title: A two-level, dynamic fitness landscape of hepatitis C virus revealed by self-organized haplotype maps

abstract:
Fitness landscapes reflect the adaptive potential of viruses. There is no information on how fitness peaks evolve when a virus replicates extensively in a controlled cell culture environment. Here we report the construction of Self-Organized Maps (SOMs), based on deep sequencing reads of three amplicons of the NS5A-NS5B-coding region of hepatitis C virus (HCV). A two-dimensional neural network was constructed and organized according to sequence relatedness. The third dimension of the fitness profile was given by the haplotype frequencies at each neuron. Fitness maps were derived for 44 HCV populations that share a common ancestor that was passaged up to 210 times in human hepatoma Huh-7.5 cells. As the virus increased its adaptation to the cells, the number of fitness peaks expanded, and their distribution shifted in sequence space. The landscape consisted of an extended basal platform, and a lower number of protruding higher fitness peaks. The function that relates fitness level and peak abundance corresponds a power law, a relationship observed with other complex natural phenomena. The dense basal platform may serve as spring-board to attain high fitness peaks. The study documents a highly dynamic, double-layer fitness landscape of HCV when evolving in a monotonous cell culture environment. This information may help interpreting HCV fitness landscapes in complex in vivo environments.

IMPORTANCEThe study provides for the first time the fitness landscape of a virus in the course of its adaptation to a cell culture environment, in absence of external selective constraints. The deep sequencing-based self-organized maps document a two-layer fitness distribution with an ample basal platform, and a lower number of protruding, high fitness peaks. This landscape structure offers potential benefits for virus resilience to mutational inputs.


title: Detection of porcine enteric viruses (Kobuvirus, Mamastrovirus and Sapelovirus) in domestic pigs in Corsica, France

abstract:
Many enteric viruses are found in pig farms around the world and can cause death of animals or important production losses for breeders. Among the wide spectrum of enteric viral species, porcine Sapelovirus (PSV), porcine Kobuvirus (PKoV) and porcine Astrovirus (PAstV) are frequently found in pig feces. In this study we investigated sixteen pig farms in Corsica, France, to evaluate the circulation of three enteric viruses (PKoV, PAstV-1 and PSV). In addition to the three viruses studied by RT-qPCR (908 pig feces samples), 26 stool samples were tested using the Next Generation Sequencing method (NGS). Our results showed viral RNA detection rates (i) of 62.0% [58.7-65.1] (n = 563/908) for PSV, (ii) of 44.8% [41.5-48.1] (n = 407/908) for PKoV and (iii) of 8.6% [6.8-10.6] (n = 78/908) for PAstV-1. Significant differences were observed for all three viruses according to age (P-value = 2.4e-13 for PAstV-1; 2.4e-12 for PKoV and 0.005 for PSV). The type of breeding was significantly associated with RNA detection only for PAstV-1 (P-value = 9.6e-6). Among the 26 samples tested with NGS method, consensus sequences corresponding to 10 different species of virus were obtained This study provides first insight on the presence of three common porcine enteric viruses in France. We also showed that they are frequently encountered in pigs born and bred in Corsica, which demonstrates endemic local circulation.

ImportanceThis study provides important information in the comprehension of the epidemiology of different viruses circulating in swine farms. We have shown the great diversity of viruses that could be present in extensive farms. Moreover, to our knowledge, this is the first detection of these different viruses in France. So far, this study has to be considered as a first step in the study of enteric viruses in Corsican pig farms.

category: molecular biology


title: Translation of cellular protein localization by generative adversarial network

abstract: The protein localization in cells had been analyzed by the fluorescent labeling by indirect immunofluorescence and fluorescent protein tagging. However, the relationships between the localizations between different proteins had not been analyzed by artificial intelligence. In this study, we applied the generative adversarial network (GAN) to generate the protein localizations each other, in which the generation was dependent on the types of cells and the relationships between the proteins. Lamellipodia are one of the actin-dependent subcellular structures involved in cell migration and are mainly generated by the Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 2 (WAVE2) and the membrane remodeling I-BAR domain protein IRSp53. Focal adhesions are another actin-based structure that contains vinculin protein and are essential for cell migration. In contrast, microtubules are not thought to be directly related to actin filaments. The GAN was trained using images of actin filaments paired with WAVE2, vinculin, IRSp53, and microtubules. Then, the generated images of WAVE2, vinculin, and IRSp53 by the GAN showed high similarity to the real images of WAVE2, vinculin, and IRSp53, respectively. However, the microtubule images generated from actin filament images were inferior, corroborating that the microscopic images of actin filaments provide more information about actin-related protein localization. Collectively, this study suggests that the image translation by the GAN can predict the localization of functionally related proteins.

category: cell biology


title: An N-terminal Fusion Allele to Study Melanin Concentrating Hormone Receptor 1

abstract: Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (mCherryMCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs.

category: neuroscience


title: A Panel of Diverse Pseudomonas aeruginosa Clinical Isolates for Research and Development

abstract:
Pseudomonas aeruginosa is a leading cause of community-acquired and hospital-acquired infections. Successful treatment is hampered by its remarkable ability to rapidly develop resistance to antimicrobial agents, mostly through mutation. In response, the World Health Organization listed carbapenem-resistant P. aeruginosa as a Priority 1 (Critical) pathogen for research and development of new treatments. A key resource in developing effective countermeasures is access to diverse and clinically relevant strains for testing. Herein we describe a panel of 100 diverse P. aeruginosa strains to support this endeavor.

Whole genome sequencing was performed on 3,785 P. aeruginosa housed in our repository. Isolates were cultured from clinical samples collected from healthcare facilities around the world between 2003 and 2017. Core-genome multi-locus sequence typing and high-resolution SNP-based phylogenetic analyses were used to select a panel of 100 strains that captured the genetic diversity of this collection. Comprehensive antibiotic susceptibility testing was also performed using 14 clinically relevant antibiotics.

This 100-strain diversity panel contained representative strains from 91 different sequence-types, including genetically distinct isolates from major epidemic clones ST-111, ST-235, ST-244, and ST-253. Seventy-one distinct antibiotic susceptibility profiles were identified ranging from pan-sensitive to pan-resistant. Known resistance alleles as well as the most prevalent mutations underlying the antibiotic susceptibilities were characterized for all isolates.

This panel provides a diverse and comprehensive set of P. aeruginosa strains for use in developing solutions to antibiotic resistance. The isolates, and all available meta-data including genome sequences, are available to industry, academic institutions, federal and other laboratories at no additional cost.

ImportancePseudomonas aeruginosa is one of the most important human pathogens and a leading target in the development of new drugs and therapeutics. This species displays a remarkable level of diversity and any potential therapeutic must contend with this characteristic to ensure it retains efficacy across different strains. To date, only limited panels of P. aeruginosa are available for testing, and none have been designed to capture the genetic diversity of the species. The panel described herein has been designed to address this shortcoming by providing a set of 100 distinct strains that greatly captures the diversity of the species. This panel will be of significant value to research groups working on this important pathogen, both for research purposes and in the development of new diagnostics and countermeasures.

category: microbiology


title: Sex-dependent effects of CYP46A1 overexpression on cognitive function during aging

abstract: Cholesterol turnover and CYP46A1 regulation are reported to be crucial for memory functions. An increasing body of evidence shows that CYP46A1 activation is able to reduce Alzheimers Disease (AD) pathological processes. In this study we report for the first time that CYP46A1 overexpression and increase of 24S-hydroxycholesterol (24OH) induces sex-specific changes in synaptic functions in aged mice, being beneficial in females while detrimental in males. The positive effects on cognition in aged CYP46A1 overexpressing female mice were accompanied by morphological changes in dendritic spines and enhancement of estrogen receptor signaling in hippocampus. In aged males, CYP46A1 overexpression leads to anxiety-like behavior and worsening of spatial memory, followed by decreased dendritic spine density and higher 5-dihydrotestosterone (DHT) levels in hippocampus. Further, analysis of cerebrospinal fluid (CSF) from AD, mild cognitive impairment and healthy patients revealed that 24OH was negatively associated to markers of neurodegeneration in women but not in men. Based on our results, CYP46A1 activation may represent a pharmacological target that could specifically enhance brain estrogen receptor signaling in women at risk of developing AD. Finally, this study highlights the importance of taking into account the sex-dimension in both preclinical and clinical studies of neurodegenerative diseases like AD.

category: animal behavior and cognition


title: Structural origins of altered spectroscopic properties upon ligand binding in proteins containing a fluorescent non-canonical amino acid.

abstract: Fluorescent non-canonical amino acids (fNCAAs) could serve as starting points for the rational design of protein-based fluorescent sensors of biological activity. However, efforts toward this goal are likely hampered by a lack of atomic-level characterization of fNCAAs within proteins. Here, we describe the spectroscopic and structural characterization of five streptavidin mutants that contain the fNCAA L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) at sites proximal to the binding site of its substrate, biotin. Many of the mutants exhibited altered fluorescence spectra in response to biotin binding, which included both increases and decreases in fluorescence intensity as well as red or blue shifted emission maxima. Structural data were also obtained for three of the five mutants. The crystal structures shed light on interactions between 7-HCAA and functional groups--contributed either by the protein or substrate--that may be responsible for the observed changes in the 7-HCAA spectra. These data could be used in future studies aimed at the rational design of fluorescent, protein-based sensors of small molecule binding or dissociation.

category: biochemistry


title: The Host Cell's Endoplasmic Reticulum Proteostasis Network Profoundly Shapes the Protein Sequence Space Accessible to HIV Envelope

abstract: The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, however, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody and drug escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s activation. This phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is activated, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.


title: Membrane surfaces regulate assembly of a ribonucleoprotein condensate

abstract:
Biomolecular condensates organize biochemistry in time and space, yet little is known about how cells control either the position or scale of these assemblies. In cells, condensates often appear as dispersed, relatively small assemblies that do not grow (coarsen) into a single droplet despite their propensity to coalesce. Here we report that ribonucleoprotein condensates of the Q-rich protein Whi3 interact with the endoplasmic reticulum, prompting us to hypothesize that membrane association controls the position and size of condensates. Reconstitution of Whi3 condensates on supported lipid bilayers reveals that association with a diffusive lipid surface promotes condensation at both physiological ionic strength and protein concentration. Notably, these assemblies rapidly arrest, matching size distributions seen in cells. The timing of the arrest is influenced by the ordering of protein-protein and protein-RNA interactions and controlled by the slow diffusion of complexes induced by the membrane. This slowed diffusion limits both transfer of small protein-RNA complexes between condensates and their coalescence, thus driving coarsening to arrest. Our experiments reveal a tradeoff between locally-enhanced protein concentration at membranes, which favors condensation, and an accompanying reduction in diffusion, which restricts coarsening. Thus, membranes can maintain a population of small condensates in the absence of active mechanisms. Given that many condensates are bound to endomembranes, we predict that the biophysical properties of lipid bilayers are key for controlling condensate sizes throughout the cell.

One sentence summaryAssembly on a membrane surface positions and scales biomolecular condensates by controlling relative diffusion rates of proteins and nucleic acids.

category: biophysics


title: Characterization of α2-Adrenergic Receptors Mediated Vasoconstriction in ex vivo model

abstract:
Introductionrat tail serves as a thermoregulatory organ by dilating or constricting tail blood vessels and rat tail lateral veins play a role in cutaneous circulation. However, this cutaneous vessel has never been examined and evaluated as a potential candidate ex vivo model to study peripheral vascular diseases. This study aims to investigate vascular tone mediated through 2-adenergic receptor on rat tail vein under different temperatures.

MethodsVasoconstriction stimulated by 2-adrenergic receptor selective agonists UK14304, guanabenz was thoroughly examined in isolated rat tail lateral vein. Susceptibility of cutaneous vessel to temperature changes was investigated under 37 {degrees}C and 28 {degrees}C. Differentiated vascular reactivity exhibited at different temperature was further explored with different 2-adrenergic receptor selective agonists and validated with antagonism by 1-adrenergic receptor and 2-adrenergic receptor selective antagonist prazosin and RX821002, respectively.

ResultsVascular tone of freshly isolated vein remained same compared with that of stored in pre-gassed physiologic buffer at 4 {degrees}C overnight. Presence of endothelium and repeated administration of UK14304 did not alter contractile property. Vessels away from torso (distal) showed significantly different contractile character compared with portion close to torso (proximal) at 28 {degrees}C but kept uniform at 37 {degrees}C. Enhanced vasoconstriction along with increased potency of 2-adrenergic receptor agonists UK14304 and guanabenz was consistently present at 28 {degrees}C, independent of temperature change orders. Potentiated vasoconstriction present at 28 {degrees}C was later proved via 2-adrenergic receptor alone.

DiscussionHarvest and preparation of rat tail lateral vein was described in details. Contractile property of venous preparations stimulated by 2-adrenergic receptor selective agonists was first time examined. Differentiated vasoconstriction at moderate cooling temperature was described and confirmed to through 2-adrenergic receptor activation. Rat tail lateral vein is found valuable for studies of cutaneous circulation altered by temperature changes.

category: pharmacology and toxicology


title: Developmental and Intergenerational Landscape of Human Circulatory Lipidome and its Association with Obesity Risk

abstract: Lipids play a vital role in human health and development, but changes to their circulatory levels during gestation and in early life are poorly understood. Here we present the first developmental and intergenerational landscape of the human circulatory lipidome, derived by profiling of 480 lipid species representing 25 lipid classes, in mothers and their offspring (n=2491). Levels of 66% of the profiled lipids increased in maternal circulation during gestation, while cord blood had higher concentrations of acylcarnitines and lysophospholipids. The offspring lipidome at age six years revealed striking similarities with postnatal maternal lipidome (adult) in its lipid composition and concentrations. Comparison of lipids associated with child and maternal adiposity identified a 92% overlap, implying intergenerational similarities in the lipid signatures of obesity risk. We also catalogued lipid signatures linked with maternal adiposity during gestation and offspring birthweight, and validated (>70% overlap) the findings in an independent birth-cohort (n=1935).

category: systems biology


title: Neural circuits for dynamics-based segmentation of time series

abstract: The brain must extract behaviorally relevant latent variables from the signals streamed by the sensory organs. Such latent variables are often encoded in the dynamics that generated the signal rather than in the specific realization of the waveform. Therefore, one problem faced by the brain is to segment time series based on underlying dynamics. We present two algorithms for performing this segmentation task that are biologically plausible, which we define as acting in a streaming setting and all learning rules being local. One algorithm is model-based and can be derived from an optimization problem involving a mixture of autoregressive processes. This algorithm relies on feedback in the form of a prediction error, and can also be used for forecasting future samples. In some brain regions, such as the retina, the feedback connections necessary to use the prediction error for learning are absent. For this case, we propose a second, model-free algorithm that uses a running estimate of the autocorrelation structure of the signal to perform the segmentation. We show that both algorithms do well when tasked with segmenting signals drawn from autoregressive models with piecewise-constant parameters. In particular, the segmentation accuracy is similar to that obtained from oracle-like methods in which the ground-truth parameters of the autoregressive models are known. We also test our methods on datasets generated by alternating snippets of voice recordings. We provide implementations of our algorithms at https://github.com/ttesileanu/bio-time-series.


title: Functional independence of endogenous mu- and delta-opioid receptors co-expressed in cholinergic interneurons

abstract: Class A G protein-coupled receptors (GPCRs) normally function as monomers, although evidence from heterologous expression systems suggests they may form homodimers and/or heterodimers. Detection of GPCR dimers in native tissues has however been challenging due to the lack of suitable tools. - and {delta}-Opioid receptors (MORs and DORs) co-expressed in transfected cells has been reported to form heterodimers. The co-localization of MORs and DORs in neurons has been studied in knock-in mice expressing genetically engineered receptors fused to fluorescent proteins. Here we report that single cholinergic neurons in the mouse striatum endogenously express both MORs and DORs. The receptors were fluorescently labeled in live brain slices with a ligand-directed labeling reagent, NAI-A594. The selective activation of MORs and DORs, with DAMGO (-agonist) and deltorphin ({delta}-agonist) inhibited spontaneous firing in all cells examined. In the continued presence of agonist, the firing rate returned to baseline with the application of deltorphin but was persistently inhibited with the application of DAMGO. In addition, agonist-induced internalization of DORs but not MORs was detected. When MORs and DORs were activated simultaneously with [Met5]enkephalin, desensitization of MORs was facilitated but internalization was not increased. Together, these results indicate that while MORs and DORs are expressed in single striatal cholinergic interneurons, the two receptors function independently.


title: Sex-specific blood-brain barrier alterations and vascular biomarkers underlie chronic stress responses in mice and human depression.

abstract: Prevalence, symptoms, and treatment of depression all point toward major sex differences. Social stress-induced neurovascular pathology is associated with depressive symptoms in male mice however it remains unknown if it contributes to this sexual dimorphism. Here, we report that chronic social and subchronic variable stress promoted sex-specific blood-brain barrier (BBB) molecular and morphological alterations in mood-related brain regions. Viral-mediated functional manipulation leading to a targeted disruption of the BBB induced anxiety- and depression-like behaviors including social avoidance and anhedonia. Endothelium cell-specific transcriptomic profiling revealed key pathways and novel genes involved in maladaptive stress responses vs resilience. We also confirmed BBB leakiness in the brain of stressed females which led us to explore and identify circulating vascular biomarkers of chronic stress that could inform on diagnosis and treatment. Importantly, these pre-clinical findings were validated in human blood and postmortem brain samples from depressed women, thus highlighting their translational value. By revealing a sex-specific causal role of BBB dysfunction in stress responses and depression, our results implicate vascular impairment as a major factor underlying mood disorders.


title: Convergent, functionally independent signaling by mu and delta opioid receptors in hippocampal parvalbumin interneurons

abstract: Functional interactions between G protein-coupled receptors are poised to enhance neuronal sensitivity to neuromodulators and therapeutic drugs. Mu and Delta opioid receptors (MORs and DORs) can interact when overexpressed in the same cells, but whether co-expression of endogenous MORs and DORs in neurons leads to functional interactions is unclear. Here, we show that both MORs and DORs inhibit parvalbumin-expressing basket cells (PV-BCs) in hippocampal CA1 through partially occlusive signaling pathways that terminate on somato-dendritic potassium channels and presynaptic calcium channels. Using photoactivatable opioid neuropeptides, we find that DORs dominate the response to enkephalin in terms of both ligand-sensitivity and kinetics, which may be due to relatively low expression levels of MOR. Opioid-activated potassium channels do not show heterologous desensitization, indicating that MORs and DORs signal independently. In a direct test for heteromeric functional interactions, the DOR antagonist TIPP-Psi does not alter the kinetics or potency of either the potassium channel or synaptic responses to photorelease of the MOR agonist DAMGO. Thus, despite largely redundant and convergent signaling, MORs and DORs do not functionally interact in PV-BCs. These findings imply that crosstalk between MORs and DORs, either in the form of physical interactions or synergistic intracellular signaling, is not a preordained outcome of co-expression in neurons.


title: A ganglioside-based senescence-associated immune checkpoint

abstract: Senescent cells accumulate in aging tissues, and their elimination can favor healthy aging1-4. Therefore, therapeutic interventions targeting cellular senescence may be promising strategies for delaying or reversing a vast range of age-related diseases5. As cells of the immune system are responsible for senescent cell elimination6-11, a possible anti-aging and pro-healthspan treatment is the specific activation of the immune system to induce senescent cell clearance. However, whether this elimination is limited by an immune checkpoint leading to tolerance of senescence cells is currently unknown. Here, we show that cellular senescence, elicited by various stressors other than oncogenic activation, triggers immune escape toward natural killer (NK) cells, which may thus limit the use of anti-senescence immunotherapies. Moreover, using mass spectrometry, we reveal that senescent cells reshuffle their glycosphingosine composition, toward a marked increase in the ganglioside content, including the appearance of disialylated ganglioside GD3. This senescence associated GD3 overexpression results from transcriptional upregulation of the gene encoding the enzyme ST8SIA1, which is responsible for GD3 synthesis. The high level of GD3 leads to a strong immunosuppressive signal affecting NK cell-mediated immunosurveillance. In a mouse model of lung fibrosis, senescent cell-dependent NK cell immunosuppression is blunted by in vivo administration of anti-GD3 monoclonal antibodies leading to a clear anti-fibrotic effect. These results demonstrate that GD3 upregulation in senescent cells drives a switch from immune clearance toward immune tolerance of senescent cells. Therefore, we propose that GD3 level acts as a senescence-associated immune checkpoint (SIC) that regulates NK cell functions toward senescent cells. Thus, targeting GD3 with specific antibodies may be a promising strategy for the development of effective anti-senescence immunotherapies.

category: immunology


title: Functional degeneracy in translationally active Nitrospira populations confers resilience to out-selection in partial nitritation activated sludge

abstract:
Managing and engineering activated sludge wastewater treatment microbiomes for low-energy nitrogen removal requires process control strategies to stop the oxidation of ammonium at nitrite. Our ability to out-select nitrite oxidizing bacteria (NOB) from activated sludge is challenged by their metabolic and physiological diversity, warranting measurements of their in situ physiology and activity under selective growth pressures. Here, we examined the stability of nitrite oxidation in activated sludge during a press disturbance induced by treating a portion of return activated sludge with a sidestream flow containing free ammonia (FA) at 200 mg NH3-N/L. The nitrite accumulation ratio peaked at 42% by day 40 in the experimental bioreactor with the press disturbance, while it did not increase in the control bioreactor. A subsequent decrease in nitrite accumulation within the experimental bioreactor coincided with shifts in dominant Nitrospira 16S rRNA amplicon sequence variants (ASVs). We applied bioorthogonal non-canonical amino acid tagging (BONCAT) coupled with fluorescence activated cell sorting (FACS) to investigate changes in the translational activity of NOB populations throughout batch exposure to FA. BONCAT-FACS confirmed that the single Nitrospira ASV washed-out of the experimental bioreactor had reduced translational activity following exposure to FA, whereas the two Nitrospira ASVs that emerged after process acclimation were not impacted by FA. Thus, the coexistence of functionally degenerate and physiologically resistant Nitrospira populations provided resilience to the nitrite-oxidizing function during the press disturbance. These results highlight how BONCAT-FACS can resolve ecological niche differentiations within activated sludge and inform strategies to engineer and control microbiome function.

ImportanceNitrogen removal from activated sludge wastewater treatment systems is an energy-intensive process due to the large aeration requirement for nitrification. This energy footprint could be minimized with engineering control strategies that wash-out nitrite oxidizing bacteria (NOB) to limit oxygen demands. However, NOB populations can have a high degree of physiological diversity, and it is currently difficult to decipher the behavior of individual taxa during applied selective pressures. Here, we utilized a new substrate analog probing approach to measure the activity of NOB at the cellular translational level in the face of an applied press disturbance to the activated sludge process. Substrate analog probing corroborated the time-series reactor sampling, showing that coexisting and functionally redundant Nitrospira provided resilience to the nitrite oxidation process. Taken together, these results highlight how substrate analog approaches can illuminate in situ ecophysiologies within shared niches, and can inform strategies to improve microbiome engineering and management.


title: Activity-based cell sorting reveals resistance of functionally degenerate Nitrospira during a press disturbance in nitrifying activated sludge


title: Fluoroquinolone-Resistant Escherichia coli Originating on Dairy Farms are an Infrequent Cause of Bacteriuria in Humans Living in the Same Geographical Region

abstract:
SynopsisO_ST_ABSBackgroundC_ST_ABSOur primary aim was to test whether cattle-associated fluoroquinolone-resistant (FQ-R) Escherichia coli found on dairy farms were a significant cause of bacteriuria in humans living in the same 50 x 50 km geographical region located in South West England. Another aim was to identify risk factors for the presence of FQ-R E. coli on dairy farms.

MethodsFQ-R E. coli were isolated during 2017-18 from 42 dairy farms and from community urine samples. Forty-two cattle and 489 human urinary isolates were subjected to WGS, allowing phylogenetic comparisons. Risk factors were identified using a Bayesian regularisation approach.

ResultsOf 489 FQ-R human isolates, 255 were also 3rd generation cephalosporin-resistant (3GC-R), with strong genetic linkage between aac(6)Ib-cr and blaCTX-M-15. We identified possible farm-to-human sharing for pairs of ST744 and ST162 isolates, but core genome SNP distances (71 and 63, respectively) were smaller in pairs of ST744 and ST162 isolates from different farms (7 and 3 SNPs, respectively). Total farm fluoroquinolone use showed a positive association with the odds of isolating FQ-R E. coli while total dry cow therapy use showed a negative association.

ConclusionsThis work suggests that FQ-R E. coli found on dairy farms have a limited impact on community bacteriuria within the local human population, however, this appears greater than observed for 3GC-R E. coli when studied in parallel. Reducing fluoroquinolone use may reduce the on-farm prevalence of FQ-R E. coli, and this reduction may be greater when dry cow therapy is targeted to the ecology of resistant E. coli on the farm.


title: Limited Phylogenetic Overlap Between Fluoroquinolone-Resistant Escherichia coli Isolated on Dairy Farms and those Causing Bacteriuria in Humans Living in the Same Geographical Region


title: Diet Potentially Drives the Differentiation of Eating Behaviours via Alterations to the Gut Microbiome in Infants

abstract: Certain infant eating behaviours are associated with adverse health outcomes such as obesity. While a diet consisting of infant formula has been linked to higher-risk eating behaviours and changes in the gut microbiome, little is known about what role the gut microbiome plays in mediating eating behaviours. Using 16S rRNA sequences extracted from 96 fecal samples collected from 58 infants, we identified a subset of bacterial taxa that were more abundant in formula-fed infants, primarily composed of the phylum Firmicutes. The presence of these taxa correlated with a lower drive to eat (i.e., lower food responsiveness). Furthermore, short-chain fatty acid production pathways were significantly more abundant in formula-fed infants, negatively correlated with food responsiveness, and positively associated with relative abundance of the Firmicutes subset. Our results suggest that higher abundances of Firmicutes in formula-fed infants may decrease their food responsiveness through short-chain fatty acid production in the first four months of life. Taken together, these findings suggest a potential role for the infants diet in impacting eating behaviour via changes to the gut microbiome, which may lead to the development of novel interventions for the prevention of childhood obesity.


title: Transcriptional activation of Il36A by C/EBPβ via a half-CRE*C/EBP element in murine macrophages is independent of its CpG methylation level

abstract: Interleukin-36(Il-36) is a member of the novel Il-1-like proin-flammatory cytokine family that is highly expressed in epithelial tissues and several myeloid-derived cell types. We have recently shown that CCAAT enhancer binding protein{beta} (C/EBP{beta}) binds specifically to an essential half cAMP response element (half-CRE)*C/EBP motif in the Il36A promoter to induce Il36A expression upon LPS stimulation. C/EBPs are transcription factors belonging to the basic leucine zipper (bZIP) family of transcriptional regulators. C/EBP proteins can form homo- and heterodimers and regulate gene expression by binding to C/EBP specific recognition sequences and composite sites that can contain 5'-cytosine-phosphate-guanine-3'dinucleotides (CpG). CpG methylation of such elements has been shown to influence transcription factor binding and gene expression. Here we show that the half-CREoC/EBP element in the Il36A promoter is differentially methylated in the murine RAW264.7 macrophage cell line and in primary murine macrophages. By using electrophoretic mobility gel shift and fluorescence polarization assays we demonstrate that C/EBP{beta} binding to the half-CRE*C/EBP element in the Il36A promoter following LPS stimulation is insensitive to CpG methylation. Transfection assays also show that methylation of the CpG in the half-CRE*C/EBP element does not alter LPS-induced Il36A promoter activity. A direct comparison of Il36A mRNA copy numbers as well as the pro-Il-36 protein level in RAW264.7 and primary macrophages revealed similar amounts in both cell types. Taken together, our data suggest that C/EBP{beta} binding to the half-CRE*C/EBP element and C/EBP{beta} mediated gene activation occurs independently of the CpG methylation status of the target DNA sequence and underline the potential of C/EBP{beta} to recognize methylated as well as unmethylated binding sites.


title: Multiple drug resistance in hookworms infecting greyhound dogs in the USA

abstract: The hookworm Ancylostoma caninum is the most prevalent nematode parasite of dogs. Recently, we confirmed multiple-drug resistance (MDR) in several A. caninum isolates to all anthelmintic drug classes approved for the treatment of hookworms in dogs in the United States (USA). Cases of MDR hookworms appear to be highly overrepresented in greyhounds, suggesting that the MDR worms evolved on racing greyhound farms/kennels. The aims of this study were to evaluate the range of drug-resistant phenotypes and genotypes of the A. caninum infecting greyhounds. Fecal samples from recently retired greyhounds originating from geographically diverse areas of the USA were acquired from two greyhound adoption kennels, one active greyhound racing kennel, and three veterinary practices that work with adoption kennels. Fecal egg counts (FECs) were performed on fecal samples from 219 greyhounds, and despite almost all the dogs having been treated with one or more anthelmintics in the previous two to four weeks, the mean FEC was 822.4 eggs per gram (EPG). Resistance to benzimidazoles and macrocyclic lactones were measured using the egg hatch assay (EHA) and the larval development assay (LDA) respectively. We performed 23 EHA and 22 LDA on either individual or pooled feces, representing 81 animals. Mean and median IC50 and IC95 values for the EHA were 5.3 uM, 3.6 uM, and 24.5 uM, 23.4 uM respectively. For the LDA, mean and median IC50 values were 749.8 nM, >1000 nM respectively. These values range from 62 to 68 times higher than those we measured in our susceptible laboratory isolates. Pre-treatment fecal samples could not be obtained, however, post-treatment samples representing 219 greyhounds were collected. For samples collected <10 days post-treatment with albendazole, moxidectin, or a combination of febantel-pyrantel-moxidectin, the mean FEC were 349, 333, and 835 EPG, respectively. Samples collected 10-21 days post-treatment with albendazole, moxidectin, or pyrantel, yielded mean FEC of 1874, 335, and 600 EPG, respectively. Samples collected >21 days post-treatment with albendazole or moxidectin yielded mean FEC of 1819 and 1117 EPG, respectively. We obtained DNA from hookworm eggs isolated from 70 fecal samples, comprised of 60 individual dogs and 10 pools from multiple dogs. Deep sequencing of the isotype 1 {beta}-tubulin gene revealed the presence of the F167Y (TTC>TAC) resistance polymorphism in 99% of these samples, with 69% having [≥]75% resistant allele frequency. No resistance-associated polymorphisms were seen at any of the other {beta}-tubulin codons previously reported as associated with benzimidazole resistance in Strongylid nematodes. These clinical, in vitro, and genetic data provide strong evidence that racing and recently retired greyhound dogs in the USA are infected with MDR A. caninum at very high levels in terms of both prevalence and infection intensity.


title: MYC drives aggressive prostate cancer by disrupting transcriptional pause release at androgen receptor targets

abstract:
c-MYC (MYC) is a major driver of prostate cancer tumorigenesis and progression. Although MYC is overexpressed in both early and metastatic disease and associated with poor survival, its impact on prostate transcriptional reprogramming remains elusive. We demonstrate that MYC overexpression significantly diminishes the androgen receptor (AR) transcriptional program (the set of genes directly targeted by the AR protein) in luminal prostate cells without altering AR expression. Importantly, analyses of clinical specimens revealed that concurrent low AR and high MYC transcriptional programs accelerate prostate cancer progression toward a metastatic, castration-resistant disease. Data integration of single-cell transcriptomics together with ChIP-seq revealed an increased RNA polymerase II (Pol II) promoter-proximal pausing at AR-dependent genes following MYC overexpression without an accompanying deactivation of AR-bound enhancers. Altogether, our findings suggest that MYC overexpression antagonizes the canonical AR transcriptional program and contributes to prostate tumor initiation and progression by disrupting transcriptional pause release at AR-regulated genes.

STATEMENT OF SIGNIFICANCEAR and MYC are key to prostate cancer etiology but our current understanding of their interplay is scarce. Here we show that the oncogenic transcription factor MYC can pause the transcriptional program of the master transcription factor in prostate cancer, AR, while turning on its own, even more lethal program.

category: genomics


title: Multi-Ancestry Meta-Analysis yields novel genetic discoveries and ancestry-specific associations

abstract: We present a new method, Multi-Ancestry Meta-Analysis (MAMA), which combines genome-wide association study (GWAS) summary statistics from multiple populations to produce new summary statistics for each population, identifying novel loci that would not have been discovered in either set of GWAS summary statistics alone. In simulations, MAMA increases power with less bias and generally lower type-1 error rate than other multi-ancestry meta-analysis approaches. We apply MAMA to 23 phenotypes in East-Asian- and European-ancestry populations and find substantial gains in power. In an independent sample, novel genetic discoveries from MAMA replicate strongly.


title: Online spike sorting via deep contractive autoencoder

abstract: Spike sorting - the process of separating spikes from different neurons - is often the first and most critical step in the neural data analysis pipeline. Spike-sorting techniques isolate a single neurons activity from background electrical noise based on the shapes of the waveforms (WFs) obtained from extracellular recordings. Despite several advancements in this area, an important remaining challenge in neuroscience is online spike sorting, which has the potential to significantly advance basic neuroscience research and the clinical setting by providing the means to produce real-time perturbations of neurons via closed-loop control. Current approaches to online spike sorting are not fully automated, are computationally expensive and are often outperformed by offline approaches. In this paper, we present a novel algorithm for fast and robust online classification of single neuron activity. This algorithm is based on a deep contractive autoencoder (DCAE) architecture. DCAEs are deep neural networks that can learn a latent state representation of their inputs. The main advantage of DCAE approaches is that they are less sensitive to noise (i.e., small perturbations in their inputs). We therefore reasoned that they can form the basis for robust online spike sorting algorithms. Overall, our DCAE-based online spike sorting algorithm achieves over 90% accuracy at sorting previously-unseen spike waveforms. Moreover, our approach produces superior results compared to several state-of-the-art offline spike-sorting procedures.

category: paleontology


title: Design of a new effector recognition specificity in a plant NLR immune receptor by molecular engineering of its integrated decoy domain

abstract: Plant nucleotide-binding and leucine-rich repeat domain proteins (NLRs) are immune sensors that specifically recognize pathogen effectors and induce immune responses. Designing artificial NLRs with new effector recognition specificities is a promising prospect for sustainable, knowledge-driven crop protection. However, such strategies are hampered by the complexity of NLR function. Here, we tested whether molecular engineering of the integrated decoy domain (ID) of an NLR could extend its recognition spectrum to a new effector. To this aim, we relied on the detailed molecular knowledge of the recognition of distinct Magnaporthe oryzae MAX (Magnaporthe AVRs and ToxB-like) effectors by the rice NLRs RGA5 and Pikp-1. For both NLRs, effector recognition involves physical binding to their HMA (Heavy Metal-Associated) IDs. However, AVR-PikD, the effector recognized by Pikp-1, binds to a completely different surface of the HMA domain compared to AVR-Pia and AVR1-CO39, recognized by RGA5. By introducing into the HMA domain of RGA5 the residues of the Pikp-1 HMA domain involved in AVR-PikD binding, we created a high-affinity binding surface for this new effector. In the Nicotiana benthamiana heterologous system, RGA5 variants carrying this engineered binding surface still recognize AVR-Pia and AVR1-CO39, but also perceive the new ligand, AVR-PikD, resulting in the activation of immune responses. Therefore, our study provides a proof of concept for the design of new effector recognition specificities in NLRs through molecular engineering of IDs. However, it pinpoints significant knowledge gaps that limit the full deployment of this NLR-ID engineering strategy and provides hypotheses for future research on this topic.

category: plant biology


title: Ribosomal RNA degradation induced by the bacterial RNA polymerase inhibitor rifampicin.

abstract: Rifampicin, a broad-spectrum antibiotic, inhibits bacterial RNA polymerase. Here we show that rifampicin treatment of Escherichia coli results in a 50% decrease in cell size due to a terminal cell division. This decrease is a consequence of inhibition of transcription as evidenced by an isogenic rifampicin-resistant strain. There is also a 50% decrease in total RNA due mostly to a 90% decrease in 23S and 16S rRNA levels. Control experiments showed this decrease is not an artifact of our RNA purification protocol and therefore due to degradation in vivo. Since chromosome replication continues after rifampicin treatment, ribonucleotides from rRNA degradation could be recycled for DNA synthesis. Rifampicin-induced rRNA degradation occurs under different growth conditions and in different strain backgrounds. However, rRNA degradation is never complete thus permitting the re-initiation of growth after removal of rifampicin. The orderly shutdown of growth under conditions where the induction of stress genes is blocked by rifampicin is noteworthy. Inhibition of protein synthesis by chloramphenicol resulted in a partial decrease in 23S and 16S rRNA levels whereas kasugamycin treatment had no effect. Analysis of temperature-sensitive mutant strains implicate RNase E, PNPase and RNase R in rifampicin-induced rRNA degradation. We cannot distinguish between a direct role for RNase E in rRNA degradation versus an indirect role involving a slowdown of mRNA degradation. Since mRNA and rRNA appear to be degraded by the same ribonucleases, competition by rRNA is likely to result in slower mRNA degradation rates in the presence of rifampicin than under normal growth conditions.


title: Transposable element profiles reveal cell line identity and loss of heterozygosity in Drosophila cell culture

abstract: Cell culture systems allow key insights into biological mechanisms yet suffer from irreproducible outcomes in part because of cross-contamination or mislabelling of cell lines. Cell line misidentification can be mitigated by the use of genotyping protocols, which have been developed for human cell lines but are lacking for many important model species. Here we leverage the classical observation that transposable elements (TEs) proliferate in cultured Drosophila cells to demonstrate that genome-wide TE insertion profiles can reveal the identity and provenance of Drosophila cell lines. We identify multiple cases where TE profiles clarify the origin of Drosophila cell lines (Sg4, mbn2, and OSS_E) relative to published reports, and also provide evidence that insertions from only a subset of LTR retrotransposon families are necessary to mark Drosophila cell line identity. We also develop a new bioinformatics approach to detect TE insertions and estimate intra-sample allele frequencies in legacy whole-genome shotgun sequencing data (called ngs_te_mapper2), which revealed copy-neutral loss of heterozygosity as a mechanism shaping the unique TE profiles that identify Drosophila cell lines. Our work contributes to the general understanding of the forces impacting metazoan genomes as they evolve in cell culture and paves the way for high-throughput protocols that use TE insertions to authenticate cell lines in Drosophila and other organisms.


title: Narratives as Networks: Predicting Memory from the Structure of Naturalistic Events

abstract: Human life consists of a multitude of diverse and interconnected events. However, extant research has focused on how humans segment and remember discrete events from continuous input, with far less attention given to how the structure of connections between events impacts memory. We conducted an fMRI study in which subjects watched and recalled a series of realistic audiovisual narratives. By transforming narratives into networks of events, we found that more central events--those with stronger semantic or causal connections to other events--were better remembered. During encoding, central events evoked larger hippocampal event boundary responses associated with memory consolidation. During recall, high centrality predicted stronger activation in cortical areas involved in episodic recollection, and more similar neural representations across individuals. Together, these results suggest that when humans encode and retrieve complex real-world experiences, the reliability and accessibility of memory representations is shaped by their location within a network of events.


title: Basal forebrain cholinergic neurons selectively drive coordinated motor learning in mice

abstract: Motor control requires precise temporal and spatial encoding across distinct motor centers that is refined through the repetition of learning. The coordination of circuit refinement across motor regions requires modulatory input to shape circuit activity. Here we identify a role for the basocortical cholinergic pathway in the acquisition of a coordinated motor skill in mice. Targeted depletion of basal forebrain cholinergic neurons results in significant impairments in training on the rotarod task of coordinated movement. Cholinergic neuromodulation is required during training sessions as chemogenetic inactivation of cholinergic neurons also impairs task acquisition. Rotarod learning drives coordinated refinement of corticostriatal neurons arising in both medial prefrontal cortex (mPFC) and motor cortex, and we have found that cholinergic input to both motor regions is required for task acquisition. Critically, the effects of cholinergic neuromodulation are restricted to the acquisition stage, as depletion of basal forebrain cholinergic neurons after learning does not affect task execution. Our results indicate a critical role for cholinergic neuromodulation of distant cortical motor centers during coordinated motor learning.


title: Costs and benefits of using rhythmic rate codes

abstract:
Neural oscillations are one of the most well-known macroscopic phenomena observed in the nervous system, and the benefits of oscillatory coding have been the topic of frequent analysis. Many of these studies focused on communication between populations which were already oscillating, and sought to understand how synchrony and communication interact. In this paper, take an alternative approach. We focus on measuring the costs, and benefits, of moving to an from an aperiodic code to a rhythmic one. We utilize a Linear-Nonlinear Poisson model, and assume a rate code. We report that no one factor seems to predict the costs, or benefits, of translating into a rhythmic code. Instead the synaptic connection type, strength, population size, and stimulus and oscillation firing rates interact in nonlinear ways. We suggest a number of experiments that might be used to confirm these predictions.

Author summaryIts good to oscillate, sometimes.


title: Developmental HCN channelopathy results in decreased neural progenitor proliferation and microcephaly in mice

abstract:
The development of the cerebral cortex relies on the controlled division of neural stem and progenitor cells. The requirement for precise spatiotemporal control of proliferation and cell fate places a high demand on the cell division machinery, and defective cell division can cause microcephaly and other brain malformations. Cell-extrinsic and intrinsic factors govern the capacity of cortical progenitors to produce large numbers of neurons and glia within a short developmental time window. In particular, ion channels shape the intrinsic biophysical properties of precursor cells and neurons and control their membrane potential throughout the cell cycle. We found that hyperpolarization-activated cyclic nucleotide-gated cation (HCN)-channel subunits are expressed in mouse, rat, and human neural progenitors. Loss of HCN-channel function in rat neural stem cells impaired their proliferation by affecting the cell-cycle progression, causing G1 accumulation and dysregulation of genes associated with human microcephaly. Transgene-mediated, dominant-negative loss of HCN-channel function in the embryonic mouse telencephalon resulted in pronounced microcephaly. Together, our findings suggest a novel role for HCN-channel subunits as a part of a general mechanism influencing cortical development in mammals.

Significance StatementImpaired cell cycle regulation of neural stem and progenitor cells can affect cortical development and cause microcephaly. During cell cycle progression, the cellular membrane potential changes through the activity of ion channels and tends to be more depolarized in proliferating cells. HCN channels, which mediate a depolarizing current in neurons and cardiac cells, are linked to neurodevelopmental diseases, also contribute to the control of cell-cycle progression and proliferation of neuronal precursor cells. In this study, HCN-channel deficiency during embryonic and fetal brain development resulted in marked microcephaly of mice designed to be deficient in HCN-channel function in dorsal forebrain progenitors. The findings suggest that HCN-channel subunits are part of a general mechanism influencing cortical development in mammals.


title: Egalitarian feeds forward to Staufen to inhibit Dynein during mRNP transport

abstract: Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including ribonucleoprotein complexes (RNP). Here we show that orchestration of oskar RNP transport in the Drosophila germline relies on the interplay of two double-stranded RNA binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). Following delivery of nurse cell-synthesized oskar mRNA into the oocyte by dynein, enhanced recruitment of Staufen to the RNPs results in dissociation of Egl and reduced minus end-directed transport without overtly affecting overall dynein levels on the cargo. This mechanism allows kinesin-1 to translocate the mRNA to its final destination at the posterior pole of the oocyte. Using in vitro motility assays with purified components, we find that Staufen directly antagonizes Egl-mediated activation of oskar mRNA transport by dynein. We additionally show that Egl associates with staufen mRNA in the nurse cells and mediates enrichment of the mRNA and its protein product in the ooplasm. Our observations identify a feed-forward mechanism, whereby dynein-dependent accumulation of staufen mRNA and protein in the oocyte enables motor switching during oskar mRNA transport by downregulating dynein activity.


title: An RNA-based feed-forward mechanism ensures motor switching in oskar mRNA transport


title: Binding mechanism of neutralizing Nanobodies targeting SARS-CoV-2 Spike Glycoprotein

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human cells upon binding of its spike (S) glycoproteins to ACE2 receptors. Several nanobodies neutralize SARS-CoV-2 infection by binding to the receptor-binding domain (RBD) of S protein, but the underlying mechanism is not well understood. Here, we identified an extended network of pairwise interactions between RBD and nanobodies H11-H4, H11-D4, and Ty1 by performing all-atom molecular dynamics (MD) simulations. Simulations of the nanobody-RBD-ACE2 complex revealed that H11-H4 more strongly binds to RBD without overlapping with ACE2 and triggers dissociation of ACE2 due to electrostatic repulsion. In comparison, Ty1 binding results in dissociation of ACE2 from RBD due to an overlap with the ACE2 binding site, whereas H11-D4 binding does not trigger ACE2 dissociation. Mutations in SARS-CoV-2 501Y.V1 and 501.V2 variants resulted in a negligible effect on RBD-ACE2 binding. However, the 501.V2 variant weakened H11-H4 and H11-D4 binding while strengthening Ty1 binding to RBD. Our simulations indicate that all three nanobodies can neutralize 501Y.V1 while Ty1 is more effective against the 501.V2 variant.


title: Autoantibodies against Progranulin and IL-1 receptor antagonist in critically ill COVID-19

abstract:
STRUCTURED ABSTRACTHyperinflammation is frequently observed in patients with severe COVID-19. Inadequate and defective IFN type I responses against SARS-CoV-2, associated with autoantibodies in a proportion of patients, lead to severe courses of disease. In addition, hyperactive responses of the humoral immune system have been described.

In the current study we investigated a possible role of neutralizing autoantibodies against antiinflammatory mediators. Plasma from adult patients with severe and critical COVID-19 was screened by ELISA for antibodies against PGRN, IL-1-Ra, IL-10, IL-18BP, IL-22BP, IL-36-Ra, CD40, IFN-2, IFN-{gamma}, IFN-{omega} and serpinB1. Autoantibodies were characterized and the antigens were analyzed for immunogenic alterations.

In a discovery cohort with severe to critical COVID-19 high titers of PGRN-autoantibodies were detected in 11 of 30 (36.7%), and of IL-1-Ra-autoantibodies in 14 of 30 (46.7%) patients. In a validation cohort of 64 patients with critical COVID-19 high-titer PGRN-Abs were detected in 25 (39%) and IL-1-Ra-Abs in 32 of 64 patients (50%). PGRN-Abs and IL-1-Ra-Abs belonged to IgM and several IgG subclasses. In separate cohorts with non-critical COVID-19, PGRN-Abs and IL-1-Ra-Abs were detected in low frequency (i.e. in < 5% of patients) and at low titers. Neither PGRN-nor IL-1-Ra-Abs were found in 40 healthy controls vaccinated against SARS-CoV-2 or 188 unvaccinated healthy controls. PGRN-Abs were not cross-reactive against SARS-CoV-2 structural proteins nor against IL-1-Ra. Plasma levels of both free PGRN and free IL-1-Ra were significantly decreased in autoantibody-positive patients compared to Ab-negative and non-COVID-19 controls. In vitro PGRN-Abs from patients functionally reduced PGRN-dependent inhibition of TNF- signaling, and IL-1-Ra-Abs from patients reduced IL-1-Ra- or anakinra-dependent inhibition of IL-1{beta} signaling. The pSer81 hyperphosphorylated PGRN isoform was exclusively detected in patients with high-titer PGRN-Abs; likewise, a hyperphosphorylated IL-1-Ra isoform was only found in patients with high-titer IL-1-Ra-Abs. Thr111 was identified as the hyperphophorylated amino acid of IL-1-Ra. In longitudinally collected samples hyperphosphorylated isoforms of both PGRN and IL-1-Ra emerged transiently, and preceded the appearance of autoantibodies. In hospitalized patients, the presence of IL-1-Ra-Abs or IL-1-Ra-Abs in combination with PGRN-Abs was associated with a higher morbidity and mortality.

To conclude, neutralizing autoantibodies to IL-1-Ra and PGRN occur in a significant portion of patients with critical COVID-19, with a concomitant decrease in circulating free PGRN and IL-1-Ra, indicative of a misdirected, proinflammatory autoimmune response. The break of self-tolerance is likely caused by atypical hyperphosphorylated isoforms of both antigens, whose appearances precede autoantibody induction. Our data suggest that these immunogenic secondary modifications are induced by the SARS-CoV-2-infection itself or the inflammatory environment evoked by the infection and predispose for a critical course of COVID-19.


title: Autoantibodies against Progranulin and IL-1 receptor antagonist due to immunogenic posttranslational isoforms contribute to hyperinflammation in critically ill COVID-19


title: A synthetic peptide CTL vaccine targeting nucleocapsid confers protection from SARS-CoV-2 challenge in rhesus macaques

abstract:
BackgroundPersistent transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has given rise to a COVID-19 pandemic. Several vaccines, evoking protective spike antibody responses, conceived in 2020, are being deployed in mass public health vaccination programs. Recent data suggests, however, that as sequence variation in the spike genome accumulates, some vaccines may lose efficacy.

MethodsUsing a macaque model of SARS-CoV-2 infection, we tested the efficacy of a peptide-based vaccine targeting MHC Class I epitopes on the SARS-CoV-2 nucleocapsid protein. We administered biodegradable microspheres with synthetic peptides and adjuvants to rhesus macaques. Unvaccinated control and vaccinated macaques were challenged with 1 x 108 TCID50 units of SARS-CoV-2, followed by assessment of clinical symptoms, viral load, chest radiographs, sampling of peripheral blood and bronchoalveolar lavage (BAL) fluid for downstream analysis.

ResultsVaccinated animals were free of pneumonia-like infiltrates characteristic of SARS-CoV-2 infection and presented with lower viral loads relative to controls. Gene expression in cells collected from BAL samples of vaccinated macaques revealed a unique signature associated with enhanced development of adaptive immune responses relative to control macaques.

ConclusionsWe demonstrate that a room temperature stable peptide vaccine based on known immunogenic HLA Class I bound CTL epitopes from the nucleocapsid protein can provide protection against SARS-CoV-2 infection in non-human primates.

Graphical Abstract

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title: Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies

abstract: Many anti-SARS-CoV-2 neutralizing antibodies target the ACE2-binding site on viral spike receptor-binding domains (RBDs). The most potent antibodies recognize exposed variable epitopes, often rendering them ineffective against other sarbecoviruses and SARS-CoV-2 variants. Class 4 anti-RBD antibodies against a less-exposed, but more-conserved, cryptic epitope could recognize newly-emergent zoonotic sarbecoviruses and variants, but usually show only weak neutralization potencies. We characterized two class 4 anti-RBD antibodies derived from COVID-19 donors that exhibited broad recognition and potent neutralization of zoonotic coronavirus and SARS-CoV-2 variants. C118-RBD and C022-RBD structures revealed CDRH3 mainchain H-bond interactions that extended an RBD {beta}-sheet, thus reducing sensitivity to RBD sidechain changes, and epitopes that extended from the cryptic epitope to occlude ACE2 binding. A C118-spike trimer structure revealed rotated RBDs to allow cryptic epitope access and the potential for intra-spike crosslinking to increase avidity. These studies facilitate vaccine design and illustrate potential advantages of class 4 RBD-binding antibody therapeutics.


title: Household transmission of SARS-CoV-2 from humans to dogs in Washington and Idaho: burden and risk factors

abstract: SARS-CoV-2 is believed to have emerged from an animal reservoir; however, the frequency of and risk factors for inter-species transmission remain unclear. We carried out a community-based study of pets in households with one or more confirmed SARS-CoV-2 infection in humans. Among 119 dogs and 57 cats with completed surveys, clinical signs consistent with SARS-CoV-2 were reported in 20 dogs (21%) and 19 cats (39%). Out of 81 dogs and 32 cats sampled for testing, 40% of dogs and 43% of cats were seropositive, and 5% of dogs and 8% of cats were PCR positive; this discordance may be due to delays in sampling. Respondents commonly reported close human-animal contact and willingness to take measures to prevent transmission to their pets. Reported preventative measures showed a slightly protective trend for both illness and seropositivity in pets, while sharing of beds and bowls had slight harmful effects.


title: Household transmission of SARS-CoV-2 from humans to pets in Washington and Idaho: burden and risk factors


title: A divergent protein kinase A in the human pathogen Leishmania is associated with developmental morphogenesis

abstract: Parasitic protozoa of the genus Leishmania cause human leishmaniasis. They cycle between the phagolysosome of mammalian macrophages, where they reside as round intracellular amastigotes, and the mid-gut of female sand flies, which they colonize as elongated extracellular promastigotes. Shifting promastigotes to a lysosome-like environment (pH 5.5 and 37C, 5% CO) initiates their development into amastigotes. Previous studies suggested a role for protein kinase A (PKA) in this differentiation process. Here, we describe a new, divergent, regulatory subunit (PKAR3) found only in a limited member of the family Kinetoplastidae. In L. donovani, phosphorylation of PKAR3 was regulated by the differentiation signal and coincided with parasite morphogenesis during stage development. LdPKAR3 was bound to the subpellicular microtubules cell cortex via a formin homology (FH2)-like domain at the tip of a large and divergent N-terminal domain. Immunoprecipitation, fluorescence resonance energy transfer (FRET), proteomics analyses, and structural modeling showed that PKAR3 selectively binds the C3 isoform of the PKA catalytic subunit in a holoenzyme complex. In promastigotes, PKAR3 recruited PKAC3 to the subpellicular microtubules at the central cortex. After exposure to a differentiation signal, PKAR3 homogenously distributed across the entire cortex, in concert with cell rounding. Deleting the genes encoding either the R3 or C3 subunit resulted in premature rounding of the promastigote population, indicating that PKA determines the normal elongated shape. Regulation of Leishmania developmental morphogenesis by interaction with the subpellicular microtubule corset is a novel function for a unique PKA complex not present in the host cell.


title: Enhancer RNAs stimulate Pol II pause release by harnessing multivalent interactions to NELF

abstract: Enhancer RNAs (eRNAs) are long non-coding RNAs that originate from enhancers. Although eRNA transcription is a canonical feature of activated enhancers, the molecular features required for eRNA function and the mechanism of how eRNAs impinge on target gene transcription have not been established. Thus, using eRNA-dependent RNA polymerase II (Pol II) pause release as a model, we examined the requirement of sequence, structure and length of eRNAs for their ability to stimulate Pol II pause release by detaching NELF from paused Pol II. We found eRNA not to exert their function through common structural or sequence motifs. Instead, efficient NELF release requires a single eRNA molecule that must contain unpaired guanosines to make multiple, allosteric contacts with several NELF subunits. By revealing the molecular determinants for eRNA function, our study mechanistically links eRNAs to Pol II pause release and provides new insight into the regulation of metazoan transcription.


title: Droplet sample preparation for single-cell proteomics applied to the cell cycle

abstract:
Many biological processes, such as the cell division cycle, are reflected in protein covariation across single cells. This covariation can be quantified and interpreted by single-cell proteomics with sufficiently high throughput and accuracy. Toward this goal, we developed the nano-ProteOmic sample Preparation (nPOP) method for single-cell proteomics. nPOP uses piezo acoustic dispensing to isolate individual cells in 300 picoliter volumes and performs all subsequent preparation steps in small droplets on a fluorocarbon-coated slide. This design enables simultaneous sample preparation of thousands of single cells, including lysing, digesting, and labeling individual cells in volumes below 20 nl. We used nPOP to prepare 1,888 single cells and 128 negative controls in a single batch. Their analysis enabled quantifying the covariation between thousands of proteins and cell-cycle protein markers. Many protein sets covaried with the cell cycle similarly across all cell types and states, reflecting cell-type independent cell cycle functions. However, the cell cycle covariation of other protein sets differed markedly between cell types, even within subpopulation of melanoma cells expressing markers for drug-resistance priming. The cells expressing these markers accumulated in the G1 phase of the cell cycle and exhibited different covariation of enzymes catabolizing glucose. These results demonstrate that protein covariation across single cells may reveal functionally concerted biological differences between closely related cell states.

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category: bioengineering


title: Exploring functional protein covariation across single cells using nPOP


title: Adherence to minimal experimental requirements for defining extracellular vesicles and their functions: a systematic review

abstract:
Rigorous measures are required to cope with the advance of extracellular vesicle (EV) research, from 183 to 2,309 studies/year, between 2012-2020. The  MISEV guidelines requested standardizing methods, thereby assuring and improving of EV research quality. We investigated how EV research improved over time.

We conducted a keyword search in 5,093 accessible publications over the period 2012-2020 and analyzed the methodology used for EV isolation and characterization. We found a significant improvement over the years particularly regarding EV characterization where recent papers used a higher number of methods and EV markers to check for quantity and purity. Interestingly, we also found that EV papers using more methods and EV markers were cited more frequently cited. Papers citing MISEV criteria were more prone to use a higher number of characterization methods.

We therefore established a concise checklist summarizing MISEV criteria to support EV researchers towards reaching the highest standards in the field.

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category: bioinformatics


title: Spatial analysis of tumor infiltrating lymphocytes based on deep learning using histopathology image to predict progression-free survival in colorectal cancer

abstract:
PurposeThis study aimed to explore the prognostic impact of spatial distribution of tumor infiltrating lymphocytes (TILs) quantified by deep learning (DL) approaches based on digitalized whole slide images stained with hematoxylin and eosin in patients with colorectal cancer (CRC).

MethodsThe prognostic impact of spatial distributions of TILs in patients with CRC was explored in the Yonsei cohort (n=180) and validated in the TCGA cohort (n=268). Concurrently, two experienced pathologists manually measured TILs at the most invasive margin as 0-3 by the Klintrup-Makinen (KM) grading method and compared to DL approaches. Interobserver agreement for TILs was measured using Cohens kappa coefficient.

ResultsOn multivariate analysis of spatial TILs features derived by DL approaches and clinicopathological variables including tumor stage, Microsatellite instability, and KRAS mutations, TILs densities within 200 m of the invasive margin (f_im200) was remained as the most significant prognostic factor for progression-free survival (PFS) (HR 0.004 [95% CI, 0.0001-0.1502], p=.002) in the Yonsei cohort. On multivariate analysis using the TCGA dataset, f_im200 retained prognostic significance for PFS (HR 0.031, [95% CI 0.001-0.645], p=.024). Interobserver agreement of manual KM grading based on Cohens kappa coefficient was insignificant in the Yonsei ({kappa}=.109) and the TCGA ({kappa}=.121), respectively. The survival analysis based on KM grading showed statistically significant different PFS from the TCGA cohort, but not the Yonsei cohort.

ConclusionsAutomatic quantification of TILs at the invasive margin based on DL approaches showed a prognostic utility to predict PFS, and could provide robust and reproducible TILs density measurement in patients with CRC.

Data and Code AvailabilitySource code and data used for this study is available at the following link: https://github.com/hwanglab/TILs_Analysis


title: Predicted structural mimicry of spike receptor-binding motifs from highly pathogenic human coronaviruses

abstract:
Viruses often encode proteins that mimic host proteins in order to facilitate infection. Little work has been done to understand the potential mimicry of the SARS-CoV-2, SARS-CoV, and MERS-CoV spike proteins, particularly the receptor-binding motifs, which could be important in determining tropism of the virus. Here, we use structural bioinformatics software to characterize potential mimicry of the three coronavirus spike protein receptor-binding motifs. We utilize sequence-independent alignment tools to compare structurally known or predicted three-dimensional protein models with the receptor-binding motifs and verify potential mimicry with protein docking simulations. Both human and non-human proteins were found to be similar to all three receptor-binding motifs. Similarity to human proteins may reveal which pathways the spike protein is co-opting, while analogous non-human proteins may indicate shared host interaction partners and overlapping antibody cross-reactivity. These findings can help guide experimental efforts to further understand potential interactions between human and coronavirus proteins.

HighlightsO_LIPotential coronavirus spike protein mimicry revealed by structural comparison
C_LIO_LIHuman and non-human protein potential interactions with virus identified
C_LIO_LIPredicted structural mimicry corroborated by protein-protein docking
C_LIO_LIEpitope-based alignments may help guide vaccine efforts
C_LI

Graphical abstract

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title: Structure-activity mapping of the force- and sequence-dependent landscape of T cell activation

abstract: Adaptive immunity relies on T lymphocytes that use {beta} T-cell receptors (TCRs) to discriminate amongst peptides presented by MHC molecules (pMHCs). An enhanced ability to screen for pMHCs capable of inducing robust T-cell responses could have broad applications in diagnosing and treating immune diseases. T cell activation relies on biomechanical forces to initiate triggering of the TCR. Yet, most in vitro screening technologies for antigenic peptides test potential pMHCs for T cell binding without force and thus are often not predictive of activating peptides. Here, we present a technology that uses biomechanical force to initiate T cell triggering in high throughput. BATTLES (Biomechanically-Assisted T-cell Triggering for Large-scale Exogenous-pMHC Screening) displays candidate pMHCs on spectrally encoded  smart beads capable of applying physiological loads to T cells, facilitating exploration of the force- and sequence-dependent landscape of T-cell responses. BATTLES can be used to explore basic T-cell mechanobiology and T cell-based immunotherapies.


title: Single-cell landscapes of primary glioblastomas and matched organoids and cell lines reveal variable retention of inter- and intra-tumor heterogeneity

abstract: Glioblastomas (GBMs) are aggressive primary malignant brain tumors characterized by extensive levels of inter- and intra-tumor genetic and phenotypic heterogeneity. Patient-derived organoids (PDOs) have recently emerged as useful models to study such heterogeneity. Here, we present bulk exome as well as single-cell genome and transcriptome profiles of primary IDH wild type GBMs from ten patients, including two recurrent tumors, as well as PDOs and brain tumor-initiating cell (BTIC) lines derived from these patients. We find that PDOs are genetically similar to and variably retain gene expression characteristics of their parent tumors. At the phenotypic level, PDOs appear to exhibit similar levels of transcriptional heterogeneity as their parent tumors, whereas BTIC lines tend to be enriched for cells in a more uniform transcriptional state. The datasets introduced here will provide a valuable resource to help guide experiments using GBM-derived organoids, especially in the context of studying cellular heterogeneity.

category: cancer biology


title: Cannabidiol as an add-on therapy to overcome the slow-onset and, possibly, resistance to antidepressant treatment: involvement of NAPE-PLD in the medial prefrontal cortex

abstract:
Antidepressants such as serotonin uptake inhibitors are the first-line pharmacological treatment for chronic stress-related psychiatric disorders. However, their late-onset therapeutic action and frequent side effects, however, are important challenges for clinicians and patients. Besides, around 30% of major depression patients are considered treatment-resistant. Cannabidiol (CBD) is a non-psychotomimetic phytocannabinoid with a wide range of psychopharmacological effects, but its mechanism of action remains unclear. Here, we found that in male mice submitted to two different repeated stress protocols (chronic unpredictable and social defeat stress), low doses of CBD (7.5mg/Kg) caused an early-onset behavioral effect when combined to the antidepressant escitalopram (ESC-10mg/Kg). The behavioral effects of the ESC+CBD combination depended on the expression/activity of the N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD, responsible for synthesizing the endocannabinoid anandamide), but not the DAGL, enzyme in the ventromedial prefrontal cortex. In addition, we described a case series with three treatment-resistant depression that were successfully treated with CBD as adjuvant therapy, as evaluated by standardized clinical rating scales. After 12 weeks of treatment, two patients were considered depression remitted (MADRS score lower than 10) while one patient successfully responded to CBD as add-on treatment (more than 50% decrease from the baseline MADRS). Our results suggest that CBD might be useful as an add-on therapy for optimizing the action of antidepressants. They also suggest that CBDs beneficial actions depends on the facilitation of N-acylethanolamines actions in the medial prefrontal cortex.

HighlightsO_LIIn mice, cannabidiol (CBD), but not escitalopram, induced a fast-onset anti-stress action.
C_LIO_LICombinations of sub-effective doses of CBD and escitalopram produce anti-stress effects after only 7 days.
C_LIO_LIThe Escitalopram + CBD treatment modulated synaptic protein markers in the medial prefrontal cortex.
C_LIO_LICRISPR-Cas9-mediated knockdown of NAPE-PLD in the medial PFC prevents the anti-stress effect of the Escitalopram + CBD.
C_LIO_LIAdding CBD to an antidepressants regimen successfully treated three patients with treatment resistant depression.
C_LI

Graphical abstract

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title: Synthetic cell-based materials extract positional information from morphogen gradients

abstract: Dynamic biomaterials composed of synthetic cellular structures have the potential to adapt and functionally differentiate guided by physical and chemical cues from their environment. Inspired by developing biological systems, which efficiently extract positional information from chemical morphogen gradients in the presence of environmental uncertainties, we here investigate the analogous question: how well can a synthetic cell determine its position within a synthetic multicellular structure? In order to calculate positional information in such systems, we created and analyzed a large number of replicas of synthetic cellular assemblies, which were composed of emulsion droplets connected via lipid bilayer membranes. The droplets contained cell-free two-node feedback gene circuits that responded to gradients of a genetic inducer acting as a morphogen. We found that in our system, simple anterior-posterior differentiation is possible, but positional information is limited by gene expression noise, and is also critically affected by the temporal evolution of the morphogen gradient and the life-time of the cell-free expression system contained in the synthetic cells. Using a 3D printing approach, we demonstrate morphogen-based differentiation also in larger tissue-like assemblies.

category: synthetic biology


title: Engineering a super-secreting strain of Escherichia coli by directed co-evolution of the multiprotein Tat translocation machinery

abstract: Escherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease of genetic manipulation. It is often desirable to export recombinant proteins into the periplasmic space for reasons related to proper disulfide bond formation, prevention of aggregation and proteolytic degradation, and ease of purification. One such system for expressing heterologous secreted proteins is the twin-arginine translocation (Tat) pathway, which has the unique advantage of delivering correctly folded proteins into the periplasm. However, transit times for proteins through the Tat translocase, comprised of the TatABC proteins, are much longer than for passage through the SecYEG pore, the translocase associated with the more widely utilized Sec pathway. To date, a high protein flux through the Tat pathway has yet to be demonstrated. To address this shortcoming, we employed a directed co-evolution strategy to isolate mutant Tat translocases for their ability to deliver higher quantities of heterologous proteins into the periplasm. Three super-secreting translocases were selected that each exported a panel of recombinant proteins at levels that were significantly greater than that observed for wildtype TatABC or SecYEG translocases. Interestingly, all three of the evolved Tat translocases exhibited quality control suppression, suggesting that increased translocation flux was gained by relaxation of substrate proofreading. Overall, our discovery of highly efficient translocase variants paves the way for the use of the Tat system as a powerful complement to the Sec pathway for secreted production of both commodity and high value-added proteins.


title: Network controllability mediates the relationship between rigid structure and flexible dynamics

abstract:
Precisely how the anatomical structure of the brain supports a wide range of complex functions remains a question of marked importance in both basic and clinical neuroscience. Progress has been hampered by the lack of theoretical frameworks explaining how a structural network of relatively rigid inter-areal connections can produce a diverse repertoire of functional neural dynamics. Here, we address this gap by positing that the brains structural network architecture determines the set of accessible functional connectivity patterns according to predictions of network control theory. In a large developmental cohort of 823 youths aged 8 to 23 years, we found that the flexibility of a brain regions functional connectivity was positively correlated with the proportion of its structural links extending to different cognitive systems. Notably, this relationship was mediated by nodes boundary controllability, suggesting that a regions strategic location on the boundaries of modules may underpin the capacity to integrate information across different cognitive processes. Broadly, our study provides a mechanistic framework that illustrates how temporal flexibility observed in functional networks may be mediated by the controllability of the underlying structural connectivity.

AUTHOR SUMMARYPrecisely how the relatively rigid white matter wiring of the human brain gives rise to a diverse repertoire of functional neural dynamics is not well understood. In this work, we combined tools from network science and control theory to address this question. Capitalizing on a large developmental cohort, we demonstrated that the ability of a brain region to flexibly change its functional module allegiance over time (i.e., its modular flexibility), was positively correlated with its proportion of anatomical edges projecting to multiple cognitive networks (i.e., its structural participation coefficient). Moreover, this relationship was strongly mediated by the regions boundary controllability, a metric capturing its capacity to integrate information across multiple cognitive domains.


title: Jetlag Expectations, not Circadian Parameters, Predict Jetlag Symptom Severity in Travelers

abstract:
After a flight across multiple time zones, most people show a transient state of circadian misalignment causing temporary malaise known as jetlag disorder. The severity of the elicited symptoms is postulated to depend mostly on circadian factors such as the number of time zones crossed and the direction of travel. Here, we examined the influence of prior expectation on symptom severity, compared to said "classic" determinants, in order to gauge potential psychosocial effects in jetlag disorder.

To this end, we monitored jetlag symptoms in travel-inexperienced individuals (n=90, 18-37y) via detailed questionnaires twice daily for one week before and after flights crossing >3 time zones. We found pronounced differences in individual symptom load that could be grouped into 4 basic symptom trajectories. Both traditional and newly devised metrics of jetlag symptom intensity and duration (accounting for individual symptom trajectories) recapitulated previous results of jetlag prevalence at about 50-60% as well as general symptom dynamics.

Surprisingly, however, regression models showed very low predictive power for any of the jetlag outcomes. The classic circadian determinants, including number of time zones crossed and direction of travel, exhibited little to no link with jetlag symptom intensity and duration. Only expectation emerged as a parameter with systematic, albeit small, predictive value.

These results suggest expectation as a relevant factor in jetlag experience - hinting at potential placebo effects and new treatment options. Our findings also caution against jetlag recommendations based on circadian principles but insufficient evidence linking circadian re-synchronization dynamics with ensuing symptom intensity and duration.

Significance StatementJetlag disorder afflicts millions of travelers each year - a nuisance on holiday trips but also a danger in safety and performance-critical operations. For effective prevention and treatment, it is critical to understand what influences jetlag severity, i.e. jetlag symptom intensity and duration. In contrast to what guidelines state, in our study, we did not find that symptom severity could be explained by the number of time zones crossed or travel direction. Rather, travelers expectations about how long and strongly they will suffer from jetlag symptoms was the only factor systematically predicting jetlag severity. If this holds true not only for subjective but also objective symptoms, we need to revisit assumptions about how circadian desynchronization relates to experienced jetlag symptoms.


title: Reconciling contrast invariance and non-linear computation in cortical circuits

abstract: Network selectivity for orientation is invariant to changes in the stimulus contrast in the primary visual cortex. Similarly, the selectivity for odor identity is invariant to changes in odorant concentration in the piriform cortex. Interestingly, invariant network selectivity appears robust to local changes in synaptic strength induced by synaptic plasticity, even though: i) synaptic plasticity can potentiate or depress connections between neurons in a feature-dependent manner, and ii) in networks with balanced excitation and inhibition, synaptic plasticity is a determinant for the network non-linearity. In this study, we investigate whether network contrast invariance is consistent with a variety of synaptic states and connectivities in balanced networks. By using mean-field models and spiking network simulations, we show how the synaptic state controls the non-linearity in the network response to contrast and how it can lead to the emergence of contrast-invariant or contrast-dependent selectivity. Different forms of synaptic plasticity sharpen or broaden the network selectivity, while others do not affect it. Our results explain how the physiology of individual synapses is linked to contrast-invariant selectivity at the network level.


title: Kaposi's Sarcoma-associated Herpesvirus Promotes Mesenchymal-to-Endothelial Transition by Resolving the Bivalent Chromatin of PROX1 Gene

abstract: Human mesenchymal stem cells (MSCs) are highly susceptible to Kaposis sarcoma-associated herpesvirus (KSHV) infection, and the infection promotes mesenchymal-to-endothelial transition (MEndT) and acquisition of Kaposis sarcoma (KS)-like phenotypes. Increasing evidence suggests that KS may arise from KSHV-infected MSCs. To understand how KSHV induces MEndT and transforms MSCs to KS cells, we investigated the mechanism underlying KSHV-mediated MSC endothelial lineage differentiation. Like embryonic stem cells, MSC differentiation and fate determination are under epigenetic control. Prospero homeobox 1 (PROX1) is a master regulator that controls lymphatic vessel development and endothelial differentiation. We found that the PROX1 gene in MSCs harbors a distinctive bivalent epigenetic signature consisting of both active marker H3K4me3 and repressive marker H3K27me3, which poises expression of the genes, allowing timely activation upon differentiation signals or environmental stimuli. KSHV infection effectively resolved the bivalent chromatin by decreased H3K27me3 and increased H3K4me3 to activate the PROX1 gene. vIL-6 signaling leads to the recruitment of MLL2 and Set1 complexes to the PROX1 promoter to increase H3K4me3, and the vGPCR-VEGF-A axis is responsible for removing PRC2 from the promoter to reduce H3K27me3. Therefore, through a dual signaling process, KSHV activates PROX1 gene expression and initiates MEndT, which renders MSC tumorigenic features including angiogenesis, invasion and migration.


title: Independent Promoter Recognition by TcpP Precedes Cooperative Promoter Activation by TcpP and ToxR

abstract: Cholera is a diarrheal disease caused by the Gram-negative bacterium Vibrio cholerae. To reach the surface of intestinal epithelial cells, proliferate, and cause disease, V. cholerae tightly regulates the production of virulence factors such as cholera toxin (ctxAB) and the toxin co-regulated pilus (tcpA-F). ToxT is directly responsible for regulating these major virulence factors while TcpP and ToxR indirectly regulate virulence factor production by stimulating toxT expression. TcpP and ToxR are membrane-localized transcription activators (MLTAs) required to activate toxT expression. To gain a deeper understanding of how MLTAs identify promoter DNA while in the membrane, we tracked the dynamics of single TcpP-PAmCherry molecules in live cells using photoactivated localization microscopy and identified heterogeneous diffusion patterns. Our results provide evidence that: 1) TcpP exists in three biophysical states (fast diffusion, intermediate diffusion, and slow diffusion); 2) TcpP transitions between these different diffusion states; 3) TcpP molecules in the slow diffusion state are interacting with the toxT promoter; and 4) ToxR is not essential for TcpP to localize the toxT promoter. These data refine the current model of cooperativity between TcpP and ToxR in stimulating toxT expression and demonstrate that TcpP locates the toxT promoter independent of ToxR.


title: Bacterial chaperone protein Hfq facilitates the annealing of sponge RNAs to small regulatory RNAs

abstract: Bacterial small RNAs (sRNAs) in association with the chaperone protein Hfq regulate the expression of many target mRNAs. Since sRNAs action is crucial to engender a response to changing environmental conditions, their activity needs to be regulated. One such mechanism occurs at posttranscriptional level and involves sponge RNAs (or anti-sRNAs) which sequester sRNAs affecting their regulatory output. Both types of RNAs were identified on Hfq, but it is not known how Hfq interacts with RNA sponges and stimulates their base-pairing with sRNAs. Here, we used biochemical methods to demonstrate that anti-sRNAs resemble sRNAs by their structure and their modes of Hfq binding. Hfq facilitates sponge RNA annealing to sRNA, and each surface of the protein plays a particular role in the process. Moreover, we found that the efficiency of sponge RNA interactions with sRNAs can be improved, therefore, we propose that natural RNA sponges might not sequester sRNAs optimally.

category: molecular biology


title: The RNA helicase DHX36/G4R1 modulates C9orf72 GGGGCC repeat-associated translation

abstract: GGGGCC (G4C2) hexanucleotide repeat expansions (HRE) in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-associated non-AUG (RAN) translation of this expansion generates toxic proteins that accumulate in patient brains and contribute to disease pathogenesis. The DEAH-Box Helicase 36 (DHX36/G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. We found that DHX36 depletion suppresses RAN translation from reporter constructs in a repeat length dependent manner while overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Taken together, these results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeats expansion disorders.


title: iPLA2-VIA is required for healthy aging in neurons, muscle, and female germline in Drosophila melanogaster

abstract: Neurodegenerative disease (ND) is a growing health burden worldwide, but its causes and treatments remain elusive. Although most cases of ND are sporadic, rare familial cases have been attributed to single genes, which can be investigated in animal models. We have generated a new mutation in the calcium-independent phospholipase A2 (iPLA2) VIA gene CG6718, the Drosophila melanogaster ortholog of human PLA2G6/PARK14, mutations in which cause a suite of NDs collectively called PLA2G6-associated neurodegeneration (PLAN). Our mutants display age-related loss of climbing ability, a symptom of neurodegeneration in flies. Although phospholipase activity commonly is presumed to underlie iPLA2-VIA function, locomotor decline in our mutants is rescued by a transgene carrying a serine-to-alanine mutation in the catalytic residue, suggesting that important functional aspects are independent of phospholipase activity. Additionally, we find that iPLA2-VIA knockdown in either muscle or neurons phenocopies locomotor decline with age, demonstrating its necessity in both neuronal and non-neuronal tissues. Furthermore, RNA in situ hybridization shows high endogenous iPLA2-VIA mRNA expression in adult germ cells, and transgenic HA-tagged iPLA2-VIA colocalizes with mitochondria there. Mutant males are fertile with normal spermatogenesis, while fertility is reduced in mutant females. Mutant female germ cells display age-related mitochondrial aggregation, loss of mitochondrial potential, and elevated cell death. These results suggest that iPLA2-VIA is important for germline mitochondrial integrity in Drosophila, which may be relevant for understanding how PLAN develops.

category: cell biology