title: Higher Baseline Lateral Habenula Firing Rate Negatively Correlates with Increase in Ethanol Intake over Days in Adult Long Evans Rats

abstract: While many adults consume alcohol, yet only some individuals are at a risk to develop alcohol use disorder (AUD). Variability in the risk for alcohol abuse is multifactorial and includes differences in behavioral and neuronal traits. The lateral habenula (LHb) has been shown to mediate aversive state-related behavioral responses. Interestingly, in both humans and rodents, depression-like symptoms are associated with high LHb activity. Additionally, there is a high co-morbidity between major depressive disorder and AUD. However, LHb lesions in rodents increase ethanol intake over time. Thus, we wanted to determine how baseline LHb activity correlates with ethanol intake over days. Specifically, we wanted to test whether individual variation in baseline LHb activity in ethanol-naive rats is related to home-cage ethanol drinking patterns. Hence, in this study, we determined the correlation between individual variability in baseline LHb neural activity, the negative-affective state-related ultrasonic vocalizations (USVs; 22-28 kHz), and the extent of ethanol intake over days. We first surgically implanted a unilateral 16-wire electrode array in the LHb of adult male Long Evans rats (n=11). Following recovery from surgery, rats were placed in sound-insulated chambers for two hours, where they were free to explore while we simultaneously recorded neuronal signals from their LHb and the USVs emitted by them. Next, these rats underwent an intermittent access to ethanol (IAE) paradigm, where they received 20% ethanol for 24 hours on alternate days (Monday-Wednesday-Friday) and ad-libitum water in their home cages for four weeks. The change in ethanol intake over days differed between rats, with some rats escalating their ethanol intake in the four weeks, while other rats showing no meaningful change in ethanol intake over days as compared to the first session. We found a significant negative correlation between average baseline LHb firing rates in rats and the changes in ethanol intake in the first week of IAE. Specifically, rats with higher baseline LHb firing rats, unlike rats with lower baseline firing rates, did not escalate their ethanol intake from the first session to the second and fourth ethanol sessions of the IAE paradigm. We also found a moderate positive correlation between the number of 22-28 kHz USVs and the average LHb firing rates of these rats. These results indicate that higher baseline LHb neuronal activity in normal adult ethanol naive rats is associated with decreased motivation to seek and consume ethanol during the early stages of ethanol consumption.


title: Baseline Lateral Habenula Firing Rate Negatively Correlates with Increase in Ethanol Intake over Days in Adult Long Evans Rats

category: neuroscience


title: Dynamics of global gene expression and chromatin accessibility of the peripheral nervous system in animal models of persistent pain

abstract:
Efforts to understand genetic variability involved in an individuals susceptibility to chronic pain support a role for upstream regulation by epigenetic mechanisms. To examine the transcriptomic and epigenetic basis of chronic pain that resides in the peripheral nervous system, we used RNA-seq and ATAC-seq of the rat dorsal root ganglion (DRG) to identify novel molecular pathways associated with pain hypersensitivity in two well-studied persistent pain models induced by Chronic Constriction Injury (CCI) of the sciatic nerve and intra-plantar injection of Complete Freunds Adjuvant (CFA) in rats. Our RNA-seq studies identify a variety of biological process related to synapse organization, membrane potential, transmembrane transport, and ion binding. Interestingly, genes that encode transcriptional regulators were disproportionately downregulated in both models. Our ATAC-seq data provide a comprehensive map of chromatin accessibility changes in the DRG. A total of 1123 regions showed changes in chromatin accessibility in one or both models when compared to the naive and 31 shared differentially accessible regions (DAR)s. Functional annotation of the DARs identified disparate molecular functions enriched for each pain model which suggests that chromatin structure may be altered differently following sciatic nerve injury and hind paw inflammation. Motif analysis identified 17 DNA sequences known to bind transcription factors in the CCI DARs and 33 in the CFA DARs. Two motifs were significantly enriched in both models. Our improved understanding of the changes in chromatin accessibility that occur in chronic pain states may identify regulatory genomic elements that play essential roles in modulating gene expression in the DRG.

SummaryShared transcriptomic and epigenetic changes in two animal models improves our understanding of how chromatin structural changes alter DRG gene expression under persistent pain conditions.


title: Enhancing motor learning by increasing stability of newly formed dendritic spines in motor cortex

abstract: Dendritic spine dynamics of Layer 5 Pyramidal neurons (L5PNs) are thought to be physical substrates for motor learning and memory of motor skills and altered spine dynamics are frequently correlated with poor motor performance. Here we describe an exception to this rule by studying mice lacking Paired immunoglobulin receptor B (PirB-/-). Using chronic two-photon imaging of primary motor cortex (M1) of PirB-/-;Thy1-YFP-H mice, we found a significant increase in the survival of spines on apical dendritic tufts of L5PNs, as well as increased spine formation rates and spine density. Surprisingly and contrary to expectations, adult PirB-/- mice learn a skilled reaching task more rapidly compared to wild type (WT) littermate controls. Conditional excision of PirB from forebrain pyramidal neurons in adult mice replicated these results. Furthermore, chronic imaging of L5PN dendrites throughout the learning period revealed that the stabilization of learning-induced newly formed spines is significantly elevated in PirB-/- mice. The degree of survival of newly formed spines in M1 yielded the strongest correlation with task performance, suggesting that this increased spine stability is advantageous and can translate into enhanced acquisition and maintenance of motor skills. Notably, inhibiting PirB function acutely in M1 of adult WT mice throughout training increases the survival of spines formed during early training and enhances motor learning. These results suggest that increasing the stability of newly formed spines is sufficient to improve long-lasting learning and motor performance and demonstrate that there are limits on motor learning that can be lifted by manipulating PirB, even in adulthood.


title: Comprehensive in situ mapping of human cortical transcriptomic cell types

abstract: The ability to spatially resolve the cellular architecture of human cortical cell types over informative areas is essential to understanding brain function. We combined in situ sequencing gene expression data and single-nucleus RNA-sequencing cell type definitions to spatially map cells in sections of the human cortex via probabilistic cell typing. We mapped and classified a total of 59,816 cells into all 75 previously defined subtypes to create a first spatial atlas of human cortical cells in their native position, their abundances and genetic signatures.


title: A robust method to isolate Drosophila fat body nuclei for transcriptomic analysis

abstract: Gene expression profiles are typically described at the level of the tissue or, often in Drosophila, at the level of the whole organism. Collapsing the gene expression of entire tissues into single measures averages over potentially important heterogeneity among the cells that make up that tissue. The advent of single-cell RNA-sequencing technology (sc-RNAseq) allows transcriptomic evaluation of the individual cells that make up a tissue. However, sc-RNAseq requires a high-quality suspension of viable cells or nuclei, and cell dissociation methods that yield healthy cells and nuclei are still lacking for many important tissues. The insect fat body is a polyfunctional tissue responsible for diverse physiological processes and therefore is an important target for sc-RNAseq. The Drosophila adult fat body consists of fragile cells that are difficult to dissociate while maintaining cell viability. As an alternative, we developed a method to isolate single fat body nuclei for RNA-seq. Our isolation method is largely free of mitochondrial contamination and yields higher capture of transcripts per nucleus compared to other nuclei preparation methods. Our method works well for single cell nuclei sequencing and potentially can be implemented for bulk RNA-seq.

category: molecular biology


title: Cooperativity boosts affinity and specificity of proteins with multiple RNA-binding domains

abstract: Numerous cellular processes rely on the binding of proteins with high affinity to specific sets of RNAs. Yet most RNA binding domains display low specificity and affinity, to the extent that for most RNA-binding domains, the enrichment of the best binding motif measured by high-throughput RNA SELEX or RNA bind-n-seq is usually below 10-fold, dramatically lower than that of DNA-binding domains. Here, we develop a thermodynamic model to predict the binding affinity for proteins with any number of RNA-binding domains given the affinities of their isolated domains. For the four proteins in which affinities for individual domains have been measured the model predictions are in good agreement with experimental values. The model gives insight into how proteins with multiple RNA-binding domains can reach affinities and specificities orders of magnitude higher than their individual domains. Our results contribute towards resolving the conundrum of missing specificity and affinity of RNA binding proteins and underscore the need for bioinformatic methods that can learn models for multi-domain RNA binding proteins from high-throughput in-vitro and in-vivo experiments.


title: Genome-wide, genic, and site-specific changes associated with the convergent evolution of sociality in spiders

abstract:
Sociality is a striking phenotypic innovation that independently evolved dozens of times across animals. Sociogenomic approaches have begun to elucidate the molecular underpinnings of social life in social insects and vertebrates, but the degree to which the convergent evolution of sociality involves convergent molecular evolution remains controversial and largely unknown. Spiders are a powerful system for identifying the genomic causes and consequences of social life because sociality is estimated to have independently evolved 15 times, and each origin likely occurred recently, within the past few million years. To determine if there are statistically supported genomic signatures of protein-coding sequence evolution associated with the convergent evolution of sociality in spiders, we compared the genomes or transcriptomes of 24 spider species that vary in social organization and represent at least seven independent origins of sociality. We identified hundreds of genes that experienced shifts in patterns of molecular evolution during the convergent evolution of sociality, and these genes were enriched for several annotated functions, including neural function, neurogenesis, and behavior, as well as immune function, growth, and metabolism. We also found evidence that directional selection for specific substitutions repeatedly occurred in social species for several genes, in particular the calcium channel gene TPC1. Finally, supporting previous results, we found elevated genome-wide rates of molecular evolution in social species, resulting mainly from relaxation of selection. Altogether, we identify genome-wide, genic, and site-specific changes that repeatedly occurred during the evolution of sociality in spiders.

Significance StatementThe transition from solitary to social life is a major transition in evolution that repeatedly occurred, but the genetic underpinnings are largely unknown. To identify genomic changes associated with sociality, we compared the genomes of 24 spider species, representing seven recent and independent origins of sociality. We identified hundreds of genes and many functional classes of genes that tended to experience shifts in molecular evolution, including genes that have previously been implicated in animal social behavior and human behavioral disorders. Our study shows that while the precise genetic details vary, the repeated evolution of sociality in spiders predictably leaves genome-wide signatures and involves sets of genes with conserved functions that may play general roles in the evolution of social behavior.

category: evolutionary biology


title: Fine mapping without phenotyping: Identification of selection targets in secondary Evolve and Resequence experiments

abstract:
Evolve and Resequence (E&R) studies investigate the genomic selection response of populations in an Experimental Evolution setup. Despite the popularity of E&R, empirical studies in sexually reproducing organisms typically suffer from an excess of candidate loci due to linkage disequilibrium, and single gene or SNP resolution is the exception rather than the rule. Recently, so-called "secondary E&R" has been suggested as promising experimental follow-up procedure to confirm putatively selected regions from a primary E&R study. Secondary E&R provides also the opportunity to increase mapping resolution by allowing for additional recombination events, which separate the selection target from neutral hitchhikers. Here, we use computer simulations to assess the effect of different crossing schemes, population size, experimental duration, and number of replicates on the power and resolution of secondary E&R. We find that the crossing scheme and population size are crucial factors determining power and resolution of secondary E&R: a simple crossing scheme with few founder lines consistently outcompetes crossing schemes where evolved populations from a primary E&R experiment are mixed with a complex ancestral founder population. Regardless of the experimental design tested, a population size of at least 4,800 individuals, which is roughly 5 times larger than population sizes in typical E&R studies, is required to achieve a power of at least 75%. Our study provides an important step towards improved experimental designs aiming to characterize causative SNPs in Experimental Evolution studies.

SignificanceDespite the popularity of Evolve and Resequence (E&R) to investigate genomic selection responses, most studies that use sexually reproducing organisms have broad selection signatures and an excess of candidate loci due to linkage disequilibrium. In this study, we use computer simulations and statistical modelling to evaluate the effects of different experimental and population genetic parameters on the success of potential follow-up experiments (=secondary E&R) aiming to validate and fine-map selection signatures of primary studies. We found that a large population size in combination with a simple crossing scheme is key to the success of secondary E&R in Drosophila.


title: DROSOPHILA MTOR COMPLEX 2 PRESERVES MITOCHONDRIAL AND CARDIAC FUNCTION UNDER HIGH FAT DIET TREATMENT

abstract: High fat diet (HFD)-associated lipotoxicity is one of the major causes of cardiovascular diseases. The mechanistic target of rapamycin (mTOR) pathway, especially mTOR complex 1 (mTORC1), has been previously implicated in HFD-induced heart dysfunction. In the present study, we find that unlike mTORC1, mTOR complex 2 (mTORC2) protects hearts from HFD-induced cardiomyopathy and mitochondrial dysfunction in Drosophila. We show that HFD feeding induces contractile dysfunction along with altered mitochondrial morphology and function. Upon HFD feeding, the mitochondria of cardiomyocytes exhibit fragmentation, loss of membrane potential, and calcium overload. Interestingly, HFD feeding also reduces the activity of cardiac mTORC2. In line with this finding, the flies with cardiac-specific knockdown of rictor, the key subunit of mTORC2, show cardiac and mitochondrial dysfunction similar to what is observed in HFD-fed wild-type flies. Conversely, cardiac-specific activation of mTORC2 by overexpressing rictor attenuates HFD-induced mitochondrial and cardiac dysfunction. Thus, our findings suggest that mTORC2 is a cardioprotective factor and regulates mitochondrial homeostasis upon HFD feeding.

category: genetics


title: MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution

abstract: Technical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolution given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of long-range genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure, and enable the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

category: genomics


title: Social and physical environment independently affect oviposition decisions in Drosophila melanogaster

abstract: In response to environmental stimuli, including variation in the presence of conspecifics, animals show highly plastic responses in behavioural and physiological traits influencing reproduction. These responses have been extensively documented in males, but equivalent study of females is so far lacking. We expect females to be highly responsive to environmental variation, with significant impacts on fitness given females direct impact on offspring number, size, and developmental conditions. Using Drosophila melanogaster as a model, we manipulate (a) exposure to conspecific females, expected to influence their expectation of number of potential mates and larval density for their own offspring, and (b) test how prior consexual population density interacts with the spatial distribution of potential oviposition sites, with females expected to prefer clustered food resources that can support a larger number of eggs and larvae. After exposure to competition, females were slower to start copulating and reduced their copulation duration - the opposite effect to that observed in males previously exposed to rivals. There was a parallel and perhaps related effect on egg production, with females previously housed in groups laying fewer eggs than those that were housed in solitude. The spatial distribution of resources also influenced oviposition behaviour: females clearly preferred aggregated patches of substrate, being more likely to lay, and laying on more of the available patches, in the clustered environment. However, we found no significant interaction between prior housing conditions and resource patchiness, indicating that females did not perceive the value of different resource distributions differently when they were expecting either high or low levels of larval competition. While exposure to consexual competition influences copulatory behaviours, it is the distribution of oviposition resources that has a greater impact on oviposition decisions.

category: animal behavior and cognition


title: Mechanism of activation and regulation of Deubiquitinase activity in MINDY1 and MINDY2

abstract:
Of the eight distinct polyubiquitin chains that can be assembled, K48-linked ubiquitin is the most well-understood linkage and modification of proteins with K48 chains targets the modified protein for degradation. By removing ubiquitin from substrates or trimming ubiquitin chains, deubiquitinases (DUBs) can modulate the outcome of ubiquitylation. MINDY1 and MINDY2 are members of the MINDY family of DUBs that have exquisite specificity for cleaving K48-linked polyubiquitin. Being recently discovered DUBs, we have a poor understanding of their catalytic mechanism. By analysing crystal structures of MINDY1 alone and in complex with monoubiquitin or K48-linked ubiquitin chains, we here reveal how substrate interaction relieves autoinhibition and activates the DUB. Further, our analyses reveal a non-canonical catalytic triad composed of Cys-His-Thr and explain how these DUBs sense both ubiquitin chain length and linkage type to trim K48-linked ubiquitin chains. Our findings highlight the multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2.

SynopsisO_LIStructure of MINDY1 in complex with K48-linked diUb reveals how K48-linked polyUb is recognized and cleaved
C_LIO_LIThe Cys loop mediates autoinhibition of the DUB and substrate binding at the S1 and S1 sites relieves autoinhibition and activates the enzyme for catalysis
C_LIO_LIMINDY1 uses a non-canonical catalytic triad composed of Cys-His-Thr
C_LIO_LIMINDY1 has five ubiquitin binding sites within its catalytic domain and switches from exo to endo cleavage in a ubiquitin chain length-dependent manner
C_LI

category: biochemistry


title: Structural insights into a novel family of integral membrane siderophore reductases

abstract: Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from P. aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a novel fold with two tightly-bound heme molecules. In combination with functional studies these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.


title: Speed-related but not detrended gait variability increases with more sensitive self-paced treadmill controllers at multiple slopes

abstract: Self-paced treadmills are being used more frequently to study humans walking with their self-selected gaits on a range of slopes. There are multiple options to purchase a treadmill with a built-in controller, or implement a custom written self-paced controller, which raises questions about how self-paced controller affect treadmill speed and gait biomechanics on multiple slopes. This study investigated how different self-paced treadmill controller sensitivities affected gait parameters and variability on decline, level, and incline slopes. We hypothesized that increasing self-paced controller sensitivity would increase gait variability on each slope. We also hypothesized that detrended variability could help mitigate differences in variability that arise from differences in speed fluctuations created by the self-paced controllers. Ten young adults walked on a self-paced treadmill using three controller sensitivities (low, medium, and high) and fixed speeds at three slopes (decline, -10{degrees}; level, 0{degrees}; incline, +10{degrees}). Within each slope, average walking speeds and spatiotemporal gait parameters were similar regardless of self-paced controller sensitivity. With higher controller sensitivities on each slope, speed fluctuations, speed variance, and step length variance increased whereas step frequency variance and step width variance were unaffected. Detrended variance was not affected by controller sensitivity suggesting that detrending variability helps mitigate differences associated with treadmill speed fluctuations. Speed-trend step length variances, however, increased with more sensitive controllers. Further, detrended step length variances were similar for self-paced and fixed speed walking, whereas self-paced walking included substantial speed-trend step length variance not present in fixed speed walking. In addition, regardless of the self-paced controller, subjects walked fastest on the level slope with the longest steps, narrowest steps, and least variance. Overall, our findings suggest that separating gait variability into speed-trend and detrended variability could be beneficial for interpreting gait variability among multiple self-paced treadmill studies and when comparing self-paced walking with fixed speed walking.

category: bioengineering


title: DECODE: A Deep-learning Framework for Condensing Enhancers and Refining Boundaries with Large-scale Functional Assays

abstract:
SummaryMapping distal regulatory elements, such as enhancers, is the cornerstone for investigating genome evolution, understanding critical biological functions, and ultimately elucidating how genetic variations may influence diseases. Previous enhancer prediction methods have used either unsupervised approaches or supervised methods with limited training data. Moreover, past approaches have operationalized enhancer discovery as a binary classification problem without accurate enhancer boundary detection, producing low-resolution annotations with redundant regions and reducing the statistical power for downstream analyses (e.g., causal variant mapping and functional validations). Here, we addressed these challenges via a two-step model called DECODE. First, we employed direct enhancer activity readouts from novel functional characterization assays, such as STARR-seq, to train a deep neural network classifier for accurate cell-type-specific enhancer prediction. Second, to improve the annotation resolution ([~]500 bp), we implemented a weakly-supervised object detection framework for enhancer localization with precise boundary detection (at 10 bp resolution) using gradient-weighted class activation mapping.

ResultsOur DECODE binary classifier outperformed the state-of-the-art enhancer prediction methods by 24% in transgenic mouse validation. Further, DECODE object detection can condense enhancer annotations to only 12.6% of the original size, while still reporting higher conservation scores and genome-wide association study variant enrichments. Overall, DECODE improves the efficiency of regulatory element mapping with graphic processing units for deep-learning applications and is a powerful tool for enhancer prediction and boundary localization.

Contactpi@gersteinlab.org

category: bioinformatics


title: Bidirectional, unlike unidirectional transport, allows transporting axonal cargos against their concentration gradient

abstract: Even though most axonal cargos are synthesized in the soma, the concentration of many of these cargos is larger at the presynaptic terminal than in the soma. This requires transport of these cargos from the soma to the presynaptic terminal or other active sites in the axon. Axons utilize both bidirectional (for example, slow axonal transport) and unidirectional (for example, fast anterograde axonal transport) modes of cargo transport. Bidirectional transport seems to be less efficient because it requires more time and takes more energy to deliver cargos. In this paper, we studied a family of models which differ by the modes of axonal cargo transport (such as anterograde and retrograde motor-driven transport and passive diffusion) as well as by the presence or absence of pausing states. The models are studied to investigate their ability to describe axonal transport against the cargo concentration gradient. We argue that bidirectional axonal transport is described by a higher-order mathematical model, which allows imposing cargo concentration not only at the axon hillock but also at the axon terminal. The unidirectional transport model allows only for the imposition of cargo concentration at the axon hillock. Due to the great lengths of the axons, anterograde transport mostly relies on molecular motors, such as kinesins, to deliver cargos synthesized in the soma to the terminal and other active sites in the axon. Retrograde transport can be also motor-driven, in which case cargos are transported by dynein motors. If cargo concentration at the axon tip is higher than at the axon hillock, retrograde transport can also occur by cargo diffusion. However, because many axonal cargos are large or they assemble in multiprotein complexes for axonal transport, the diffusivity of such cargos is very small. We investigated the case of a small cargo diffusivity using a perturbation technique and found that for this case the effect of diffusion is limited to a very thin diffusion boundary layer near the axon tip. If cargo diffusivity is decreased in the model, we show that without motor-driven retrograde transport the model is unable to describe a high cargo concentration at the axon tip. To the best of our knowledge, our paper presents the first explanation for the utilization of seemingly inefficient bidirectional transport in neurons.

category: biophysics


title: A noncanonical FLT3 gatekeeper mutation disrupts gilteritinib binding and confers resistance

abstract: The recent FDA approval of the FLT3 inhibitor, gilteritinib, for AML represents a major breakthrough for treatment of FLT3 mutated AML. However, patients only respond to gilteritinib for 6-7 months due to the emergence of drug resistance. Clinical resistance to gilteritinib is often associated with expansion of NRAS mutations, and less commonly via gatekeeper mutations in FLT3, with F691L being the most common. We developed an in vitro model that charts the temporal evolution of resistance to gilteritinib from early microenvironmental-mediated resistance to late intrinsic resistance mutations. Our model system accurately recapitulates the expansion of NRAS mutations and the F691L gatekeeper mutations found in AML patients. As part of this study, we also identified a novel FLT3N701K mutation that also appeared to promote resistance to gilteritinib. Using the Ba/F3 system, we demonstrate that N701K mutations effectively act like a gatekeeper mutation and block gilteritinib from binding to FLT3, thereby promoting resistance. Structural modeling of FLT3 reveals how N701K, and other reported gilteritinib resistance mutations, obstruct the gilteritinib binding pocket on FLT3. Interestingly, FLT3N701K does not block quizartinib binding, suggesting that FLT3N701K mutations are more specific for type 1 FLT3 inhibitors (gilteritinib, midostaurin, and crenolanib). Thus, our data suggests that for the FLT3N701K mutation, switching classes of FLT3 inhibitors may restore clinical response. As the use of gilteritinib expands in the clinic, this information will become critical to define clinical strategies to manage gilteritinib resistance.

category: cancer biology


title: The MEMIC: An ex vivo system to model the complexity of the tumor microenvironment

abstract: There is an urgent need for accurate, scalable, and cost-efficient models of the complexity and heterogeneity of the tumor microenvironment. Here, we detail how to fabricate and use the Metabolic Microenvironment Chamber (MEMIC) - a 3D-printed ex vivo model of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is the accessibility to the blood stream that provides key resources such as oxygen and nutrients. While some tumor cells have direct access to these resources, many others must survive under progressively more ischemic environments as they reside further from the vasculature. The MEMIC is designed to simulate the differential access to nutrients and allows co-culturing different cell types, such as tumor and immune cells. This system is optimized for live imaging and other microscopy-based approaches and it is a powerful tool to study tumor features such as the effect of nutrient scarcity on tumor-stroma interactions. Due to its adaptable design and full experimental control, the MEMIC can provide novel insights into the tumor microenvironment that would be difficult to obtain via other methods. As a proof of principle, we show that cells can sense gradual changes in metabolite concentration, and tune intracellular cell signaling to form multicellular spatial patterns of cell proliferation. We also show that ischemic macrophages reduce epithelial features in neighboring tumor cells highlighting the power of this system to study cell-cell interactions and non-cell autonomous effects of the metabolic microenvironment. We propose that the MEMIC can be easily adapted to study early development, ischemic stroke, and other systems where multiple cell types interact within heterogeneous environments.


title: Water levels affect photosynthesis and nutrient use more than salinity in a scrub Red Mangrove forest of the southeastern Florida Everglades

abstract: We investigated how mangrove-island micro-elevation (i.e., habitat: center vs. edge) affects tree physiology in a scrub mangrove forest of the southeastern Everglades. We measured leaf gas exchange rates of scrub Rhizophora mangle L. trees monthly during 2019, hypothesizing that CO2 assimilation (Anet) and stomatal conductance (gsw) would decline with increasing water levels and salinity, expecting more-considerable differences at mangrove-island edges than centers, where physiological stress is greatest. Water levels varied between 0 and 60 cm from the soil surface, rising during the wet season (May-October) relative to the dry season (November-April). Porewater salinity ranged from 15 to 30 ppt, being higher at mangrove-island edges than centers. Anet maximized at 15.1 {micro}mol m-2 s-1, and gsw was typically <0.2 mol m-2 s-1, both of which were greater in the dry than the wet season and greater at island centers than edges, with seasonal variability being roughly equal to variation between habitats. After accounting for season and habitat, water level positively affected Anet in both seasons but did not affect gsw. Our findings suggest that inundation stress (i.e., water level) is the primary driver of variation in leaf gas exchange rates of scrub mangroves in the Florida Everglades, while also constraining Anet more than gsw. The interaction between inundation stress due to permanent flooding and habitat varies with season as physiological stress is alleviated at higher-elevation mangrove-island center habitats during the dry season. Freshwater inflows during the wet season, increase water levels and inundation stress at higher-elevation mangrove-island centers, but also potentially alleviate salt and sulfide stress in soils. Thus, habitat heterogeneity leads to differences in nutrient and water acquisition and use between trees growing in island centers versus edges, creating distinct physiological controls on photosynthesis, which likely affect carbon flux dynamics of scrub mangroves in the Everglades.

category: plant biology


title: Water levels primarily drive variation in photosynthesis and nutrient use of scrub Red Mangroves in the southeastern Florida Everglades


title: Thermal laser ablation with tunable lesion size reveals multiple origins of seizure-like convulsions in Caenorhabditis elegans

abstract: Laser microsurgery has long been an important means of assessing the functions of specific cells and tissues. Most laser ablation systems use short, highly focused laser pulses to create plasma-mediated lesions with dimensions on the order of the wavelength of light. While the small size of the lesion enables ablation with high spatial resolution, it also makes it difficult to ablate larger structures. We developed an infrared laser ablation system capable of thermally lesioning tissues with spot sizes tunable by the duration and amplitude of laser pulses. We used our laser system in the roundworm C. elegans to kill single neurons and to sever the dorsal and ventral nerve cords, structures that are difficult to lesion using a plasma-based ablation system. We used these ablations to investigate the source of convulsions in a gain-of-function mutant for the acetylcholine receptor ACR-2. Severing the ventral nerve cord caused convulsions to occur independently anterior and posterior to the lesion, suggesting that convulsions can arise independently from distinct subsets of the motor circuit.


title: Genotype and age influence cortical-FKBP5 at multiple regulatory and single-cell-type levels in severe psychiatric disorders

abstract: Deducing genes capable of classifying biologically-distinct psychiatric subtypes, and their targets for treatment, is a priority approach in psychiatry. FKBP5 is one such gene with strong evidence of utility to delineate a trans-diagnostic psychiatric subtype. Yet how brain-expressed FKBP5 is affected in psychiatric disorders in humans is not fully understood and critical for propelling FKBP5-targeting treatment development. We performed a large-scale postmortem study (n=895) of FKBP5 using dorsolateral prefrontal cortex samples derived from individuals with severe psychiatric disorders with a comprehensive battery of bulk/single-cell omics and histological analyses. We observed consistently heightened FKBP5 mRNA and protein in psychopathology, moderated by genotype and age, and accompanied by DNA methylation changes in key enhancers. These effects were most prominent in superficial-layer pyramidal cells. Heightened FKBP5 was also differentially associated with downstream pathways according to age, being specifically associated with synaptic transmission in early adulthood, and neuroinflammation and neurodegeneration in later life.


title: Brain expressed FKBP5 delineates a therapeutic subtype of severe mental illness


title: Proteomic portraits reveal evolutionarily conserved and divergent responses to spinal cord injury

abstract: Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of CSF and serum samples from 111 acute SCI patients and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at six months with an AUC of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response, and establish the CSF abundance of glial fibrillary acidic protein (GFAP) as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.


title: Complexity and graded regulation of neuronal cell type-specific alternative splicing revealed by single-cell RNA sequencing

abstract:
The enormous neuronal cellular diversity in the mammalian brain, which is highly prototypical and organized in a hierarchical manner, is dictated by cell type-specific gene regulatory programs at the molecular level. Although prevalent in the brain, contribution of alternative splicing (AS) to the molecular diversity across neuronal cell types is just starting to emerge. Here we systematically investigated AS regulation across over 100 transcriptomically defined neuronal types of the adult mouse cortex using deep single cell RNA-sequencing (scRNA-seq) data. We found distinct splicing programs between glutamatergic and GABAergic neurons and between subclasses within each neuronal class, consisting of overlapping sets of alternative exons showing differential splicing at multiple hierarchical levels. Using an integrative approach, our analysis suggests that RNA-binding proteins (RBPs) Celf1/2, Mbnl2 and Khdrbs3 are preferentially expressed and more active in glutamatergic neurons, while Elavl2 and Qk are preferentially expressed and more active in GABAergic neurons. Importantly, these and additional RBPs also contribute to differential splicing between neuronal subclasses at multiple hierarchical levels, and some RBPs drive splicing dynamics that do not conform to the hierarchical structure defined by the transcriptional profiles. Thus, our results suggest graded regulation of AS across neuronal cell types, which provides a molecular mechanism orthogonal to, rather than downstream of, transcriptional regulation in specifying neuronal identity and function.

SignificanceAlternative splicing (AS) is extensively used in the mammalian brain, but its contribution to the molecular and cellular diversity across neuronal cell types remains poorly understood. Through systematic and integrative analysis of AS regulation across over 100 transcriptomically defined cortical neuronal types, we found neuronal subclass-specific splicing regulatory programs consists of overlapping alternative exons showing differential splicing at multiple hierarchical levels. This graded AS regulation is controlled by unique combinations of RNA-binding proteins (RBPs). Importantly, these RBPs also drive splicing dynamics across neuronal cell types that do not conform to the hierarchical taxonomy established based on transcriptional profiles, suggesting that the graded AS regulation provides a molecular mechanism orthogonal to transcriptional regulation in specifying neuronal identity and function.


title: Stimulating human prefrontal cortex increases reward learning

abstract:
Work in computational psychiatry suggests that mood disorders may stem from aberrant reinforcement learning processes. Specifically, it is proposed that depressed individuals believe that negative events are more informative than positive events, resulting in faster learning from negative outcomes (Pulcu & Browning, 2019). In this proof-of-concept study, we investigated whether learning rates for affective outcomes are malleable using transcranial direct current stimulation (tDCS). Healthy adults completed an established reinforcement learning task (Pulcu & Browning, 2017) in which the information content of reward and loss outcomes was manipulated by varying the volatility of stimulus-outcome associations. Learning rates on the tasks were quantified using computational models. Stimulation over dorsolateral prefrontal cortex (DLPFC) but not motor cortex (M1) specifically increased learning rates for reward outcomes. The effects of prefrontal tDCS were cognitive state-dependent: online stimulation increased learning rates for wins; offline stimulation decreased both win and loss learning rates. A replication study confirmed the key finding that online tDCS to DLPFC specifically increased learning rates for rewards relative to losses. Taken together, these findings demonstrate the potential of tDCS for modulating computational parameters of reinforcement learning relevant to mood disorders.

Significance statementDisproportionate learning from negative relative to positive outcomes has been implicated in the development and maintenance of depression. The present work demonstrates that transcranial direct current stimulation (tDCS) to dorsolateral prefrontal cortex can specifically increase learning from positive events in healthy adults. Our results provide preliminary evidence that non-invasive brain stimulation can be used to shape reinforcement learning, indicating a potential novel cognitive neurostimulation intervention strategy for affective disorders.


title: A computational model of dopaminergic modulation of hippocampal Schaffer collateral-CA1 long-term plasticity

abstract:
Dopamine plays a critical role in modulating the long-term synaptic plasticity of the hippocampal Schaffer collateral-CA1 pyramidal neuron synapses (SC-CA1), a widely accepted cellular model of learning and memory. Limited results from hippocampal slice experiments over the last four decades have shown that the timing of the activation of dopamine D1/D5 receptors relative to a high/low-frequency stimulation (HFS/LFS) in SC-CA1 synapses regulates the modulation of HFS/LFS-induced long-term potentiation/depression (LTP/LTD) in these synapses. However, the existing literature lacks a complete picture of how various concentrations of D1/D5 agonists and the relative timing between the activation of D1/D5 receptors and LTP/LTD induction by HFS/LFS, affect the spatiotemporal modulation of SC-CA1 synaptic dynamics. In this paper, we have developed a computational model, a first of its kind, to make quantitative predictions of the temporal dose-dependent modulation of the HFS/LFS induced LTP/LTD in SC-CA1 synapses by D1/D5 agonists activating cAMP-mediating biochemical pathways. Our model combines the biochemical effects with the electrical effects at the electrophysiological level. We have estimated the model parameters from the published electrophysiological data, available from diverse hippocampal CA1 slice experiments, in a Bayesian framework. Our modeling results demonstrate the capability of our model in making quantitative predictions of the available experimental results under diverse HFS/LFS protocols. The predictions from our model show a strong nonlinear dependency of the modulated LTP/LTD by D1/D5 agonists on the relative timing between the activated D1/D5 receptors and the HFS/LFS protocol as well as the applied concentration of D1/D5 agonists. Particularly, our model predicts that D1/D5 agonists could significantly boost the LTP induced by weak HFS if the agonist is applied much before the HFS protocol. Furthermore, our model predicts that specific D1/D5 agonists can convert the LFS-induced LTD in SC-CA1 synapses to LTP if D1/D5 receptors are activated before the applied LFS protocol.

Author summaryDopamine, a reward neuromodulator, plays an essential role in shaping hippocampal-dependent learning and memory of behavioral tasks. Limited experimental studies have revealed that pharmacological agents of dopaminergic receptors can significantly modulate the electrically-induced long-term potentiation/depression (LTP/LTD) of the hippocampal Schaffer collateral CA1 pyramidal (SC-CA1) synapses, a cellular model of learning and memory, in a time and dose dependent manner.

However, exploring the effect of the parameter space of various concentration levels of the applied pharmacological agent as well as the frequency-specific characteristics of the high (low) frequency stimulation (H(L)FS) protocol on the dopaminergic receptors mediated spatiotemporal modulation of LTP/LTD is a combinatorically challenging problem which is both expensive and time-consuming to address in experiments alone. Here, we develop a multi-timescale computational modeling framework to address this question. Our model integrates the slow biochemical dynamics and the fast-electrical dynamics of the CA1 pyramidal neuron and makes quantitative predictions of the experimentally observed modulation of H(L)FS-induced LTP/LTD in SC-CA1 synapses by dopaminergic (D1/D5) receptors agonists. Our modeling results complement the experimental findings and show specific predictions on the potential role of dopamine in strengthening weak synapses.


title: High contrast, moving targets in an emerging target paradigm promote fast visuomotor responses during visually guided reaching

abstract:
Humans have a remarkable capacity to rapidly interact with the surrounding environment, often by transforming visual input into motor output on a moment-to-moment basis. But what visual features promote rapid reaching? High contrast, fast-moving targets elicit strong responses in the superior colliculus (SC), a structure associated with express saccades and implicated in rapid electromyographic (EMG) responses on upper limb muscles. To test the influence of stimulus properties on rapid reaches, we had human subjects perform visually guided reaches to moving targets varied by speed (experiment 1) or speed and contrast (experiment 2), in an emerging target paradigm which has recently been shown to robustly elicit fast visuomotor responses. Our analysis focused on stimulus-locked responses (SLRs) on upper limb muscles. SLRs appear within <100 ms of target presentation, and as the first wave of muscle recruitment, they have been hypothesized to arise from the SC. Across 32 subjects studied in both experiments, 97% expressed SLRs in the emerging target paradigm, whereas only 69% expressed SLRs in an immediate response paradigm towards static targets. Faster moving targets (experiment 1) evoked large magnitude SLRs, while high contrast fast moving targets (experiment 2) evoked short latency, large magnitude SLRs. In some instances, SLR magnitude exceeded the magnitude of movement aligned activity. Both large magnitude and short latency SLRs were correlated with short latency reach reaction times. Our results support the hypothesis that, in scenarios requiring expedited responses, a subcortical pathway originating in the SC elicits the earliest wave of muscle recruitment, expediting reaction times.

New & NoteworthyHow does the brain rapidly transform vision into action? Here, by recording upper limb muscle activity, we find that high contrast and fast moving targets are highly effective at evoking rapid visually guided reaches. We surmise that a brainstem circuit originating in the superior colliculus contributes to the most rapid reaching responses. When time is of the essence, cortical areas may serve to prime this circuit, and elaborate subsequent phases of recruitment.


title: Sauna-like conditions or menthol treatment reduce tau phosphorylation through mild hyperthermia

abstract: In Alzheimers disease (AD), hyper-phosphorylation and aggregation of tau correlates with clinical progression and represents a valid therapeutic target. A recent 20-year prospective study revealed an association between moderate to high frequency of Finnish sauna bathing and a lower incidence of dementia and AD, but the molecular mechanisms underlying these benefits remain uncertain. Here, we tested the hypothesis that sauna-like conditions could lower tau phosphorylation by increasing body temperature. We observed a decrease in tau phosphorylation in wild-type and hTau mice as well as in neuron-like cells when exposed to higher temperatures. These effects were correlated with specific changes in phosphatase and kinase activities, but not with inflammatory or heat-shock responses. We also used a drug strategy to promote thermogenesis. Topical application of menthol led to a sustained increase in body temperature in hTau mice concomitant with a significant decrease in tau phosphorylation. Our results suggest that sauna-like conditions or menthol treatment could lower tau pathology through mild hyperthermia, and may provide promising therapeutic strategies for AD and other tauopathies.


title: The neural response to the temporal fine structure of continuous musical pieces is not affected by selective attention

abstract: Speech and music are spectro-temporally complex acoustic signals that a highly relevant for humans. Both contain a temporal fine structure that is encoded in the neural responses of subcortical and cortical processing centres. The subcortical response to the temporal fine structure of speech has recently been shown to be modulated by selective attention to one of two competing voices. Music similarly often consists of several simultaneous melodic lines, and a listener can selectively attend to a particular one at a time. However, the neural mechanisms that enable such selective attention remain largely enigmatic, not least since most investigations to date have focussed on short and simplified musical stimuli. Here we study the neural encoding of classical musical pieces in human volunteers, using scalp electroencephalography (EEG) recordings. We presented volunteers with continuous musical pieces composed of one or two instruments. In the latter case, the participants were asked to selectively attend to one of the two competing instruments and to perform a vibrato identification task. We used linear encoding and decoding models to relate the recorded EEG activity to the stimulus waveform. We show that we can measure neural responses to the temporal fine structure of melodic lines played by one single instrument, at the population level as well as for most individual subjects. The neural response peaks at a latency of 7.6 ms and is not measurable past 15 ms. When analysing the neural responses elicited by competing instruments, we find no evidence of attentional modulation. Our results show that, much like speech, the temporal fine structure of music is tracked by neural activity. In contrast to speech, however, this response appears unaffected by selective attention in the context of our experiment.


title: Single-cell immune profiling reveals the impact of antiretroviral therapy on HIV-1-induced immune dysfunction, T cell clonal expansion, and HIV-1 persistence in vivo

abstract: Despite antiretroviral therapy (ART), HIV-1 persists in proliferating T cell clones that increase over time. To understand whether early ART affects HIV-1 persistence in vivo, we performed single-cell ECCITE-seq and profiled 89,279 CD4+ T cells in paired samples during viremia and after immediate versus delayed ART in six people in the randomized interventional Sabes study. We found that immediate ART partially reverted TNF responses while delayed ART did not. Antigen and TNF responses persisted despite immediate ART and shaped the transcriptional landscape of CD4+ T cells, HIV-1 RNA+ cells, and T cell clones harboring them (cloneHIV-1). Some HIV-1 RNA+ cells reside in the most clonally expanded cytotoxic T cell populations (GZMB and GZMK Th1 cells). CloneHIV-1+ were larger in clone size, persisted despite ART, and exhibited transcriptional signatures of antigen, cytotoxic effector, and cytokine responses. Using machine-learning algorithms, we identified markers for HIV-1 RNA+ cells and cloneHIV-1+ as potential therapeutic targets. Overall, by combining single-cell immune profiling and T cell expansion dynamics tracking, we identified drivers of HIV-1 persistence in vivo.

category: immunology


title: Golden hamster piRNAs are necessary for early embryonic development and establishment of spermatogonia

abstract: PIWI-associated RNAs (piRNAs) support the germline by suppressing retrotransposons and genes. In mice, piRNAs are essential for spermatogenesis but not oogenesis. To test how this applies to other mammals, we deleted Mov10l1 helicase in golden hamster, whose piRNA pathway is configured more similarly to that of other mammals. Mov10l1-/- male hamsters showed impaired establishment of spermatogonia accompanied by transcriptome dysregulation and a surge in MYSERV retrotransposon expression. The rare viable spermatogenic cells showed a meiotic failure phenotype like Mov10l1-/- mice. Female Mov10l1-/- hamsters were sterile due to post-meiotic loss of developmental competence in zygotes. Unique phenotypes of Mov10l1-/- hamsters demonstrate the adaptive nature of piRNA-mediated control of genes and retrotransposons in order to confront emerging genomic threats or acquire new physiological roles.


title: Generation of a New Frizzled 2 Flox Mouse Model to Clarify Its Role in Development

abstract: It is currently accepted that Wnt receptors, Frizzleds (Fzd), have high functional redundancy, making individual receptors challenging to target therapeutically. Specifically, Fzd2 is believed to be functionally redundant with Fzd1 and Fzd7, findings which were based largely on previously published global knockout mouse studies. Conversely, a Fzd2 global knockout mouse model developed by the International Mouse Phenotype Consortium (IMPC) is early embryonic lethal, suggesting Fzd2 is critical for early embryonic development. If global deletion of Fzd2 leads to early lethality, floxed models are necessary to identify tissue-specific phenotypes. We found that a previously published Fzd2 flox model does not fully delete Fzd2 function. To reconcile the contradictory findings in Fzd2 mouse models and allow for tissue-specific studies of Fzd2, we have generated a new flox model using a modified two-cell homologous recombination CRISPR approach. We demonstrated successful simultaneous insertion of two loxP sites fully surrounding the Fzd2 gene and confirmed cre-mediated recombination deletes the sequence between the loxP sites leading to a Fzd2 null allele. Preliminary studies suggest global knockouts are early embryonic lethal and full characterization of the tissue-specific effects of Fzd2 deletion is currently underway. This work suggests Fzd2 uniquely regulates development and emphasizes the importance of thorough validation of newly generated mouse models.

category: cell biology


title: The Endoplasmic Reticulum pool of Bcl-xL dampens the Unfolded Protein Response through IP3R-dependent Calcium Release

abstract: Apoptosis plays a role in cell homeostasis in both normal development and disease. Bcl-xL, a member of the Bcl-2 family of proteins, regulates the intrinsic mitochondrial pathway of apoptosis. It is overexpressed in several cancers. Bcl-xL has a dual subcellular localization and is found at the mitochondria as well as the endoplasmic reticulum (ER). However, the biological significance of its ER localization is unclear. In order to decipher the functional contributions of the mitochondrial and reticular pools of Bcl-xL, we generated genetically modified mice expressing exclusively Bcl-xL at the ER, referred to as ER-xL, or the mitochondria, referred to as Mt-xL. By performing cell death assays, we showed that ER-xL MEFs show increased vulnerability to apoptotic stimuli but are more resistant to ER stress. Furthermore, ER-xL MEFs demonstrated a reduced expression of the Unfolded Protein Response (UPR) markers upon ER stress and displayed reduced inositol trisphosphate receptor (IP3R)-mediated ER calcium release. Collectively, our data show that upon ER stress, Bcl-xL negatively regulates IP3R-mediated calcium flux from the ER, which prevents ER calcium depletion and maintains the UPR and subsequent cell death in check. This work reveals a moonlighting function of Bcl-xL at the ER, apart from its cliche regulation of apoptosis.


title: Games of risk and reward in carnivore communities

abstract:
Mesopredator release theory suggests that dominant predators suppress subordinate carnivores and ultimately shape community dynamics, but the assumption that subordinate species are only negatively affected ignores the possibility of facilitation through scavenging. We examined the interplay within a carnivore community consisting of cougars, coyotes, black bears, and bobcats using contemporaneous Global Positioning System telemetry data from 51 individuals, diet analysis from 972 DNA-metabarcoded scats, and data from 128 physical investigations of cougar kill sites, 28 of which were monitored with remote cameras. Resource provisioning from competitively-dominant cougars to coyotes through scavenging was so prolific as to be an overwhelming determinant of coyote behavior, space use, and resource acquisition. This was evident via strong attraction of coyotes to cougar kill sites, frequent scavenging of cougar-killed prey, and coyote diets that nearly matched cougars in the magnitude of ungulate consumption. Yet coyotes were often killed by cougars and used space to minimize encounters, complicating the fitness benefits gained from scavenging. We estimated that 23% (95% CI: 8-55%) of the coyote population in our study area was killed by cougars annually suggesting that coyote interactions with cougars are a complex behavioral game of risk and reward. In contrast, we found no indication that bobcat space use or diet was influenced by cougars. Black bears avoided cougars, but there was no evidence of attraction to cougar kill sites, and much lower levels of ungulate consumption and carcass visitation than for coyotes. Interspecific interactions among carnivores are multifaceted encompassing both suppression and facilitation.

Significance StatementAn incomplete understanding of the total influence competitively-dominant predators exert on subordinate species hinders our ability to anticipate the effects that changing carnivore populations will have in ecological communities. Here we show that cougars are the architects of a complex behavioral game of risk and reward, as subordinate or co-occurring carnivores are both provisioned and preyed on by the dominant predators. Each co-occurring carnivore species considered here employed a different strategy to approach the risk-reward tradeoff suggesting there are multiple viable solutions to the game. By not considering the multitude of effects dominant predators have on other carnivores, we are missing important linkages in terrestrial food webs.

category: ecology


title: Exploring a local genetic interaction network using evolutionary replay experiments

abstract: Understanding how genes interact is a central challenge in biology. Experimental evolution provides a useful, but underutilized, tool for identifying genetic interactions, particularly those that involve non-loss-of-function mutations or mutations in essential genes. We previously identified a strong positive genetic interaction between specific mutations in KEL1 (P344T) and HSL7 (A695fs) that arose in an experimentally-evolved Saccharomyces cerevisiae population. Because this genetic interaction is not phenocopied by gene deletion, it was previously unknown. Using "evolutionary replay" experiments we identified additional mutations that have positive genetic interactions with the kel1-P344T mutation. We replayed the evolution of this population 672 times from six timepoints. We identified 30 populations where the kel1-P344T mutation reached high frequency. We performed whole-genome sequencing on these populations to identify genes in which mutations arose specifically in the kel1-P344T background. We reconstructed mutations in the ancestral and kel1-P344T backgrounds to validate positive genetic interactions. We identify several genetic interactors with KEL1, we validate these interactions by reconstruction experiments, and we show these interactions are not recapitulated by loss-of-function mutations. Our results demonstrate the power of experimental evolution to identify genetic interactions that are positive, allele specific, and not readily detected by other methods, and sheds light on a previously under-explored region of the yeast genetic interaction network.


title: Low-cost, scalable, and automated fluid sampling for fluidics applications

abstract: We present colosseum, a low-cost, modular, and automated fluid sampling device for scalable fluidic applications. The colosseum fraction collector uses a single motor, can be built for less than $100 using off-the-shelf and 3D-printed components, and can be assembled in less than an hour. Build Instructions and source files are available at https://github.com/pachterlab/colosseum.


title: Graph Contextualized Attention Network for Predicting Synthetic Lethality in Human Cancers

abstract:
MotivationSynthetic Lethality (SL) plays an increasingly critical role in the targeted anticancer therapeutics. In addition, identifying SL interactions can create opportunities to selectively kill cancer cells without harming normal cells. Given the high cost of wet-lab experiments, in silico prediction of SL interactions as an alternative can be a rapid and cost-effective way to guide the experimental screening of candidate SL pairs. Several matrix factorization-based methods have recently been proposed for human SL prediction. However, they are limited in capturing the dependencies of neighbors. In addition, it is also highly challenging to make accurate predictions for new genes without any known SL partners.

ResultsIn this work, we propose a novel graph contextualized attention network named GCATSL to learn gene representations for SL prediction. First, we leverage different data sources to construct multiple feature graphs for genes, which serve as the feature inputs for our GCATSL method. Second, for each feature graph, we design node-level attention mechanism to effectively capture the importance of local and global neighbors and learn local and global representations for the nodes, respectively. We further exploit multi-layer perceptron (MLP) to aggregate the original features with the local and global representations and then derive the feature-specific representations. Third, to derive the final representations, we design feature-level attention to integrate feature-specific representations by taking the importance of different feature graphs into account. Extensive experimental results on three datasets under different settings demonstrate that our GCATSL model outperforms 14 state-of-the-art methods consistently. In addition, case studies further validate the effectiveness of our proposed model in identifying novel SL pairs.

AvailabilityPython codes and dataset are available at:

Contactluojiawei@hnu.edu.cn and xlli@i2r.a-star.edu.sg


title: The environment topography alters the transition from single-cell populations to multicellular structures in Myxococcus xanthus

abstract: The social soil-dwelling bacteria Myxococcus xanthus can form multicellular structures, known as fruiting bodies. Experiments in homogeneous environments have shown that this process is affected by the physico-chemical properties of the substrate, but they have largely neglected the role of complex topographies. We experimentally demonstrate that the topography alters single-cell motility and multicellular organization in M. xanthus. In topographies realized by randomly placing silica particles over agar plates, we observe that the cells interaction with particles drastically modifies the dynamics of cellular aggregation, leading to changes in the number, size and shape of the fruiting bodies, and even to arresting their formation in certain conditions. We further explore this type of cell-particle interaction in a minimal computational model. These results provide fundamental insights into how the environment topography influences the emergence of complex multicellular structures from single cells, which is a fundamental problem of biological, ecological and medical relevance.


title: Epigenetic state determines inflammatory sensing in neuroblastoma

abstract: Immunotherapy has revolutionized cancer treatment, but many cancers are not impacted by currently available immunotherapeutic strategies. Here, we investigated inflammatory signaling pathways in neuroblastoma, a classically "cold" pediatric cancer. By testing the functional response of a panel of 20 diverse neuroblastoma cell lines to three different inflammatory stimuli, we found that all cell lines have intact interferon signaling and all but one lack functional cGAS-STING signaling. However, toll-like receptor (TLR) signaling, particularly through TLR3, was heterogeneous. Six cell lines showed robust response, five of which are in the mesenchymal epigenetic state, while all 14 unresponsive cell lines are in the adrenergic state. Genetically switching the adrenergic BE2(c) cell line towards the mesenchymal state fully restored TLR responsiveness. In responsive cells, TLR3 activation results in the secretion of pro-inflammatory cytokines, enrichment of inflammatory transcriptomic signatures, and increased tumor killing by T-cells in vitro. Using single cell RNA sequencing data, we show that human neuroblastoma cells with stronger mesenchymal signatures have a higher basal inflammatory state, demonstrating intra-tumoral heterogeneity in inflammatory signaling that has significant implications for immunotherapeutic strategies in this aggressive childhood cancer.


title: microRNA775 targets a β-(1,3)-galactosyltransferase to regulate growth and development in Arabidopsis thaliana

abstract:
MicroRNAs are critical regulators of gene expression in plants and other organisms, and are involved in regulating plethora of developmental processes. Evolutionarily, miRNAs can be ancient and conserved across species or recently evolved and young, which are not conserved across diverse plant groups. miR775 is a non-conserved miRNA identified only in Arabidopsis thaliana. Here, we investigated the functional significance of miR775 in A. thaliana and observed that miR775 targets a probable {beta}-(1,3)-galactosyltransferase gene at post transcriptional level. Phenotypic analysis of miR775 over-expression lines and the target mutant suggested miR775 regulates rosette size by elongating petiole length and increasing leaf area. Further, the expression of miR775 was found to be up-regulated in response to UV-B and hypoxia. Our results also suggest that miR775 regulated {beta}-(1,3)-galactosyltransferase may involve in regulating the {beta}-(1,3)-galactan content of arabinogalactans. Collectively, our findings establish a role of miR775 in regulating growth and development in A. thaliana.

HighlightsO_LIThe role of an uncharacterized microRNA, miR775 has been explored
C_LIO_LImiR775 targets a probable {beta}-(1,3)-galactosyltransferase involved in complex carbohydrate biosynthesis
C_LIO_LImiR775 regulates rosette size in A. thaliana and may play role under UV light and hypoxia
C_LI


title: Genome-scale metabolic modeling reveals SARS-CoV-2-induced host metabolic reprogramming and identifies metabolic antiviral targets

abstract: Tremendous progress has been made to control the COVID-19 pandemic caused by the SARS-CoV-2 virus. However, effective therapeutic options are still rare. Drug repurposing and combination represent practical strategies to address this urgent unmet medical need. Viruses, including coronaviruses, are known to hijack host metabolism to facilitate viral proliferation, making targeting host metabolism a promising antiviral approach. Here, we describe an integrated analysis of 12 published in vitro and human patient gene expression datasets on SARS-CoV-2 infection using genome-scale metabolic modeling (GEM), revealing complicated host metabolism reprogramming during SARS-CoV-2 infection. We next applied the GEM-based metabolic transformation algorithm to predict anti-SARS-CoV-2 targets that counteract the virus-induced metabolic changes. We successfully validated these targets using published drug and genetic screen data and by performing an siRNA assay in Caco-2 cells. Further generating and analyzing RNA-sequencing data of remdesivir-treated Vero E6 cell samples, we predicted metabolic targets acting in combination with remdesivir, an approved anti-SARS-CoV-2 drug. Our study provides clinical data-supported candidate anti-SARS-CoV-2 targets for future evaluation, demonstrating host metabolism-targeting as a promising antiviral strategy.

category: systems biology


title: Genome-scale metabolic modeling reveals SARS-CoV-2-induced metabolic changes and antiviral targets


title: Acyloxyacyl Hydrolase is a Host Determinant of Gut Microbiome-Mediated Pelvic Pain

abstract: Dysbiosis of gut microbiota is associated with many pathologies, yet host factors modulating microbiota remain unclear. Interstitial cystitis/bladder pain syndrome (IC/BPS or "IC") is a debilitating condition of chronic pelvic pain often with co-morbid urinary dysfunction and anxiety/depression, and recent studies find fecal dysbiosis in IC/BPS patients. We previously identified the locus encoding acyloxyacyl hydrolase, Aoah, as a modulator of pelvic pain severity in a murine IC/BPS model. AOAH-deficient mice spontaneously develop rodent correlates of pelvic pain, increased responses to induced pelvic pain models, voiding dysfunction, and anxious/depressive behaviors. Here, we report that AOAH-deficient mice exhibit dysbiosis of GI microbiota. AOAH-deficient mice exhibit an enlarged cecum, a phenotype long associated with germ-free rodents, and reduced trans-epithelial electrical resistance consistent with a "leaky gut" phenotype. AOAH-deficient ceca showed altered gene expression consistent with inflammation, Wnt signaling, and urologic disease. 16S rRNA sequencing of stool revealed altered microbiota in AOAH-deficient mice, and GC-MS identified altered metabolomes. Co-housing AOAH-deficient mice with wild type mice resulted in converged microbiota and altered predicted metagenomes. Co-housing also abrogated the pelvic pain phenotype of AOAH-deficient mice, which was corroborated by oral gavage of AOAH-deficient mice with stool slurry of wild type mice. Converged microbiota also alleviated comorbid anxiety-like behavior in AOAH-deficient mice. Oral gavage of AOAH-deficient mice with anaerobes cultured from IC/BPS stool resulted in exacerbation of pelvic allodynia. Together, these data indicate that AOAH is a host determinant of normal gut microbiota, and the dysbiosis associated with AOAH deficiency contributes to pelvic pain. These findings suggest that the gut microbiome is a potential therapeutic target for IC/BPS.


title: Diminished miRNA activity is associated with aberrant cytoplasmic intron retention in ALS pathogenesis

abstract: Intron retention (IR) is now recognized as a dominant splicing event during motor neuron (MN) development, however the role and regulation of intron-retaining transcripts (IRTs) localized to the cytoplasm remain particularly understudied. By resolving the spatiotemporal dynamics of IR underlying distinct stages of MN lineage restriction, we identify a cytoplasmic group of IRTs that is not associated with reduced expression of their own genes but instead with an upregulation of predicted target genes of specific miRNAs, the motifs of which are enriched within the intronic sequences of this group. Next, we show that ALS-causing VCP mutations lead to a selective increase in IR of this particular class of introns. This in turn temporally coincides with an increase in the expression level of predicted target genes of these miRNAs, providing a potential mechanistic insight into ALS pathogenesis. Altogether, we propose a novel role for the cytoplasmic intronic sequences in regulating miRNA activity through miRNA sequestration, which potentially contributes to ALS pathogenesis.


title: Interneuron activity-structural plasticity association is driven by context-dependent sensory experience

abstract: Neuronal dendritic spine dynamics provide a plasticity mechanism for altering brain circuit connectivity to integrate new information for learning and memory. Previous in vivo studies in the olfactory bulb (OB) showed that regional increases in activity caused localized spine stability, at a population level, yet how activity affects spine dynamics at an individual neuron level remains unknown. In this study, we tracked in vivo the correlation between an individual neurons activity and its dendritic spine dynamics of OB granule cell (GC) interneurons. Odor experience caused a consistent correlation between individual GC activity and spine stability. Dissecting the components of the OB circuit showed that increased principal cell (MC) activity was sufficient to drive this correlation, whereas cell-autonomously driven GC activity had no effect. A mathematical model was able to replicate the GC activity-spine stability correlation and showed MC output having improved odor discriminability while retaining odor memory. These results reveal that GC spine plasticity provides a sufficient network mechanism to decorrelate odors and maintain a memory trace.


title: Response time modelling reveals evidence for multiple, distinct sources of moral decision caution

abstract: People are often cautious in delivering moral judgments of others behaviours, as falsely accusing others of wrongdoing can be costly for social relationships. Caution might further be present when making judgements in information-dynamic environments, as contextual updates can change our minds. This study investigated the processes with which moral valence and context expectancy drive caution in moral judgements. Across two experiments, participants (N = 122) made moral judgements of others sharing actions. Prior to judging, participants were informed whether contextual information regarding the deservingness of the recipient would follow. We found that participants slowed their moral judgements when judging negatively valenced actions and when expecting contextual updates. Using a diffusion decision model framework, these changes were explained by shifts in drift rate and decision bias (valence) and boundary setting (context), respectively. These findings demonstrate how moral decision caution can be decomposed into distinct aspects of the unfolding decision process.


title: IL-6 blockade suppresses the blood-brain barrier disorder, leading to prevention of onset of NMOSD

abstract:
Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune astrocytopathy caused by antibodies against the aquaporin 4(AQP4) in end-feet of astrocytes. Breakdown of the blood-brain barrier (BBB) allowing ingress of AQP4 antibodies into the central nervous system (CNS) plays a key role in NMOSD. Although IL-6 blockade therapies such as satralizumab are effective in NMOSD, the therapeutic mechanism of IL-6 blockade, especially with respect to BBB disruption, are not fully understood because of the lack of the human models that are specialized to evaluate the BBB function.

We constructed new in vitro human BBB models for evaluating continued barrier function, leukocyte transmigration and intracerebral transferability of IgGs utilizing the newly established triple co-culture system. In vitro and vivo experiments revealed that NMO-IgG increased intracerebral transferability of satralizumab, and that satralizumab suppressed the NMO-IgG-induced transmigration of T cells and barrier dysfunction. These results suggest that satralizumab, which can pass through the BBB in the presence of NMO-IgG, suppresses the barrier dysfunction and the disrupting controlled cellular infiltration at the BBB, leading to prevention of onset of NMOSD.

One sentence summarySatralizumab and IL-6 blockade prevent lymphocyte migration and barrier dysfunction induced by NMO-IgG in EAE and novel triple co-culture BBB models.


title: Unbiased interrogation of memory B cells from convalescent COVID-19 patients reveals a broad antiviral humoral response targeting SARS-CoV-2 antigens beyond the spike protein

abstract: Patients who recover from SARS-CoV-2 infections produce antibodies and antigen-specific T cells against multiple viral proteins. Here, an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent patients has been performed by generating large, stable hybridoma libraries and screening thousands of monoclonal antibodies to identify specific, high-affinity immunoglobulins (Igs) directed at distinct viral components. As expected, a significant number of antibodies were directed at the Spike (S) protein, a majority of which recognized the full-length protein. These full-length Spike specific antibodies included a group of somatically hypermutated IgMs. Further, all but one of the six COVID-19 convalescent patients produced class-switched antibodies to a soluble form of the receptor-binding domain (RBD) of S protein. Functional properties of anti-Spike antibodies were confirmed in a pseudovirus neutralization assay. Importantly, more than half of all of the antibodies generated were directed at non-S viral proteins, including structural nucleocapsid (N) and membrane (M) proteins, as well as auxiliary open reading frame-encoded (ORF) proteins. The antibodies were generally characterized as having variable levels of somatic hypermutations (SHM) in all Ig classes and sub-types, and a diversity of VL and VH gene usage. These findings demonstrated that an unbiased, function-based approach towards interrogating the COVID-19 patient memory B cell response may have distinct advantages relative to genomics-based approaches when identifying highly effective anti-viral antibodies directed at SARS-CoV-2.


title: D614G Substitution of SARS-CoV-2 Spike Protein Increases Syncytium Formation and Viral Transmission via Enhanced Furin-mediated Spike Cleavage

abstract: Since the D614G substitution in the spike (S) of SARS-CoV-2 emerged, the variant strain underwent rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage of viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614. Both the virus titer and syncytial phenotype were significantly increased in S-G614 than in S-D614 isolates. We further showed increased cleavage of S at the furin substrate site, a key event that promotes syncytium, in S-G614 isolates. These functions of the D614G substitution were validated in cells expressing S protein. The effect on syncytium was abolished by furin inhibitor treatment and mutation of the furin-cleavage site, suggesting its dependence on cleavage by furin. Our study provides a mechanistic explanation for the increased transmissibility of S-G614 containing SARS-CoV-2 through enhanced furin-mediated S cleavage, which increases membrane fusion and virus infectivity.

category: microbiology


title: Early therapy with remdesivir and antibody combinations improves COVID-19 disease in mice

abstract: Improving the standard of clinical care for individuals infected with SARS-CoV-2 variants is a global health priority. Small molecule antivirals like remdesivir (RDV) and biologics such as human monoclonal antibodies (mAb) have demonstrated therapeutic efficacy against SARS-CoV-2, the causative agent of COVID-19. However, it is not known if combination RDV/mAb will improve outcomes over single agent therapies or whether antibody therapies will remain efficacious against variants. In kinetic studies in a mouse-adapted model of ancestral SARS-CoV-2 pathogenesis, we show that a combination of two mAbs in clinical trials, C144 and C135, have potent antiviral effects against even when initiated 48 hours after infection. The same antibody combination was also effective in prevention and therapy against the B.1.351 variant of concern (VOC). Combining RDV and antibodies provided a modest improvement in outcomes compared to single agents. These data support the continued use of RDV to treat SARS-CoV-2 infections and support the continued clinical development of the C144 and C135 antibody combination to treat patients infected with SARS-CoV-2 variants.


title: Prevention and therapy of SARS-CoV-2 and the B.1.351 variant in mice


title: Phage Cocktails can Prevent the Evolution of Phage-Resistant Enterococcus

abstract:
Antibiotic resistant Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy could be an alternative to antibiotics and has shown promise in in vitro and in early clinical studies. Phage therapy is often deployed as a cocktail of phages, but there is little understanding of how to most effectively combine phages. Here we utilized a collection of 20 Enterococcus phages to test principles of phage cocktail design and determine the phenotypic effects of evolving phage resistance in Enterococcus isolates that were susceptible or resistant to antibiotics (e.g., Vancomycin Resistant Enterococcus (VRE)). We tested the ability of each phage to clear Enterococcus host cultures and prevent the emergence of phage resistant Enterococcus. We found that some phages which were ineffective individually were effective at clearing the bacterial culture when used in cocktails. To understand the dynamics within phage cocktails, we used qPCR to track which phages increased in abundance in each cocktail, and saw dynamics ranging from one dominant phage to even phage growth. Further, we isolated several phage-resistant mutants to test for altered Vancomycin sensitivity. We found that mutants tended to have no change or slightly increased resistance to Vancomycin. By demonstrating the efficacy of phage cocktails in suppressing growth of antibiotic susceptible and VRE clinical isolates when exposed to phages, this work will help to inform cocktail design for future phage therapy applications.

IMPORTANCEAntibiotic resistant Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy could be an alternative to antibiotics and has shown promise in in vitro and in early clinical studies. Phage therapy in the form of cocktails is often suggested, with similar goals as the combination therapy that has been successful in the treatment of HIV infection, but there is little understanding about how to combine phages most effectively. Here we utilized a collection of 20 Enterococcus phages to test whether several phage cocktails could prevent the host from evolving resistance to therapy and to determine whether evolving resistance to phages affected host susceptibility to antibiotics. We showed that cocktails of two or three unrelated phages often prevented the growth of phage-resistant mutants, when the same phages applied individually were not able to.


title: Pipeline for generating stable large genomic deletions in zebrafish, from small domains to whole gene excisions

abstract: Here we describe a short feasibility study and methodological framework for the production of stable, CRISPR/Cas9-based, large genomic deletions in zebrafish, ranging from several base pairs (bp) to hundreds of kilobases (kb). Using a cocktail of four sgRNAs targeting a single genomic region mixed with a marker-sgRNA against the pigmentation gene tyrosinase (tyr), we demonstrate that one can easily and accurately excise genomic regions such as promoters, protein domains, specific exons or whole genes. We exemplify this technique with a complex gene family, neurexins, composed of three duplicated genes with multiple promoters and intricate splicing processes leading to thousands of isoforms. We precisely deleted small regions such as their transmembrane domains (150bp deletion in average) to their entire genomic locus (300kb deletion for nrxn1A for instance). We find that both the concentration and ratio of Cas9/sgRNAs are critical for the successful generation of these large deletions and, interestingly, that their transmission frequency does not decrease with increasing distance between sgRNA target sites. Considering the growing reports and debate about genetically compensated small indel mutants, the use of large-deletion approaches is likely to be widely adopted in studies of gene function. This strategy will also be key to the study of non-coding genomic regions.


title: Modeling mutational effects on biochemical phenotypes using convolutional neural networks: application to SARS-CoV-2

abstract: Biochemical phenotypes are major indexes for protein structure and function characterization. They are determined, at least in part, by the intrinsic physicochemical properties of amino acids and may be reflected in the protein three-dimensional structure. Modeling mutational effects on biochemical phenotypes is a critical step for understanding protein function and disease mechanism as well as enabling drug discovery. Deep Mutational Scanning (DMS) experiments have been performed on SARS-CoV-2s spike receptor binding domain and the human ACE2 zinc-binding peptidase domain - both central players in viral infection and evolution and antibody evasion - quantifying how mutations impact binding affinity and protein expression. Here, we modeled biochemical phenotypes from massively parallel assays, using convolutional neural networks trained on protein sequence mutations in the virus and human host. We found that neural networks are significantly predictive of binding affinity, protein expression, and antibody escape, learning complex interactions and higher-order features that are difficult to capture with conventional methods from structural biology. Integrating the intrinsic physicochemical properties of amino acids, including hydrophobicity, solvent-accessible surface area, and long-range non-bonded energy per atom, significantly improved prediction (empirical p<0.01) though there was such a strong dependence on the sequence data alone to yield reasonably good prediction. We observed concordance of the DMS data and our neural network predictions with an independent study on intermolecular interactions from molecular dynamics (multiple 500 ns or 1 s all-atom) simulations of the spike protein-ACE2 interface, with critical implications for the use of deep learning to dissect molecular mechanisms. The mutation- or genetically-determined component of a biochemical phenotype estimated from the neural networks has improved causal inference properties relative to the original phenotype and can facilitate crucial insights into disease pathophysiology and therapeutic design.


title: AnnoMiner: a new web-tool to integrate epigenetics, transcription factor occupancy, and transcriptomics data to predict transcriptional regulators

abstract: Gene expression regulation requires precise transcriptional programs, led by transcription factors in combination with epigenetic events. Recent advances in epigenomic and transcriptomic techniques provided insight into different gene regulation mechanisms. However, to date it remains challenging to understand how combinations of transcription factors together with epigenetic events control cell-type specific gene expression. We developed the AnnoMiner web-server and introduce an innovative and flexible way to annotate and integrate epigenetic, and transcription factor occupancy data. First, AnnoMiner annotates user-provided peaks with gene features. Second, AnnoMiner can integrate genome binding data from two different transcriptional regulators together with gene features. Third, AnnoMiner offers to explore the transcriptional deregulation of 10 genes nearby a user-provided peak. AnnoMiners fourth function performs transcription factor or histone mark enrichment analysis for user-provided gene lists by utilizing hundreds of public, high-quality datasets from ENCODE for the model organisms human, mouse, Drosophila and C. elegans. Thus, AnnoMiner can predict transcriptional regulators for a studied process without the strict need for chromatin data from the same process. We compared AnnoMiner to existing tools and experimentally validated several transcriptional regulators predicted by AnnoMiner to indeed contribute to muscle morphogenesis in Drosophila. AnnoMiner is freely available at http://chimborazo.ibdm.univ-mrs.fr/AnnoMiner/.


title: N-terminally truncated helper NLR NRG1C antagonizes immunity mediated by its full-length neighbors NRG1A and NRG1B

abstract: Both animals and plants utilize nucleotide-binding leucine-rich repeat immune receptors (NLRs) to perceive the presence of pathogen-derived molecules and induce immune responses. NLR genes are far more abundant and diverse in higher plants. Interestingly, truncated NLRs, which lack one or more of the canonical domains, are also commonly encoded in plant genomes. However, little is known about their functions, especially regarding the N-terminally truncated ones. Here, we show that Arabidopsis thaliana (A. thaliana) N-terminally truncated helper NLR gene NRG1C (N REQUIREMENT GENE 1) is highly induced upon pathogen infection and in autoimmune mutants. The immune response and cell death conferred by some TIR (Toll/interleukin-1 receptor)-type NLRs (TNLs) are compromised in the NRG1C overexpression lines. Detailed genetic analysis revealed that NRG1C antagonizes the immunity mediated by its full-length neighbors NRG1A and NRG1B. Biochemical tests indicate that NRG1C possibly interferes with the EDS1-SAG101 complex, which likely signals together with NRG1A/1B. Interestingly, Brassicaceae NRG1Cs are functionally exchangeable, and the Nicotiana benthamiana (N. benthamiana) N-terminally truncated helper NLR NRG2 antagonizes NRG1 in tobacco. Together, our study uncovers an unexpected negative role of N-terminally truncated helper NLRs in different plants.


title: Identification of Gip as a novel phage-encoded gyrase inhibitor protein featuring a broad activity profile

abstract: Bacteriophages represent a powerful source for the identification of novel antimicrobial proteins. In this study, a screening of small cytoplasmic proteins encoded by the CGP3 prophage of Corynebacterium glutamicum, resulted in the identification of the novel gyrase-inhibiting protein Cg1978 (Gip), which shows a direct interaction with the gyrase subunit A (GyrA). In vitro supercoiling assays further suggest a stabilization of the cleavage complex by Gip. Overproduction of Gip in C. glutamicum resulted in a severe growth defect as well as an induction of the SOS response. The cells adapted to gip overexpression by increasing expression levels of gyrAB and by reducing topA expression reflecting the homeostatic control of DNA topology. Interestingly, Gip features a similar activity profile towards gyrases of Escherichia coli, Mycobacterium tuberculosis and C. glutamicum. Therefore, the detailed elucidation of the mechanism of Gip action may provide novel directions for the design of drugs targeting DNA gyrase.


title: In-frame deletion of SPECC1L microtubule binding domain results in embryonic tissue movement and fusion defects

abstract: Embryonic morphogenesis of the neural tube, palate, ventral body wall and optic fissure require precise sequence of tissue movement and fusion, which if incomplete, leads to anencephaly/exencephaly, cleft palate, omphalocele and coloboma, respectively. These are genetically heterogeneous birth defects, so there is a continued need to identify etiologic genes. Patients with autosomal dominant SPECC1L mutations show syndromic malformations, including hypertelorism, cleft palate and omphalocele. These SPECC1L mutations cluster in the second coiled-coil domain (CCD2), which facilitates association with microtubules. To study SPECC1L function in mice, we first generated a null allele (Specc1l{Delta}Ex4) lacking the entire SPECC1L protein. Homozygous mutants for these truncations died perinatally without cleft palate or exencephaly. Given the clustering of human mutations in CCD2, we hypothesized that targeted perturbation of CCD2 may be required. Indeed, homozygotes for in-frame deletions involving CCD2 (Specc1l{Delta}CCD2) resulted in ~50% exencephaly and ~50% cleft palate. Interestingly, these two phenotypes are never observed in the same embryo. Examination of embryos with and without exencephaly revealed that the oral cavity was narrower in exencephalic embryos, which allowed palatal shelves to elevate despite their defect. In contrast to an evenly distributed subcellular expression pattern, mutant SPECC1L-{Delta}CCD2 protein showed abnormal subcellular localization, decreased overlap with microtubules, increased actin bundles, and dislocated non-muscle myosin II to the cell cortex. Thus, we show that perturbations of CCD2 in the context of full SPECC1L protein affects tissue fusion dynamics, indicating that human SPECC1L CCD2 mutations are gain-of-function. Improper SPECC1L subcellular localization appears to disrupt connections between actomyosin and microtubule networks, which in turn may affect cell alignment and coordinate movement during tissue morphogenesis.

category: developmental biology


title: Vasopressin V2 is a promiscuous G protein-coupled receptor that is biased by its peptide ligands

abstract: Activation of the G protein-coupled receptors by agonists may result in the activation of one or more G proteins, and in the recruitment of arrestins. The balance of activation of different pathways can be influenced by the ligand. Using BRET-based biosensors, we showed that the vasopressin V2 receptor activates or at least engages many different G proteins across all G protein subfamilies in response to its native agonist arginine vasopressin (AVP). This includes members of the Gi/o and G12/13 families that have not been previously reported. These signalling pathways are also activated by the synthetic peptide desmopressin and natural homologs of AVP, namely oxytocin and the non-mammalian hormone vasotocin. They demonstrated varying degrees of functional selectivity relative to AVP, as quantified using the operational model for quantifying ligand bias. Additionally, we modelled G protein activation as a Michaelis-Menten reaction. This approach provided a complementary way to quantify signalling bias, with an added benefit of clear separation of the effects of ligand affinity from the intrinsic activity of the receptor. These results showed that V2 receptor is not only promiscuous in its ability to engage several G proteins, but also that its signalling profile could be easily biased by small structural changes in the ligand.

category: pharmacology and toxicology


title: Emergence of a smooth interface from growth of a dendritic network against a mechanosensitive contractile material

abstract: Structures and machines require smoothening of raw materials. Self-organized smoothening guides cell and tissue morphogenesis, and is relevant to advanced manufacturing. Across the syncytial Drosophila embryo surface, smooth interfaces form between expanding Arp2/3-based actin caps and surrounding actomyosin networks, demarcating the circumferences of nascent dome-like compartments used for pseudo-cleavage. We found that smoothening of the actomyosin interfaces requires Arp2/3 in vivo. To dissect the physical basis of this requirement, we reconstituted the interacting networks using node-based models. When actomyosin networks were simulated with clearances instead of Arp2/3 networks, rough boundaries persisted with low levels of myosin contractility. With addition of expanding Arp2/3 networks, network-network interfaces failed to smoothen, but accumulated myosin nodes and tension. After incorporating actomyosin mechanosensitivity, Arp2/3 network growth induced local contractility and smoothening of the interfaces, effects also evident in vivo. In this way, a smooth structure can emerge from the lateral interaction of irregular starting materials.