformational changes in transmembrane helix 1 drives substrate movement across the membrane bilayer upon ATP binding. Observation of a double substrate-bound outward-facing conformation suggests that Pgp can co-transport two compounds simultaneously.

One-Sentence SummaryCryo-EM reveals the substrate transport pathway and mechanism of P-glycoprotein from initial binding to release.


title: Genome-wide screen for enhanced noncanonical amino acid incorporation in yeast

abstract:
Numerous applications of noncanonical amino acids (ncAAs) in basic biology and therapeutic development require efficient protein biosynthesis using an expanded genetic code. However, achieving such incorporation at repurposed stop codons in cells is generally inefficient and limited by complex cellular processes that preserve the fidelity of protein synthesis. A more comprehensive understanding of the processes that contribute to ncAA incorporation would aid in the development of genomic engineering strategies for augmenting genetic code manipulation. In this work, we screened a pooled Saccharomyces cerevisiae molecular barcoded yeast knockout (YKO) collection to identify single-gene knockout strains exhibiting improved ncAA incorporation efficiency in response to the amber (TAG) stop codon. We used a series of intracellular fluorescent reporters in tandem with fluorescence activated cell sorting (FACS) to identify 55 unique candidate deletion strains. Identified genes encode for proteins that participate in diverse cellular processes; many of the genes have no known connection with protein translation. We then verified that two knockouts, yil014c-a{Delta} and alo1{Delta}, had improved incorporation efficiency using independently acquired strains possessing the knockouts. Characterizations of the activity of yil014c-a{Delta} and alo1{Delta} with additional orthogonal translation systems and ncAAs indicate that deletion of each of these genes enhances ncAA incorporation efficiency without loss of fidelity over a wide range of conditions. Our findings highlight opportunities for further modulating gene expression with genetic, genomic, and synthetic biology approaches to improve ncAA incorporation efficiency. In addition, these discoveries have the potential to enhance our fundamental understanding of protein translation. Ultimately, this study provides a foundation for future efforts to engineer cells to incorporate ncAA at greater efficiencies, which in turn will streamline the realization of applications utilizing expanded genetic codes ranging from basic biology to drug discovery.

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: synthetic biology


title: C-Jun N-terminal Kinase Promotes Stress Granule Assembly and Neurodegeneration in C9orf72-mediated ALS and FTD

abstract: Stress granules (SGs), RNA/protein condensates assembled in cells under stress, are believed to play a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, how SG assembly is regulated and related to pathomechanism is incompletely understood. Here, we show that ER stress activates JNK via IRE1 in fly and cellular models of C9orf72-mediated ALS/FTD (c9ALS/FTD), the most common genetic form of ALS/FTD. Furthermore, activated JNK promotes SG assembly induced by poly(GR) and poly(PR), two toxic proteins implicated in c9ALS/FTD, by promoting the transcription of G3BP1, a key SG protein. Consistent with these findings, JNK or IRE1 inhibition reduced SG formation, G3BP1 mRNA and protein levels, and neurotoxicity in cells overexpressing poly(GR) and poly(PR) or neurons derived from c9ALS/FTD patient induced pluripotent stem cells (iPSCs). Our findings connect ER stress, JNK, and SG assembly in a unified pathway contributing to c9ALS/FTD neurodegeneration.