title: Multiplexed single-cell proteomics using SCoPE2

abstract:
Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying over 1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Isobaric carrier based multiplexed single-cell proteomics is a scalable, reliable, and cost-effective method that can be fully automated and implemented on widely available equipment. It uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. Here we describe an automated Single Cell ProtEomics (SCoPE2) workflow that allows analyzing about 200 single cells per 24 hours using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.

SCoPE2 Protocol

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category: bioengineering


title: Langerhans islets induce anti-tumor immunity at the expense of glycemic control and predict chemotherapy response in pancreatic cancer

abstract: Induction of anti-tumor immunity in pancreatic ductal adenocarcinoma (PDA) is an unresolved challenge. Systematic investigation of the microenvironment of primary pancreatic tumors revealed a role of endocrine Langerhans islets in the coordination of immune activation. We found that intratumoral {beta}-cells, regulated via STAT3, secrete C-C motif chemokine ligand 27 (CCL27) and thereby promote a TH1 phenotype in the microenvironment resulting in an enhanced T cell infiltration and prolonged patient survival. The local effect can be abrogated in a patient-based human explant model by inhibition of the CCL27 receptor CCR10. This defense mechanism is paralleled by an impaired metabolic function of Langerhans islets with reduced insulin levels resulting in a dysregulation of glycemic control in patients. Based on these findings, screening of PDA cases (n= 2264) led to the identification of type 2 diabetes mellitus (T2DM) and extractable glycated haemoglobin (HbA1c) levels as response markers for neoadjuvant chemotherapy with fluorouracil, leucovorin, irinotecan and oxaliplatin (FOLFIRINOX). Collectively these data provide insights into the interconnection of T2DM and PDA, and link declining glycemic control to therapeutic efficacy, which can be utilized as a tool for clinical decision-making and improve patient management.

category: cancer biology


title: Natural variants with 2D correlation genetics identify domains coordinating sarcomere proteins during contraction

abstract: Muscle proteins assemble in a sarcomere then by coordinated action produce contraction force to shorten muscle. In the human heart ventriculum, cardiac myosin motor ({beta}mys) repetitively converts ATP free energy into work. Cardiac myosin binding protein C (MYBPC3) in complex with {beta}mys regulates contraction power generation. Their bimolecular complex {beta}mys/MYBPC3 models the contractile system and is used here to study protein coupling. The database for single nucleotide variants (SNVs) in {beta}mys and MYBPC3 surveys human populations worldwide. It consistently records SNV physical characteristics including substituted residue location in the protein functional domain, the side chain substitution, substitution frequency, and human population group, but inconsistently records SNV implicated phenotype and pathology outcomes. A selected consistent subset of the data trains and validates a feed-forward neural network modeling the contraction mechanism. The full database is completed using the model then interpreted probabilistically with a discrete Bayes network to give the SNV probability for a functional domain location given pathogenicity and human population. Co-domains, intra-protein domains coupling {beta}mys and MYBPC3, are identified by their population correlated SNV probability product for given pathogenicity. Divergent genetics in human populations identify co-domain correlates in this method called 2D correlation genetics. Pathogenic and benign SNV data identify three critical regulatory sites, two in MYBPC3 with links to several domains across the {beta}mys motor, and, one in {beta}mys with links to the known MYBPC3 regulatory domain. Critical sites in MYBPC3 are hinges (one known another proposed) sterically enabling regulatory interactions with {beta}mys. The critical site in {beta}mys is the actin binding C-loop, a contact sensor triggering actin-activated myosin ATPase and contraction velocity modulator coordinating also with actin bound tropomyosin. C-loop and MYBPC3 regulatory domain linkage potentially impacts multiple functions across the contractile system. Identification of co-domains in a binary protein complex implies a capacity to estimate spatial proximity constraints for specific dynamic protein interactions in vivo opening another avenue for protein complex structure/function determination.

category: biochemistry


title: Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Glycopeptide Data

abstract: Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project - Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance for N- and O-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved "high coverage" and "high accuracy" glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.


title: Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Serum N- and O-Glycopeptide Data


title: How do biofilms feel their environment?

abstract: The ability of bacteria to colonize and grow on different surfaces is an essential process for biofilm development and depends on complex biomechanical interactions between the biofilm and the underlying substrate. Changes in the physical properties of the underlying substrate are known to alter biofilm expansion, but the mechanisms by which biofilms sense and respond to physical features of their environment are still poorly understood. Here, we report the use of synthetic polyacrylamide hydrogels with tunable stiffness and controllable pore size to assess physical effects of the substrate on biofilm development. Using time lapse microscopy to track the growth of expanding Serratia marcescens colonies, we find that biofilm colony growth can increase with increasing substrate stiffness on purely elastic substrates, unlike what is found on traditional agar substrates. Using traction force microscopy, we find that biofilms exert transient stresses correlated over length scales much larger than a single bacterium. Our results are consistent with a model of biofilm development in which the interplay between osmotic pressure arising from the biofilm and the poroelastic response of the underlying substrate controls biofilm growth and morphology.

category: biophysics


title: In vitro and in vivo comparison of the MRI glucoCEST properties between native glucose and 3OMG in a murine tumor model

abstract:
PurposeD-Glucose and 3-O-Methyl-D-glucose (3OMG) have been shown to provide contrast in MRI-CEST images. However, a systematic comparison between these two molecules has not yet been performed. This study dealt with the assessment of the effect of pH, saturation power level (B1) and magnetic field strength (B0) on the MRI-CEST contrast with the aim of comparing the in vivo CEST contrast detectability of these two agents in the glucoCEST procedure.

MethodsPhosphate buffered solutions of D-Glucose or 3OMG (20 mM) were prepared at different pH values and Z-spectra acquired at several B1 levels and at 37{degrees}C. In vivo glucoCEST images were obtained at 3 T and 7 T over a period of 30 min after injection of D-Glucose or 3OMG (at the doses of 1.5 and 3 g/kg) in a murine melanoma tumour model.

ResultsA markedly different pH dependence of CEST response was observed in vitro for D-Glucose and 3OMG. The glucoCEST contrast enhancement in the tumour region following the intravenous administration (at the dose 3 g/kg) resulted to be comparable for both the molecules: 1-2% at 3 T and 2-3% at 7 T. The ST% resulted almost constant for 3OMG over the 30 min period, whereas a significant increase in the case of D-Glucose was detected.

ConclusionOur results show similar CEST contrast efficiency but different temporal kinetics for the metabolizable and the non-metabolizable glucose derivatives in tumour murine models when administered at the same doses.


title: A mechanistic model captures the emergence and implications of non-genetic heterogeneity and reversible drug resistance in ER+ breast cancer cells

abstract: Resistance to anti-estrogen therapy is an unsolved clinical challenge in successfully treating ER+ breast cancer patients. Acquisition of mutations can confer heritable resistance to cancer cells, enabling their clonal selection to establish a drug-resistant population. Recent studies have demonstrated that cells can tolerate drug treatment without any genetic alterations too; however, the mechanisms and dynamics of such non-genetic adaptation remain elusive. Here, we investigate coupled dynamics of epithelial-mesenchymal transition (EMT) in breast cancer cells and emergence of reversible drug resistance. Our mechanism-based model for the underlying regulatory network reveals that these two axes can drive one another, thus conferring bidirectional plasticity. This network can also enable non-genetic heterogeneity in a population of cells by allowing for six co-existing phenotypes: epithelial-sensitive, mesenchymal-resistant, hybrid E/M-sensitive, hybrid E/M-resistant, mesenchymal-sensitive and epithelial-resistant, with the first two ones being most dominant. Next, in a population dynamics framework, we exemplify the implications of phenotypic plasticity (both drug-induced and intrinsic stochastic switching) and/or non-genetic heterogeneity in promoting population survival in a mixture of sensitive and resistant cells, even in the absence of any cell-cell cooperation. Finally, we propose the potential therapeutic use of MET (mesenchymal-epithelial transition) inducers besides canonical anti-estrogen therapy to limit the emergence of reversible drug resistance. Our results offer mechanistic insights into empirical observations on EMT and drug resistance and illustrate how such dynamical insights can be exploited for better therapeutic designs.


title: Phagocytic 'teeth' and myosin-II 'jaw' power target constriction during phagocytosis

abstract: Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely mediated by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f ("teeth") that are interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, suggesting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings suggest a phagocytic cup-shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.

category: cell biology


title: In-situ fiducial markers for 3D correlative cryo- fluorescence and FIB-SEM imaging

abstract: 1.Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), which offer high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. However, correlative light and electron microscopy (CLEM) workflows combining cryo-fluorescence microscopy (cryo-FM) and FIB-SEM are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in-situ fiducial markers for correlating cryo-FM and FIB-SEM datasets, and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization and mineral deposits in cells.


title: A novel soluble ACE2 protein totally protects from lethal disease caused by SARS-CoV-2 infection

abstract: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2), which is membrane bound, as its initial cell contact receptor preceding viral entry. Here we report a human soluble ACE2 variant fused with a 5kD albumin binding domain (ABD) and bridged via a dimerization motif hinge-like 4-cysteine dodecapeptide, which we term ACE2 1-618-DDC-ABD. This protein is enzymatically active, has increased duration of action in vivo conferred by the ABD-tag, and displays 20-30-fold higher binding affinity to the SARS-CoV-2 receptor binding domain than its des-DDC monomeric form (ACE2 1-618-ABD) due to DDC-linked dimerization. ACE2 1-618-DDC-ABD was administered for 3 consecutive days to transgenic k18-hACE2 mice, a model that develops lethal SARS-CoV-2 infection, to evaluate the preclinical preventative/ therapeutic value for COVID-19. Mice treated with ACE2 1-618-DDC-ABD developed a mild to moderate disease for the first few days assessed by a clinical score and modest weight loss. The untreated control animals, by contrast, became severely ill and had to be sacrificed by day 6/7 and lung histology revealed extensive pulmonary alveolar hemorrhage and mononuclear infiltrates. At 6 days, mortality was totally prevented in the treated group, lung histopathology was improved and viral titers markedly reduced. This demonstrates for the first time in vivo the preventative/ therapeutic potential of a novel soluble ACE2 protein in a preclinical animal model.

category: pathology


title: Brain areas associated with visual spatial attention display topographic organization during auditory spatial attention

abstract: The existence of a cortical "attentional filter" in the form of spatially selective modulation of alpha power (8-14 Hz) is a dominating hypothesis in electrophysiological studies. During auditory spatial attention hemipsheric lateralized posterior alpha modulations have been reported leading to the generalization of this hypothesis. Typically, this pattern is interpreted as reflecting a top-down endogenous mechanism for suppressing distracting input from unattended directions of sound origin. The present study on auditory spatial attention rejects this interpretation by demonstrating that alpha power modulation is closely linked to oculomotor action. Towards this end, we designed an auditory paradigm in which participants were required to attend to upcoming sounds from one of 24 loudspeakers arranged in a horizontal circular array around the head. Maintaining the location of an auditory cue was associated with a topographically modulated distribution of posterior alpha power resembling the findings known from visual attention. Multivariate analyses allowed the prediction of the sound location in the horizontal plane. Importantly, this prediction was also possible, when derived from signals capturing saccadic activity. Using eye tracking, two control experiments on visual (N=122) and auditory (N=10) spatial working memory confirmed that, in absence of any visual/auditory input, lateralization of alpha power is linked to lateralized gaze direction ({+/-} 3{degrees} visual angle). Attending to an auditory stimulus engages oculomotor and visual circuits in a topographic manner akin to the retinotopic organization in vision. We conclude that allocation of spatial attention is not sufficient to account for alpha lateralization without consideration of gaze control.

category: neuroscience


title: Sound-location specific alpha power modulation in the visual cortex in absence of visual input


title: Total number and ratio of GABAergic neuron types in the mouse lateral and basal amygdala

abstract:
GABAergic neurons are key circuit elements in cortical networks. In spite of growing evidence showing that inhibitory cells play a critical role in the lateral (LA) and basal (BA) amygdala functions, neither the number of GABAergic neurons nor the ratio of their distinct types have been determined in these amygdalar nuclei. Using unbiased stereology, we found that the ratio of GABAergic neurons in the BA (22 %) is significantly higher than in the LA (16 %) in both male and female mice. No difference was observed between the right and left hemispheres in either sexes. In addition, we assessed the ratio of the major inhibitory cell types in both amygdalar nuclei. Using transgenic mice and a viral strategy for visualizing inhibitory cells combined with immunocytochemistry, we estimated that the following cell types together compose the vast majority of GABAergic cells in the LA and BA: axo-axonic cells (5.5-6 %), basket cells expressing parvalbumin (17-20 %) or cholecystokinin (7-9 %), dendrite-targeting inhibitory cells expressing somatostatin (10-16 %), NPY-containing neurogliaform cells (14-15 %), VIP and/or calretinin-expressing interneuron-selective interneurons (29-38 %) and GABAergic projection neurons expressing somatostatin and neuronal nitric oxide synthase (nNOS, 5.5-8 %). Our results show that these amygdalar nuclei contain all major GABAergic neuron types as found in other cortical regions. Furthermore, our data offer an essential reference for future studies aiming to reveal changes in GABAergic cell number and in inhibitory cell types typically observed under different pathological conditions, and to model functioning amygdalar networks in health and disease.

Significance statementGABAergic cells in cortical structures, like in the lateral and basal nucleus of the amygdala, have a determinant role in controlling circuit operation. In this study, we provide the first estimate for the total number of inhibitory cells in these two amygdalar nuclei. In addition, our study is the first to define the ratio of the major GABAergic cell types present in these cortical networks. Taking into account that hyper-excitability in the amygdala, arising from the imbalance between excitation and inhibition typifies many altered brain functions including anxiety, posttraumatic stress disorder, schizophrenia and autism, uncovering the number and ratio of distinct amygdalar inhibitory cell types offers a solid base for comparing the changes in inhibition in pathological brain states.


title: A Synthesis of the Many Errors and Learning Processes of Visuomotor Adaptation

abstract: Visuomotor adaptation has one of the oldest experimental histories in psychology and neuroscience, yet its precise nature has always been a topic of debate. Here we offer a survey and synthesis of recent work on visuomotor adaptation that we hope will prove illuminating for this ongoing dialogue. We discuss three types of error signals that drive learning in adaptation tasks: task performance error, sensory prediction-error, and a binary target hitting error. Each of these errors has been shown to drive distinct learning processes. Namely, both target hitting errors and putative sensory prediction-errors drive an implicit change in visuomotor maps, while task performance error drives learning of explicit strategy use and non-motor decision-making. Each of these learning processes contributes to the overall learning that takes place in visuomotor adaptation tasks, and although the learning processes and error signals are independent, they interact in a complex manner. We outline many task contexts where the operation of these processes is counter-intuitive and offer general guidelines for their control, measurement and interpretation. We believe this new framework unifies several disparate threads of research in sensorimotor adaptation that often seem in conflict. We conclude by explaining how this more nuanced understanding of errors and learning processes could lend itself to the analysis of other types of sensorimotor adaptation, of motor skill learning, of the neural processing underlying sensorimotor adaptation in humans, of animal models and of brain computer interfaces.


title: Design of bacterially expressed influenza stem immunogens displayed on Msdps2 protein nanoparticles

abstract: Current influenza vaccines need to be updated annually due to mutations in the globular head of the viral surface protein, hemagglutinin (HA). To address this, vaccine candidates have been designed based on the relatively conserved HA stem domain and have shown protective efficacy in animal models. Oligomerization of the antigens either by fusion to oligomerization motifs or display on self-assembling nanoparticle scaffolds, can induce more potent immune responses compared to the corresponding monomeric antigen due to multivalent engagement of B-cells. Since nanoparticle display can increase manufacturing complexity, and often involves one or more mammalian cell expressed components, it is important to characterize and compare various display and oligomerization scaffolds. Using a structure guided approach, we successfully displayed multiple copies of a previously designed soluble, trimeric influenza stem domain immunogen, pH1HA10, on the ferritin like protein, MsDps2 (12 copies), Ferritin (24 copies) and Encapsulin (180 copies). All proteins were expressed in Escherichia coli. The nanoparticle fusion immunogens were found to be well folded and bound to the influenza stem directed broadly neutralizing antibodies with high affinity. An 8.5 [A] Cryo-EM map of Msdps2-pH1HA10 confirmed the successful design of the nanoparticle fusion immunogen. Mice immunization studies with the soluble trimeric stem and nanoparticle fusion constructs revealed that all of them were immunogenic, and protected mice against homologous (A/Belgium/145-MA/2009) and heterologous (A/Puerto Rico/8/1934) challenge with 10MLD50 mouse adapted virus. Although nanoparticle display conferred a small but statistically significant improvement in protection relative to the soluble trimer in a homologous challenge, heterologous protection was similar in both nanoparticle-stem immunized and trimeric stem immunized groups. Such rapidly producible, bacterially expressed antigens and nanoparticle scaffolds are useful modalities to tackle future influenza pandemics.

category: immunology


title: Comparative immunogenicity of bacterially expressed soluble trimers and nanoparticle displayed influenza hemagglutinin stem immunogens


title: A free-living protist that lacks canonical eukaryotic DNA replication and segregation systems

abstract: Cells must replicate and segregate their DNA with precision. In eukaryotes, these processes are part of a regulated cell-cycle that begins at S-phase with the replication of DNA and ends after M-phase. Previous studies showed that these processes were present in the last eukaryotic common ancestor and the core parts of their molecular systems are conserved across eukaryotic diversity. However, some unicellular parasites, such as the metamonad Giardia intestinalis, have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a high-quality draft genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. We found that parasitic and free-living metamonads harbor a conspicuously incomplete set of canonical proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and other replisome components, most structural kinetochore subunits including the Ndc80 complex, as well as several canonical cell-cycle checkpoint proteins. Carpediemonas is the first eukaryote known to have lost this large suite of conserved complexes, suggesting that it has a highly unusual cell cycle and that unlike any other known eukaryote, it must rely on novel or alternative set of mechanisms to carry out these fundamental processes.

category: evolutionary biology


title: BEdeepon: an in silico tool for prediction of base editor efficiencies and outcomes

abstract: Base editors enable direct conversion of one target base into another in a programmable manner, but conversion efficiencies vary dramatically among different targets. Here, we performed a high-throughput gRNA-target library screening to measure conversion efficiencies and outcome product frequencies at integrated genomic targets and obtained datasets of 60,615 and 73,303 targets for ABE and CBE, respectively. We used the datasets to train deep learning models, resulting in ABEdeepon and CBEdeepon which can predict on-target efficiencies and outcome sequence frequencies. The software is freely accessible via online web server http://www.deephf.com/#/bedeep.

category: bioinformatics


title: BEdeepoff: an in silico tool for off-target prediction of ABE and CBE base editors

abstract: Base editors, including adenine base editors (ABEs) and cytosine base editors (CBEs), are valuable tools for introducing point mutations, but they frequently induce unwanted off-target mutations. Here, we performed a high-throughput gRNA-target library screening to measure editing efficiencies at integrated genomic off-targets and obtained datasets of 48,632 and 52,429 off-targets for ABE and CBE, respectively. We used the datasets to train deep learning models, resulting in ABEdeepoff and CBEdeepoff which can predict editing efficiencies at off-targets. These tools are freely accessible via online web server http://www.deephf.com/#/bedeep.


title: Event-related and oscillatory signatures of response inhibition: A magnetoencephalography study with subclinical high and low impulsivity adults

abstract: Inhibition, the ability to withhold a response or to stop an initiated response, is a necessary cognitive function that can be vulnerable to an impairment. High levels of impulsivity have been shown to impact response inhibition and/or cognitive task performance. The present study investigated the spectral and spatio-temporal dynamics of response inhibition, during a combined go/no-go/stop-signal task, using magnetoencephalography (MEG) in a healthy undergraduate student population. Participants were divided by their level of impulsivity, as assessed by self-report measures, to explore potential differences between high (n=17) and low (n=17) impulsivity groups. Results showed that individuals scoring high on impulsivity failed significantly more NOGO and STOP trials than those scoring low, but no significant differences were found between stop-signal reaction times. During NOGO and STOP conditions, high impulsivity individuals showed significantly smaller M1 components in posterior regions, which could suggest an attentional processing deficit. During NOGO trials, the M2 component was found to be reduced in individuals scoring high, possibly reflecting less pre-motor inhibition efficiency, whereas in STOP trials, the network involved in the stopping process was engaged later in high impulsivity individuals. The high impulsivity group also engaged frontal networks more during the STOP-M3 component only, possibly as a late compensatory process. The lack of response time differences on STOP trials could indicate that compensation was effective to some degree (at the expense of higher error rates). Decreased frontal delta and theta band power was observed in high impulsivity individuals, suggesting a possible deficit in frontal pathways involved in motor suppression, however, unexpectedly, increased delta and theta band power in central and posterior sensors was also observed, which could be indicative of an increased effort to compensate for frontal deficits. Individuals scoring highly also showed decreased alpha power in frontal sensors, suggesting decreased inhibitory processing, along with reduced alpha suppression in posterior regions, reflecting reduced cue processing. These results provide evidence for how personality traits, such as impulsivity, relate to differences in the neural correlates of response inhibition.


title: A map of direct SARS-CoV-2 protein interactions implicates specific human host processes

abstract: Key steps in viral propagation, immune suppression, and pathology are mediated by direct, binary, physical interactions between viral and host proteins. To understand the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated an unbiased systematic map of binary interactions between viral and host proteins, complementing previous co-complex association maps by conveying more direct mechanistic understanding and potentially enabling targeted disruption of direct interactions. To this end, we deployed two parallel strategies, identifying 205 virus-host and 27 intraviral binary interactions amongst 171 host and 19 viral proteins, and confirming high quality of these interactions via a calibrated orthogonal assay. Host proteins interacting with SARS-CoV-2 proteins are enriched in various cellular processes, including immune signaling and inflammation, protein ubiquitination, and membrane trafficking. Specific subnetworks provide new hypotheses related to viral modulation of host protein homeostasis and T-cell regulation. The binary virus-host protein interactions we identified can now be prioritized as targets for therapeutic intervention. More generally, we provide a resource of systematic maps describing which SARS-CoV-2 and human proteins interact directly.

category: systems biology


title: A map of binary SARS-CoV-2 protein interactions implicates host immune regulation and ubiquitination


title: Structure, Mechanism and Crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase

abstract: The global COVID-19 pandemic is caused by the SARS-CoV-2 virus and has infected over 100 million and caused over 2 million fatalities worldwide at the point of writing. There is currently a lack of effective drugs to treat people infected with SARS-CoV-2. The SARS-CoV-2 Non-structural protein 13 (NSP13) is a superfamily1B helicase that has been identified as a possible target for anti-viral drugs due to its high sequence conservation and essential role in viral replication. In this study we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and the non-hydrolysable ATP analogue (AMP-PNP). Comparisons of these structures reveal details of global and local conformational changes that are induced by nucleotide binding and hydrolysis and provide insights into the helicase mechanism and possible modes of inhibition. Structural analysis reveals two pockets on NSP13 that are classified as "druggable" and include one of the most conserved sites in the entire SARS-CoV-2 proteome. To identify possible starting points for anti-viral drug development we have performed a crystallographic fragment screen against SARS-CoV-2 NSP13 helicase. The fragment screen reveals 65 fragment hits across 52 datasets, with hot spots in pockets predicted to be of functional importance, including the druggable nucleotide and nucleic acid binding sites, opening the way to structure guided development of novel antiviral agents.


title: Purine auxotrophic starvation evokes quiescence like phenotype in the budding yeast

abstract:
Purine auxotrophy is a typical marker for many laboratory yeast strains. Supplementation of additional purine source (like adenine) is necessary to cultivate these strains. If not supplied in adequate amounts, purine starvation sets in. We tested purine starvation effects in budding yeast Saccharomyces cerevisiae ade8 knockout. We explored effects brought by purine starvation in cellular, central carbon metabolism and in the global transcriptome level.

We observed that cells cultivated in purine depleted media became significantly more tolerant to severe thermal, oxidative and desiccation stresses when compared to the cells cultivated in media with all necessary supplements. When starved for purine, cells stop their cell cycle in G1 or G0 state; intracellular concentration of ATP, ADP and AMP decreases, but adenylate charge remains stable. Intracellular RNA concentration decreases and massive downregulation of ribosomal RNA occurs.

We think that purine auxotrophic starvation in a way mimics "natural" nitrogen or carbon starvations and therefore initiates elements of a transcriptional program typical for stationary phase cells (cell cycle arrest, increased stress resistance). Therefore our results demonstrate that organised metabolic response is initiated not only via "natural starvations", but also when starving for metabolic intermediates, like purines.


title: Effect of nandrolone decanoate on healing of experimentally induced radial nonunion in rabbits (Oryctolagus cuniculus)

abstract:
The aim of this study was to evaluate the effect of nandrolone decanoate (ND) on treatment of bone nonunion in the radius of rabbits. Thirty-one, young adult, New Zealand White rabbits (Oryctolagus cuniculus) were allocated to one of four groups: nandrolone males (NMG), nandrolone females (NFG), placebo males (NPM), and placebo females (NPF). After bone nonunion of a 10 mm ostectomy of the radius was confirmed (45 days after surgery), the animals in the NMG and NFG groups received 10 mg/kg ND intramuscular once a week for four weeks, while placebo groups received intramuscular 0.9% NaCl solution. Radiographic, histopathologic, and densitometric parameters (DXA) were used to compared groups.

ResultsNo significant differences were observed radiographically. However, ND groups showed greater area (P=0.0258) and BMC (P=0.0140) in the densitometric evaluation. Histologically, the placebo group showed a predominance of primary bone tissue. Whereas, lamellary organizations of secondary bone and the presence of fibrocartilage were found in the ND group (P =0.006). In conclusion, ND promoted bone regeneration after the creation of a large defect in the radius of rabbits.


title: Diversification of Transcription Factor NF-κB in Protists

abstract:
In this report, we investigate the evolution of transcription factor NF-{kappa}B by examining its structure, activity, and regulation in two protists using phylogenetic, cellular, and biochemical techniques. In Capsaspora owczarzaki (Co), we find that full-length NF-{kappa}B has an N-terminal DNA-binding domain and a C-terminal Ankyrin (ANK) repeat inhibitory domain, and its DNA-binding activity is more similar to metazoan NF-{kappa}B rather than Rel proteins. As with mammalian NF-{kappa}B proteins, removal of the ANK repeats is required for Co-NF-{kappa}B to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-{kappa}B is not induced by co-expression of IKK in human cells. Exogenously expressed Co-NF-{kappa}B localizes to the nucleus in Co cells. NF-{kappa}B mRNA and DNA-binding levels differ across three life stages of Capsaspora, suggesting distinct roles for NF-{kappa}B in these life stages. RNA-seq and GO analyses identify possible gene targets and biological functions of Co-NF-{kappa}B. We also show that three NF-{kappa}B-like proteins from the choanoflagellate Acanthoeca spectabilis (As) all consist of primarily the N-terminal conserved Rel Homology domain sequences of NF-{kappa}B, and lack C-terminal ANK repeats. All three As-NF-{kappa}B proteins constitutively enter the nucleus of human and Co cells, but differ in their DNA-binding and transcriptional activation activities. Furthermore, all three As-NF-{kappa}B proteins can form heterodimers, indicating that NF-{kappa}B diversified into multi-subunit families at least two times during evolution. Overall, these results present the first functional characterization of NF-{kappa}B in a taxonomic kingdom other than Animalia and provide information about the evolution and diversification of this biologically important transcription factor.

SignificanceThese results represent the first functional characterization of the biologically important transcription factor NF-{kappa}B in a taxonomic kingdom other than Animalia. As such, they provide information on the evolutionary origins and basal diversification of NF-{kappa}B outside of metazoans. These results suggest that NF-{kappa}B plays life stage-specific roles in Capsaspora, the closest unicellular ancestor to all metazoans. Finally, the analysis of three NF-{kappa}B proteins in a single choanoflagellate indicates that choanoflagellates have subclasses of NF-{kappa}Bs, which can form heterodimers, suggesting that NF-{kappa}B subunit expansion and diversification has occurred at least twice in evolution.


title: S-acylation controls SARS-Cov-2 membrane lipid organization and enhances infectivity

abstract: SARS-CoV-2 virions are surrounded by a lipid bilayer which contains membrane proteins such as Spike, responsible for target-cell binding and virus fusion, the envelope protein E and the accessory protein Orf3a. Here, we show that during SARS-CoV-2 infection, all three proteins become lipid modified, through action of the S-acyltransferase ZDHHC20. Particularly striking is the rapid acylation of Spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics and biochemical approaches, we show that this massive lipidation controls Spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid rich lipid nanodomains, in the early Golgi where viral budding occurs. ZDHHC20-mediated acylation allows the formation of viruses with enhanced fusion capacity and overall infectivity. Our study points towards S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.


title: Chromosome counting in the mouse and human zygote using low-invasive super-resolution live-cell imaging

abstract:
In preimplantation embryos, an abnormal chromosome number causes developmental failure and a reduction in the pregnancy rate. Conventional chromosome testing methods requiring biopsy reduce the risk of associated genetic diseases; nevertheless, the reduction in cell number also reduces the pregnancy rate. Therefore, we attempted to count the chromosomes in mouse (Slc:ICR) embryos using super-resolution live-cell imaging as a new method of chromosome counting that does not reduce the cell number or viability. We counted the forty chromosomes at the first mitosis by injecting embryos with histone H2B-mCherry mRNA under conditions by which pups could be obtained; however, the results were often an underestimation of chromosome number and varied by embryo and time point. Therefore, we developed a method to count the chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization targeting the sequence of the centromere region, enabling us to count the chromosomes more accurately in mouse embryos. The methodology presented here may provide useful information for assisted reproductive technologies, such as those used in livestock animals/humans, as a technique for assessing the chromosomal integrity of embryos prior to transfer.

Impact StatementLow-toxic super-resolution observation enables chromosome counting in preimplantation embryos without cell collection.

category: developmental biology


title: Chromosome counting in the mouse zygote using low-invasive super-resolution live-cell imaging


title: Morphometry of newborn piglets and its relevance at weaning

abstract:
This study aimed to evaluate the development of suckling piglets using morphometric parameters. Different models were created to predict the probability to occur any of the three weight classes (light, medium, and heavy) based on the piglets weaning weight. The variables in this research were birth weight (PWB), lactation length (Lac), and morphometric parameters- body length (BL), heart girth (HG), body mass index (BMI), ponderal index (PI), surface-mass ratio (SM), and birth order (BO). An adjustment of the ordinal regression was proposed to predict the weight classifications. The model with a significant effect of the Lac variables was selected. The light and heavy piglets, regardless of their morphometry, have a high chance of staying in the same weight class at weaning. However, this does not occur in medium piglets with diverse morphometry.

Summary statementThis manuscript demonstrates how morphometry can influence the development of animals, even with similar weights.


title: Twenty years of change in benthic communities across the Belizean Barrier Reef

abstract: Disease, ocean warming, and pollution have caused catastrophic declines in the cover of living coral on reefs across the Caribbean. Subsequently, reef-building corals have been replaced by invertebrates and macroalgae, leading to changes in ecological functioning. We describe changes in benthic community composition and cover at 15 sites across the Belizean Barrier Reef (BBR) following numerous major disturbances--bleaching, storms, and disease outbreaks--over the 20-year period 1997-2016. We tested the role of potential drivers of change on coral reefs, including local human impacts and ocean temperature. From 1997 to 2016, mean coral cover significantly declined from 26.3% to 10.7%, while macroalgal cover significantly increased from 12.9% to 39.7%. We documented a significant decline over time of the reef-building corals Orbicella spp. and described a major shift in benthic composition between early sampling years (1997-2005) and later years (2009-2016). The covers of hard-coral taxa, including Acropora spp., M. cavernosa, Orbicella spp., and Porites spp., were negatively related to marine heatwave frequency. Only gorgonian cover was related, negatively, to our metric of the magnitude of local impacts (the Human Influence Index). Changes in benthic composition and cover were not associated with local protection or fishing. This result is concordant with studies throughout the Caribbean that have documented living coral decline and shifts in reef-community composition following disturbances, regardless of local fisheries restrictions. Our results suggest that benthic communities along the BBR have experienced disturbances that are beyond the capacity of the current management structure to mitigate. We recommend that managers devote greater resources and capacity to enforce and expand existing marine protected areas and that government, industry, and the public act to reduce global carbon emissions.

category: ecology


title: Infective prey leads to a partial role reversal in a predator-prey interaction

abstract: An infective prey has the potential to infect, kill and consume its predator. Such a prey-predator relationship fundamentally differs from the classical Lotka-Volterra predator-prey premise because the prey can directly profit from the predator as a growth resource. Here we present a population dynamics model of partial role reversal in the predator-prey interaction. We parametrize the model to represent the predator-prey interaction of sea cucumber Apostichopus japonicus and bacterium Vibrio splendidus. We observe that two major factors stabilize the predator-prey interaction. First, the partial role reversal in the predator-prey community stabilizes the predator-prey interaction. Second, if the predator is a generalist and follows the type I functional response in attacking the prey, the predator-prey interaction is stable. We also analysed the conditions for species extinction. The extinction of the prey, V. splendidus, may occur when its growth rate is low, or in the absence of infectivity. The extinction of the predator, A. japonicus, may follow if either the infectivity of the prey is high or a moderately infective prey is abundant. We conclude that partial role reversal is an underestimated subject in predator-prey studies.


title: Phylogeography and Proline amino acid usage of Asian tiger mosquito Aedes albopictus (Skuse 1894) populations along landscape gradients in Indonesia

abstract: Asian tiger mosquito Aedes albopictus (Skuse 1894) is one of mosquito-borne vector that globally distributed including Southeast Asia. Despite its wide distributions, information on phylogeography and its determinant factors influencing A. albopictus genetic diversity and speciation is still lacking, mainly in SE Asia country. Considering this, the phylogeography of A. albopictus along with its genetic attributes in Indonesia has been assessed. The results identify that there are 2 distinct clades of A. albopictus phylogeography. First clade was related to the human-mediated dissemination. Relative Synonymous Codon Usage (RSCU) shows the TTG, CTG, GTG, ACG, GCG, and GGG codons were seldom represented. While GGA, CGA, and followed by TAT, CAT, CCT, TCT, AAC, and TTC codons were common. From a total of 60 codons, 40% codon has RSCU > 1and 21.6 % has RSCU < 1. The differences between west and east part populations can be observed in proline amino acid signaled by CCT and CCC codons. In this amino acid, east A. albopictus has higher CCC than CCT codons. Since proline is functioned to provide energy for flight, then the differences of this proline related codon among A. albopictus populations were related to the landscape variations in west and east parts of Indonesia with east parts have more rugged landscape and this condition is quite demanding for aerial animal distribution since it requires more flight energy.


title: Seasonal occurrence of Japanese pygmy octopus Octopus parvus in the intertidal zone

abstract: The Japanese pygmy octopus Octopus parvus is a small octopus that occurs commonly along the coast of southern Japan, and is caught using traditional fishing methods. To date, however, there have been no studies on the ecology of this species. In this study, we investigated the annual seasonal occurrence of O. parvus in the intertidal zone of Fukue Island, Nagasaki, Japan, and examined the growth, maturation, and feeding habits of this octopus. We accordingly found that the octopus inhabits the intertidal zone of the study site from August to January, during which time it appears to move from the high to low intertidal zone, and subsequently migrates to the subtidal zone. During low tide at night in the intertidal zone, we observed that the octopuses actively captured and fed on prey, such as fish, crabs, hermit crabs, shrimp, snails, and bivalves. Both males and females were found to be characterized by similar body sizes and growth, although gonadal development occurs relatively earlier in males. However, any mating or brooding behaviors were not observed during the course of the study. Our observations thus indicate that O. parvus uses intertidal zones as feeding grounds for rapid growth and maturation, and may thereafter move to the subtidal zone for spawning.


title: Complex population dynamics in a spatial microbial ecosystem with Physarum polycephalum

abstract: This research addresses the interactions between the unicellular slime mold Physarum polycephalum and a red yeast in a spatial ecosystem over week-long imaging experiments. An inverse relationship between the growth rates of both species is shown, where P. polycephalum has positive growth when the red yeast has a negative growth rate and vice versa. The data also captures successional/oscillatory dynamics between both species. An advanced image analysis methodology for semantic segmentation is used to quantify population density over time, for all components of the ecosystem. We suggest that P. polycephalum is capable of exhibiting a sustainable feeding strategy by depositing a nutritive slime trail, allowing yeast to serve as a periodic food source. This opens a new direction of P. polycephalum research, where the population dynamics of spatial ecosystems can be readily quantified and complex ecological dynamics can be studied.


title: A signature of Neanderthal introgression on molecular mechanisms of environmental responses

abstract: Ancient human migrations led to the settlement of population groups in varied environmental contexts worldwide. The extent to which adaptation to local environments has shaped human genetic diversity is a longstanding question in human evolution. Recent studies have suggested that introgression of archaic alleles in the genome of modern humans may have contributed to adaptation to environmental pressures such as pathogen exposure. Functional genomic studies have demonstrated that variation in gene expression across individuals and in response to environmental perturbations is a main mechanism underlying complex trait variation. We considered gene expression response to in vitro treatments as a molecular phenotype to identify genes and regulatory variants that may have played an important role in adaptations to local environments. We investigated if Neanderthal introgression in the human genome may contribute to the transcriptional response to environmental perturbations. To this end we used eQTLs for genes differentially expressed in a panel of 52 cellular environments, resulting from 5 cell types and 26 treatments, including hormones, vitamins, drugs, and environmental contaminants. We found that SNPs with introgressed Neanderthal alleles (N-SNPs) disrupt binding of transcription factors important for environmental responses, including ionizing radiation and hypoxia, and for glucose metabolism. We identified an enrichment for N-SNPs among eQTLs for genes differentially expressed in response to 8 treatments, including glucocorticoids, caffeine, and vitamin D. Using Massively Parallel Reporter Assays (MPRA) data, we validated the regulatory function of 21 introgressed Neanderthal variants in the human genome, corresponding to 8 eQTLs regulating 15 genes that respond to environmental perturbations. These findings expand the set of environments where archaic introgression may have contributed to adaptations to local environments in modern humans and provide experimental validation for the regulatory function of introgressed variants.


title: A ROR2 coding variant is associated with craniofacial variation in domestic pigeons

abstract: Vertebrate craniofacial morphogenesis is a highly orchestrated process that is directed by evolutionarily conserved developmental pathways 1,2. Within species, canalized developmental programs typically produce only modest morphological variation. However, as a result of millennia of artificial selection, the domestic pigeon (Columba livia) displays radical variation in craniofacial morphology within a single species. One of the most striking cases of pigeon craniofacial variation is the short beak phenotype, which has been selected in numerous breeds. Classical genetic experiments suggest that pigeon beak length is regulated by a small number of genetic factors, one of which is sex-linked (Ku2 locus) 3-5. However, the molecular genetic underpinnings of pigeon craniofacial variation remain unknown. To determine the genetic basis of the short beak phenotype, we used geometric morphometrics and quantitative trait loci (QTL) mapping on an F2 intercross between a short-beaked Old German Owl (OGO) and a medium-beaked Racing Homer (RH). We identified a single locus on the Z-chromosome that explains a majority of the variation in beak morphology in the RH x OGO F2 population. In complementary comparative genomic analyses, we found that the same locus is also strongly differentiated between breeds with short and medium beaks. Within the differentiated Ku2 locus, we identified an amino acid substitution in the non-canonical Wnt receptor ROR2 as a putative regulator of pigeon beak length. The non-canonical Wnt (planar cell polarity) pathway serves critical roles in vertebrate neural crest cell migration and craniofacial morphogenesis 6,7. In humans, homozygous ROR2 mutations cause autosomal recessive Robinow syndrome, a rare congenital disorder characterized by skeletal abnormalities, including a widened and shortened facial skeleton 8,9. Our results illustrate how the extraordinary craniofacial variation among pigeons can reveal genetic regulators of vertebrate craniofacial diversity.

category: genetics


title: Complex genetic architecture of three-dimensional craniofacial shape variation in domestic pigeons

abstract: Deciphering the genetic basis of vertebrate craniofacial variation is a longstanding biological problem with broad implications in evolution, development, and human pathology. One of the most stunning examples of craniofacial diversification is the adaptive radiation of birds, in which the beak serves essential roles in virtually every aspect of their life histories. The domestic pigeon (Columba livia) provides an exceptional opportunity to study the genetic underpinnings of craniofacial variation because of its unique balance of experimental accessibility and extraordinary phenotypic diversity within a single species. We used traditional and geometric morphometrics to quantify craniofacial variation in an F2 laboratory cross derived from the straight-beaked Pomeranian Pouter and curved-beak Scandaroon pigeon breeds. Using a combination of genome-wide quantitative trait locus scans and multi-locus modeling, we identified a set of genetic loci associated with complex shape variation in the craniofacial skeleton, including beak curvature, braincase shape, and mandible shape. Some of these loci control coordinated changes between different structures, while others explain variation in the size and shape of specific skull and jaw regions. We find that in domestic pigeons, a complex blend of both independent and coupled genetic effects underlie three-dimensional craniofacial morphology.


title: Recurrent structural variations of lncRNA gene CCDC26 in diffuse intrinsic pontine glioma

abstract: We report recurrent somatic structural variations (SVs) involving long noncoding RNA (lncRNA) CCDC26 in 13% of Diffuse Intrinsic Pontine Glioma (DIPG) patients. We validate our findings using whole genome sequencing data from two independent patient cohorts. CCDC26 SVs cause increased expression of CCDC26 gene in patients. In addition, CCDC26 expression is associated with elevated expression of MYC and proliferation signature. Our findings identify CCDC26 as a novel significantly mutated gene in DIPG and highlight the importance of structural variations in pediatric brain cancer.

category: genomics


title: Structural insights into the role of Dicer-related helicase 3 in RNAi in Caenorhabditis elegans.

abstract: DRH-3 belongs to the family of duplex RNA-dependent ATPases (DRAs), which include Dicer and RIG-I-like receptors (RLRs). DRH-3 is critically involved in germline development and RNAi-facilitated chromosome segregation via the 22G-siRNA pathway in C. elegans. The molecular understanding of DRH-3 and its function in endogenous RNAi pathways remains elusive. In this study, we solved the crystal structures of the DRH-3 N-terminal domain (NTD) and the C-terminal domains (CTDs) in complex with 5-triphosphorylated RNAs. The NTD of DRH-3 adopts a distinct fold of tandem Caspase Activation and Recruitment Domains (CARDs) structurally similar to the CARDs of RIG-I and MDA5, suggesting a signaling function in the endogenous RNAi biogenesis. The CTD preferentially recognizes 5-triphosphorylated double-stranded RNAs bearing the typical features of secondary siRNA transcripts. The full-length DRH-3 displays unique structural dynamics upon binding to RNA duplexes that differ from RIG-I or MDA5. These unique molecular features of DRH-3 help explain its function in RNAi in worms and the evolutionary divergence of the Dicer-like helicases.


title: The Hsp90 chaperone system from the African trypanosome, Trypanosoma brucei

abstract: African Trypanosomiasis is a neglected tropical disease caused by Trypanosoma brucei (T. brucei) and is spread by the tsetse fly in sub-Saharan Africa. The disease is fatal if left untreated and the currently approved drugs for treatment are toxic and difficult to administer. The trypanosome must survive in the insect vector and its mammalian host, and to adapt to these different conditions, the parasite relies on molecular chaperones called heat shock proteins. Heat shock proteins mediate the folding of newly synthesized proteins as well as prevent misfolding of proteins under normal conditions and during stressful conditions. Heat shock protein 90 (Hsp90) is one of the major molecular chaperones of the stress response at the cellular level. It functions with other chaperones and co-chaperones and inhibition of its interactions is being explored as a potential therapeutic target for numerous diseases. This study provides an in-silico overview of Hsp90 and its co-chaperones in both T. brucei brucei and T. brucei gambiense in relation to human and other kinetoplastid parasites. The evolutionary, functional, and structural analyses of Hsp90 were also shown. The updated information on Hsp90 and its co-chaperones from recently published proteomics on T. brucei was examined for the different life cycle stages and subcellular localisations. The results show a difference between T. b. brucei and T. b. gambiense with T. b. brucei encoding 12 putative Hsp90 genes, 10 of which are cytosolic and located on a single chromosome while T. gambiense encodes 5 Hsp90 genes, 3 of which are located in the cytosol. Eight putative co-chaperones were identified in this study, 6 TPR-containing and 2 non-TPR-containing co-chaperones. This study provides an updated context for studying the biology of the African trypanosome and evaluating Hsp90 and its interactions as potential drug targets.


title: Structural basis for late maturation steps of the human mitoribosomal large subunit

abstract: Mitochondrial ribosomes (mitoribosomes) synthezise a critical set of proteins essential for oxidative phosphorylation. Therefore, their function is vital to cellular energy supply and mitoribosomal defects give rise to a large and diverse group of human diseases 1. The architecture of mitoribosomes is strikingly different from that of their bacterial and eukaryotic cytosolic counterparts and display high divergence between species 2-6. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determined the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5. This allowed us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis that involve normally short-lived assembly intermediates. Our structures provide important insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangments leading to PTC formation. Moreover, our data reveal an unexpected role for the elongation factor mtEF-Tu in mt-LSU assembly, in which mt-EF-Tu interacts with GTPBP5 in a manner similar to its interaction with tRNA during translational elongation. Together, our approaches provide detailed understanding of the last stages of mt-LSU biogenesis that are unique to mammalian mitochondria.


title: Development of novel MOG analogues with increased stability to explore MCT2 and α-ketoglutarate biology in vivo

abstract: -ketoglutarate (KG) is a central metabolic node with a broad influence on cellular physiology. The KG analogue N-oxalylglycine (NOG) and its membrane-permeable pro-drug derivative dimethyl-oxalylglycine (DMOG) have been extensively used as tools to study prolyl hydroxylases (PHDs) and other KG-dependent processes. In cell culture media, DMOG is rapidly converted to MOG, which enters cells through monocarboxylate transporter MCT2, leading to intracellular NOG concentrations that are sufficiently high to inhibit glutaminolysis enzymes and cause cytotoxicity. Therefore, the degree of (D)MOG instability together with MCT2 expression levels determine the intracellular targets NOG engages with and, ultimately, its effects on cell viability. Here we designed and characterised a series of MOG analogues with the aims of improving stability and exploring the functional requirements for interaction with MCT2, a relatively understudied member of the SLC16 family. We report MOG analogues that maintain ability to enter cells via MCT2, and identify compounds that do not inhibit glutaminolysis or cause cytotoxicity but can still inhibit PHDs. We use these analogues to show that glutaminolysis-induced activation of mTORC1 can be uncoupled from PHD activity. Therefore, these new compounds can help deconvolute cellular effects that result from the polypharmacological action of NOG.


title: Novel MOG analogues to explore the MCT2 pharmacophore, α-ketoglutarate biology and cellular effects of N-oxalylglycine


title: A structure-derived mechanism reveals how capping protein promotes nucleation in branched actin networks

abstract: Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP stimulates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we report the structure of capped actin filament barbed ends, which reveals how CP not only prevents filament elongation, but also controls access to both terminal filament subunits. In addition to its primary binding site that blocks the penultimate subunit, we find that the CP sterically occludes the central interaction site of the terminal actin protomer through one of its C-terminal "tentacle" extensions. Deletion of this {beta} tentacle only modestly impairs capping. However in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors (NPFs) by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes -capping and nucleation- in branched actin network assembly.


title: Preparation of E. coli RNA polymerase transcription elongation complexes for systematic RNA assays

abstract: RNA folds into secondary and tertiary structures that can mediate diverse cellular functions. Understanding how RNA sequence directs the formation of biologically active structures requires approaches that can comprehensively assess how changes in an RNA sequence affect its structure and function. Towards this goal, I have developed a general method for purifying E. coli RNA polymerase (RNAP) transcription elongation complexes (TECs) for use in systematic RNA assays. My approach depends on two constituent technologies: First, I have designed an E. coli {sigma} 70 promoter that can be efficiently barcoded using a one-pot series of enzymatic reactions. Second, I have developed a strategy for purifying promoter-initiated E. coli RNAP TECs by selective photo-elution from streptavidin-coated magnetic beads. Together, these methods establish a platform for the development of TEC Display assays in which the functional properties of RNA sequence variants can be recorded by fractionating and quantitatively barcoding a TEC library.


title: Hydroxycarboxylic acid receptor 2 (HCAR2/GPR109A) expression and signaling promotes the maintenance of an immunoinhibitory retinal environment

abstract:
BackgroundExcessive oxidative stress and related chronic, sub-clinical inflammation is linked causally to the development and progression of degenerative diseases of the retina including diabetic retinopathy, age-related macular degeneration and glaucoma, leading causes of blindness worldwide. The above responses may be related directly to dysregulated retinal immunity and are potentiated by the combined actions of native retinal cells (e.g., retinal pigment epithelial (RPE) and microglial cells) and immune cells infiltrating from the periphery. Maintaining tight regulation of these cells such that effective control of pathogens is accomplished yet uncontrolled inflammation and consequent tissue damage is prevented is extremely important. However, the molecular mechanisms that control this delicate balance are poorly understood. We hypothesize that the hydroxycarboxylic acid receptor 2 (HCAR2/GPR109A) may play an important role. HCAR2/GPR109A has been shown to regulate immune cell responses that potentiate anti-inflammatory signaling upon its activation in various tissues as evidenced principally by suppressed pro-inflammatory cytokine secretion in various experimental model systems. We have demonstrated HCAR2/GPR109A expression in RPE, microglia and endothelial cells and, our in vitro studies support that the receptor elicits anti-inflammatory signaling in these cell types. However, the functional relevance of HCAR2/GPR109A expression and its activation in the retina of the living animal has not been demonstrated definitively. This is the purpose of the present study.

MethodsRetinal function was evaluated temporally in wildtype (Hcar2/Gpr109a +/+, WT) and knockout (Hcar2/Gpr109 -/-, KO) mice via electroretinography (ERG). Fundoscopic imaging, spectral domain-optical coherence tomography (OCT), fluorescein angiography and post-mortem histological analyses were additionally performed to evaluate retinal health. Gene microarray, RT-qPCR studies, ingenuity analyses and proteome pathway mapping were performed to evaluate potential key differences in the molecular signatures of WT and KO mouse retinas. Leukostasis and flow cytometric assays were performed to demonstrate the in vivo impact of HCAR2/GPR109A expression and its therapeutic activation on pro-inflammatory immune cell trafficking in retina.

ResultsLongitudinal studies revealed progressive anomalies in retinal morphology and function in HCAR2/GPR109A knockout mice that impacted the entire retina. Gene expression and protein interactome analyses revealed differences in gene and protein expression consistent with the increased immune reactivity and infiltration of bone-marrow derived immune cells detected in KO mouse retinas. Studies conducted in an acute model of retinal (endotoxin-induced) inflammation revealed that targeting the receptor via intraperitoneal administration of agonist, beta-hydroxybutyrate, limits immune cell activation, infiltration and related inflammation in WT retinas.

ConclusionsThe present studies demonstrate a central role for HCAR2/GPR109A in regulating the complex interplay between resident retinal cells and peripheral immune cells and, the potential therapeutic utility that targeting the receptor holds with respect to preventing and treating inflammatory retinal diseases.

HighlightsOxidative stress and inflammation are major causative factors in degenerative retinal diseases stemming from numerous causes (e.g., aging, diabetes, sickle cell). Thus, identifying new targets and developing strategies to counter these factors to prevent and treat retinal degeneration is important. The present in vivo study demonstrates convincingly the principal role of the hydroxycarboxylic acid receptor 2 (HCAR2/GPR109A) as a major regulator of retinal immune responses under normal conditions and therefore, as a target with extremely high potential for therapeutic modulation of these responses in retinal disease.


title: One-shot identification of SARS-CoV-2 S RBD escape mutants using yeast screening

abstract:
The potential emergence of SARS-CoV-2 Spike (S) escape mutants is a threat to reduce the efficacy of existing vaccines and neutralizing antibody (nAb) therapies. An understanding of the antibody/S escape mutations landscape is urgently needed to preemptively address this threat. Here we describe a rapid method to identify escape mutants for nAbs targeting the S receptor binding site. We identified escape mutants for five nAbs, including three from the public germline class VH3-53 elicited by natural COVID-19 infection. Escape mutations predominantly mapped to the periphery of the ACE2 recognition site on the RBD with K417, D420, Y421, F486, and Q493 as notable hotspots. We provide libraries, methods, and software as an openly available community resource to accelerate new therapeutic strategies against SARS-CoV-2.

One Sentence SummaryWe present a facile method to identify antibody escape mutants on SARS-CoV-2 S RBD.


title: Analysis of the PcrA-RNA polymerase complex reveals a helicase interaction motif and a role for PcrA/UvrD helicase in the suppression of R-loops.

abstract: The PcrA/UvrD helicase binds directly to RNA polymerase (RNAP) but the structural basis for this interaction and its functional significance have remained unclear. In this work we used biochemical assays and hydrogen-deuterium exchange coupled to mass spectrometry to study the PcrA-RNAP complex. We find that PcrA binds tightly to a transcription elongation complex in a manner dependent on protein:protein interaction with the conserved PcrA C-terminal Tudor domain. The helicase binds predominantly to two positions on the surface of RNAP. The PcrA C-terminal domain engages a conserved region in a lineage-specific insert within the {beta} subunit which we identify as a helicase interaction motif present in many other PcrA partner proteins, including the nucleotide excision repair factor UvrB. The catalytic core of the helicase binds near the RNA and DNA exit channels and blocking PcrA activity in vivo leads to the accumulation of R-loops. We propose a role for PcrA as an R-loop suppression factor that helps to minimise conflicts between transcription and other processes on DNA including replication.


title: Microbial diversity in raw milk and Sayram Ketteki from southern of Xinjiang, China

abstract: Raw milk and fermented milk are rich in microbial resources, which are essential for the formation of texture, flavor and taste. In order to gain a deeper knowledge of the bacterial and fungal community diversity in local raw milk and home-made yogurts from Sayram town, Baicheng county, Akesu area, southern of Xinjiang, China,30 raw milk and 30 home-made yogurt samples were collected and experiment of high-throughput sequencing was implemented.The results of experiments revealed the species of fungi in raw milk was the most, and the species of bacteria in fermented milk was the least.Based on principal component analysis (PCA), it was found that the bacterial and fungal community structure differed in samples from two types of dairy products.And the presence of 15 bacterial and 12 fungal phyla, comprising 218 bacterial and 495 fungal genera respectively, among all samples. Firmicutes and Ascomycota,Lactobacillus and Candida were the predominant phyla and genera of bacteria and fungi, respectively. The results indicated that the microbial community of raw milk differs from home-made yogurts due to sampling location and manufacturing process. The study suggested that high-throughput sequencing could provide a better understanding of microbiological diversity as well as lay a theoretical foundation for selecting beneficial microbial resources from this natural yogurt.


title: GTP and lipids control self-assembly and functional promiscuity of Dynamin2 molecular machinery

abstract:
Dynamin2 GTPase (Dyn2) is a crucial player in clathrin-mediated endocytosis. Dyn2 is tetrameric in cytoplasm and self-assembles into functional units upon membrane binding. How the curvature activities and functionality of Dyn2 emerge during self-assembly and are regulated by lipids remains unknown. Here we reconstituted the Dyn2 self-assembly process using membrane nanotubes (NT) and vesicles and characterized it using single- molecule fluorescence microscopy, optical tweezers force spectroscopy and cryo-electron microscopy. On NTs, Dyn2 first forms small subhelical oligomers, which are already curvature active and display pronounced curvature sensing properties. Conical lipids and GTP promote their further self-assembly into helical machinery mediating the NT scission. In the presence of large unilamellar vesicles (LUVs), an alternative self- assembly pathway emerges where the subhelical oligomers form membrane tethering complexes mediating LUV-NT binding. Reconstitution of tethering in the LUV system revealed that lipid mixing is controlled by conical lipid species, divalents, GTP, and SH3 binding partners of Dyn2. In membranes with a high content of lipids with negative intrinsic curvature, cryo-EM revealed putative membrane contact sites made by Dyn2 clusters. On such membranes, with GTP lowered to 0.2 mM, both membrane fission and tethering activities become possible, indicating functional promiscuity of Dyn2.

We conclude that GTP and lipids control both extent and topology of Dyn2 functional self-assembly. The function of Dyn2 oligomers evolves from curvature sensing, seen in subhelical Dyn2 oligomers, to curvature creation and fission, seen in Dyn2 helices. Under specific circumstances, such as downregulation of SH3 partners of Dyn2 and GTP depletion, membrane tethering activity can emerge in membrane systems enriched with conical lipids. Hence the Dyn2 functionality is actively adapted to lipidome, explaining its large habitat in the cells and tissues.


title: DNA tension-modulated translocation and loop extrusion by SMC complexes revealed by molecular dynamics simulations

abstract: Structural Maintenance of Chromosomes (SMC) complexes play essential roles in genome organization across all domains of life. To determine how the activities of these large ( 50 nm) complexes are controlled by ATP binding and hydrolysis, we developed a molecular dynamics model that accounts for conformational motions of the SMC and DNA. The model combines DNA loop capture with an ATP-induced "power stroke" to translocate the SMC complex along DNA. This process is sensitive to DNA tension: at low tension (0.1 pN), the model makes loop-capture steps of average 60 nm and up to 200 nm along DNA (larger than the complex itself), while at higher tension, a distinct inchworm-like translocation mode appears. By tethering DNA to an experimentally-observed additional binding site ("safety belt"), the model SMC complex can perform loop extrusion (LE). The dependence of LE on DNA tension is distinct for fixed DNA tension vs. when fixed DNA end points: LE reversal occurs above 0.5 pN for fixed tension, while LE stalling without reversal occurs at about 2 pN for fixed end points. Our model matches recent experimental results for condensin and cohesin, and makes testable predictions for how specific structural variations affect SMC function.


title: Modeling the three-way feedback between cellular contractility, actin polymerization, and adhesion turnover resolves the contradictory effects of RhoA and Rac1 on endothelial junction dynamics

abstract: The formation and recovery of gaps in the vascular endothelium governs a wide range of physiological and pathological phenomena, from angiogenesis to atherosclerosis and tumor cell extravasation. However, the interplay between the mechanical and signaling processes that drive dynamic behavior in vascular endothelial cells is not well understood. In this study, we propose a chemo-mechanical model to investigate the maintenance of endothelial junctions as dependent on the crosstalk between actomyosin contractility, VE-cadherin bond turnover, and actin polymerization, which mediate the forces exerted on the cell-cell interface. Our theoretical model reveals that active cell tension can stabilize cadherin bonds within an adhesion, but excessive RhoA signaling can drive bond dissociation and junction failure. While Rac1-mediated actin polymerization aids gap closure, high levels of Rac1 may also facilitate junction weakening. Combining the modeling framework with novel experiments, we identify how dynamic rupture and heal cycles emerge and, further, describe why gaps tend to localize at multi-cell contacts. Beyond, our analysis also indicates that a critical balance between RhoA and Rac1 expression is required to maintain junction stability and limit endothelial dysfunction. The model predicts how pharmacological modulation of actin polymerization and cell contractility impacts junction stability, with predictions subsequently validated experimentally. Our proposed framework can help guide the development of therapeutics that target the Rho family of GTPases and downstream active mechanical processes.


title: Effects of Mutations in the Receptor-Binding Domain of SARS-CoV-2 Spike on its Binding Affinity to ACE2 and Neutralizing Antibodies Revealed by Computational Analysis

abstract: SARS-CoV-2 causing coronavirus disease 2019 (COVID-19) is responsible for one of the most deleterious pandemics of our time. The interaction between the ACE2 receptors at the surface of human cells and the viral Spike (S) protein triggers the infection making the receptor-binding domain (RBD) of the SARS-CoV-2 S-protein a focal target for the neutralizing antibodies (Abs). Despite the recent progress in the development and deployment of vaccines, the emergence of novel variants of SARS-CoV-2 insensitive to Abs produced in response to the vaccine administration and/or monoclonal ones represents upcoming jeopardy. Here, we assessed the possible effects of single and multiple mutations in the RBD of SARS-CoV-2 S-protein on its binding energy to various antibodies and the human ACE2 receptor. The performed computational analysis indicates that while single amino acid replacements in RBD may only cause partial impairment of the Abs binding, moreover, limited to specific epitopes, some variants of SARS-CoV-2 (with as few as 8 mutations), which are already present in the population, may potentially result in a much broader antigenic escape. We also identified a number of point mutations, which, in contrast to the majority of replacements, reduce RBD affinity to various antibodies without affecting its binding to ACE2. Overall, the results provide guidelines for further experimental studies aiming at the identification of the high-risk RBD mutations allowing for an antigenic escape.


title: Automated Modeling of Protein Accumulation at DNA Damage Sites using qFADD.py

abstract:
Cells are exposed to a plethora of influences that can cause damage to DNA and alter the genome, often with detrimental consequences for health. Cells mitigate this damage through a variety of repair protein pathways, and accurate measurement of the accumulation, action, and dissipation timescales of these repair proteins is required to fully understand the DNA damage response. Recently, we described the Q-FADD (Quantitation of Fluorescence Accumulation after DNA Damage) method, which enhances the analytical power of the widely used laser microirradiation technique. In that study, Q-FADD and its preprocessing operations required licensed software and a significant amount of user overhead to find the model of best fit. Here, we present "qFADD.py", an open-source implementation of the Q-FADD algorithm that is available as both a stand-alone software package and on a publicly accessible webserver (https://qfadd.colorado.edu/). Furthermore, we describe significant improvements to the fitting and preprocessing methods that include corrections for nuclear drift and an automated grid-search for the model of best fit. To improve statistical rigor, the grid-search algorithm also includes automated simulation of replicates. As an example, we discuss the recruitment dynamics of the signaling protein PARP1 to DNA damage sites, and we show how to compare different populations of qFADD.py models.

Statement of SignificanceCells are constantly bombarded by factors that can alter or damage their genome, and they have evolved a variety of proteins that can identify and fix this damage. To fully understand how these proteins interact in repair pathways, we need robust methods to quantify the timescales between the initial identification of the DNA damage event and the subsequent protein-protein interactions that lead to repair. Laser microirradiation is a popular method for studying these repair protein cascades in vivo, and methods for quantifying the timescales of recruitment in these experiments have historically been simple to implement but lacking in physical interpretation. Here, we present qFADD.py, the next iteration of the Q-FADD method, which uses Monte Carlo diffusion models to interpret repair protein recruitment timescales to sites of DNA damage. By moving towards automated fitting procedures with minimal bias from the user, qFADD.py provides a statistically robust but low-effort means to analyze laser microirradiation experiments through a biophysical framework.


title: A minimal robophysical model of quadriflagellate self-propulsion

abstract: Locomotion at the microscale is remarkably sophisticated. Microorganisms have evolved diverse strategies to move within highly viscous environments, using deformable, propulsion-generating appendages such as cilia and flagella to drive helical or undulatory motion. In single-celled algae, these appendages can be arranged in different ways around an approximately 10 {micro}m cell body, and coordinated in distinct temporal patterns. Inspired by the observation that some quadriflagellates (bearing four flagella) have an outwardly similar morphology and flagellar beat pattern, yet swim at different speeds, this study seeks to determine whether variations in swimming performance could arise solely from differences in swimming gait. Robotics approaches are particularly suited to such investigations, where the phase relationships between appendages can be readily manipulated. Here, we developed autonomous, algae-inspired robophysical models that can self-propel in a viscous fluid. These macroscopic robots (length and width = 8.5 cm, height = 2 cm) have four independently actuated  flagella that oscillate back and forth under low-Reynolds number conditions (Re[~] [O] (10-1)). We tested the swimming performance of these robot models with appendages arranged in one of two distinct configurations, and coordinated in one of three distinct gaits. The gaits, namely the pronk, the trot, and the gallop, correspond to gaits adopted by distinct microalgal species. When the appendages are inserted perpendicularly around a central  body, the robot achieved a net performance of 0.15 - 0.63 body lengths per cycle, with the trot gait being the fastest. Robotic swimming performance was found to be comparable to that of the algal microswimmers across all gaits. By creating a minimal robot that can successfully reproduce cilia-inspired drag-based swimming, our work paves the way for the design of next-generation devices that have the capacity to autonomously navigate aqueous environments.


title: Using fecal immunochemical tubes for the analysis of gut microbiome has potential to improve colorectal cancer screening

abstract:
BackgroundColorectal cancer (CRC) is an important and challenging public health problem which successful treatment depends on the early detection of the disease. Recently, colorectal cancer specific microbiome signatures have been proposed as an additional marker for CRC detection. A desirable aim would be the possibility to analyze microbiome from the fecal samples collected during CRC screening programs into FIT tubes for fecal occult blood testing.

MethodsWe investigated the impact of the Fecal Immunohistochemical Test (FIT) and stabilization buffer on the microbial community structure in stool samples from 30 volunteers and compared their communities to fresh-frozen samples highlighting also the previously published cancer-specific communities. Altogether 214 samples were analyzed including positive and negative controls using 16S rRNA gene sequencing.

ResultsThe variation between individuals is greater than the differences introduced by collection strategy. The vast majority of the genera are stable for up to 7 days. None of the changes observed between fresh frozen samples and FIT tubes are related to previously shown colorectal-cancer specific bacteria.

ConclusionsOverall, our results show that FIT tubes can be used for profiling the gut microbiota in colorectal cancer screening programs as the community is similar to fresh frozen samples and stable at least for 7 days.

ImpactSample material from FIT tubes could be used in addition to fecal immunochemical tests for future investigations into the role of gut microbiota in colorectal cancer screening programs circumventing the need to collect additional samples and possibly improving the sensitivity of FIT.


title: Gα13 loss promotes tumor progression in the KPC transgenic mouse model of advanced pancreatic cancer

abstract: G13 transduces signals from G protein-coupled receptors. G13 is pro-tumorigenic in epithelial cancer cell lines, which contrasts with its tumor-suppressive function in transgenic mouse models of lymphomas. Here we show that while loss of G13 in pancreatic cell lines decreases tumor growth in vivo, G13 loss in the Kras-driven (KC) mouse model of pancreatic tumor initiation does not affect tumor development or survival. Instead, G13 loss in the Kras/Tp53 (KPC) transgenic mouse model of advanced pancreatic cancer promotes well-differentiated tumors with increased tumor burden and reduced survival. Mechanistically, G13 loss in the KPC mouse model enhances E-cadherin-mediated cell-cell junctions and mTOR signaling. Importantly, human pancreatic cancers with low G13 expression exhibit increased E-cadherin protein expression and mTOR signaling. This work establishes a context-dependent role of G13 in pancreatic tumorigenesis, demonstrating a tumor-suppressive role in transgenic mouse models of advanced pancreatic cancer.


title: First isolation of virulent Tenacibaculum maritimum strains from diseased orbicular batfish (Platax orbicularis) farmed in Tahiti Island

abstract:
The orbicular batfish (Platax orbicularis), also called  Paraha peue in Tahitian, is the most important marine fish species reared in French Polynesia. Sudden and widespread outbreaks of severe  white-patch disease have occurred since 2011 in batfish farms one to three weeks after the transfer of juveniles from bio-secured hatcheries to lagoon cages. With cumulative mortality ranging from 20 to 90%, the sustainability of aquaculture of this species is severely threatened.

In this study, we describe for the first time the isolation from diseased batfish of several strains belonging to the species Tenacibaculum maritimum, a major pathogen of many marine fish species. Histopathological analysis, an experimental bath challenge and a field monitoring study showed that T. maritimum is associated with white-patch disease. Moreover, molecular and serological analyses performed on representative strains revealed some degree of genetic diversity among the isolates, a finding of primary importance for epidemiological studies and for the development of management and control strategies such as vaccination.


title: A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum

abstract: Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grassspecific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.

category: plant biology


title: Structural Covariance Networks in Post-Traumatic Stress Disorder: A Multisite ENIGMA-PGC Study

abstract:
IntroductionCortical thickness (CT) and surface area (SA) are established biomarkers of brain pathology in posttraumatic stress disorder (PTSD). Structural covariance networks (SCN) constructed from CT and SA may represent developmental associations, or unique interactions between brain regions, possibly influenced by a common causal antecedent. The ENIGMA-PGC PTSD Working Group aggregated PTSD and control subjects data from 29 cohorts in five countries (n=3439).

MethodsUsing Destrieux Atlas, we built SCNs and compared centrality measures between PTSD subjects and controls. Centrality is a graph theory measure derived using SCN.

ResultsNotable nodes with higher CT-based centrality in PTSD compared to controls were left fusiform gyrus, left superior temporal gyrus, and right inferior temporal gyrus. We found sex-based centrality differences in bilateral frontal lobe regions, left anterior cingulate, left superior occipital cortex and right ventromedial prefrontal cortex (vmPFC). Comorbid PTSD and MDD showed higher CT-based centrality in the right anterior cingulate gyrus, right parahippocampal gyrus and lower SA-based centrality in left insular gyrus.

ConclusionUnlike previous studies with smaller sample sizes ([≤]318), our study found differences in centrality measures using a sample size of 3439 subjects. This is the first cross-sectional study to examine SCN interactions with age, sex, and comorbid MDD. Although limited to group level inferences, centrality measures offer insights into a nodes relationship to the entire functional connectome unlike approaches like seed-based connectivity or independent component analysis. Nodes having higher centrality have greater structural or functional connections, lending them invaluable for translational treatments like neuromodulation.


title: Gel-like inclusions of C-terminal fragments of TDP-43 sequester and inhibit proteasomes in neurons

abstract: TDP-43 inclusions enriched in C-terminal fragments of ~25kDa ("TDP-25") are associated with neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we analyzed gain-of-function mechanisms of TDP-25 combining cryo-electron tomography, proteomics and functional assays. TDP-25 inclusions are amorphous with gel-like biophysical properties and sequester proteasomes adopting exclusively substrate-processing conformations. This leads to proteostasis impairment, further enhanced by pathogenic mutations. These findings bolster the importance of proteasome dysfunction in ALS/FTD.


title: Structure Can Predict Function in the Human Brain: A Graph Neural Network Deep Learning Model of Functional Connectivity and Centrality based on Structural Connectivity

abstract: Although functional connectivity and associated graph theory measures (e.g., centrality; how centrally important to the network a region is) are widely used in brain research, the full extent to which these functional measures are related to the underlying structural connectivity is not yet fully understood. Graph neural network deep learning methods have not yet been applied for this purpose, and offer an ideal model architecture for working with connectivity data given their ability to capture and maintain inherent network structure. This model applied here to predict functional connectivity and centrality from structural connectivity accounted for 89% of the variance in mean functional connectivity, 56% of the variance in individual-level functional connectivity, 99% of the variance in mean functional centrality, and 81% of the variance in individual-level functional centrality. This model provides a new benchmark for performance and represents a novel finding that functional centrality can be robustly predicted from structural connectivity. Regions of particular importance to the models performance as determined through lesioning are discussed, whereby regions with higher centrality have a higher impact on model performance. Future research on models of patient, demographic, or behavioural data can also benefit from this graph neural network method as it is ideally-suited for capturing connectivity and centrality in brain networks. These results have set a new benchmark for prediction of functional connectivity from structural connectivity, and models like this may ultimately lead to a way to predict functional connectivity in individuals who are unable to do fMRI tasks (e.g., non-responsive patients).


title: Structure Can Predict Function in the Human Brain: A Graph Neural Network Deep Learning Model of Functional Connectivity and Centrality Based on Structural Connectivity


title: Anatomical and Functional Gradients Shape Dynamic Functional Connectivity in the Human Brain

abstract: Large-scale biophysical circuit models can provide mechanistic insights into the fundamental micro-scale and macro-scale properties of brain organization that shape complex patterns of spontaneous brain activity. By allowing local synaptic properties to vary across brain regions, recent large-scale circuit models have demonstrated better fit to empirical observations, such as inter-regional synchrony averaged over several minutes, i.e. static functional connectivity (FC). However, most previous models do not capture how inter-regional synchrony patterns vary over timescales of seconds, i.e., time-varying FC dynamics. Here we developed a spatially-heterogeneous large-scale dynamical circuit model that allowed for variation in local circuit properties across the human cortex. We showed that parameterizing local circuit properties with both anatomical and functional gradients was necessary for generating realistic static and dynamical properties of resting-state fMRI activity. Furthermore, empirical and simulated FC dynamics demonstrated remarkably similar sharp transitions in FC patterns, suggesting the existence of multiple attractors. We found that time-varying regional fMRI amplitude tracked multi-stability in FC dynamics. Causal manipulation of the large-scale circuit model suggested that sensory-motor regions were a driver of FC dynamics. Finally, the spatial distribution of sensory-motor drivers matched the principal gradient of gene expression that encompassed certain interneuron classes, suggesting that heterogeneity in excitation-inhibition balance might shape multi-stability in FC dynamics.