Retraction ATPase motors from three orthologous type IVa pilus systems support promiscuous retraction of the Vibrio cholerae competence pilus Bacterial surface appendages called type IVa pili (T4aP) promote diverse activities including DNA uptake, twitching motility, and virulence. These activities rely on the ability of T4aP to dynamically extend and retract from the cell surface. Dynamic extension relies on a motor ATPase commonly called PilB. Most T4aP also rely on specific motor ATPases, commonly called PilT and PilU, to dynamically and forcefully retract. Here, we systematically assess whether motor ATPases from three orthologous T4aP can functionally complement Vibrio cholerae mutants that lack their endogenous motors. We found that the PilT and PilU retraction ATPases from the three T4aP systems tested are promiscuous and promote retraction of the V. cholerae competence T4aP despite a high degree of sequence divergence. In contrast, the orthologous extension ATPases from the same T4aP systems were not able to mediate extension of the V. cholerae competence T4aP despite exhibiting a similar degree of sequence divergence. Also, we show that one of the PilT orthologs characterized does not support PilU-dependent retraction and provide some data to indicate that the C-terminus of PilT is important for PilU-dependent retraction. Together, our data suggest that retraction ATPases may have maintained a high degree of promiscuity for promoting retraction of T4aP, while extension ATPases may have evolved to become specific for their cognate systems.

IMPORTANCEOne way that bacteria interact with their environments is via hair-like appendages called type IVa pili (T4aP). These appendages dynamically extend and retract from the cell surface via the action of distinct ATPase motors. T4aP are present in diverse bacterial species. Here, we demonstrate that retraction motors from three T4aP are promiscuous, and capable of promoting retraction of a heterologous T4aP system. By contrast, the extension ATPase motors from these same T4aP systems are specific and cannot promote extension of a heterologous T4aP. Thus, these results suggest that T4aP extension may be more tightly regulated compared to T4aP retraction.
Global diversity and distribution of prophages are lineage-specific within the Ralstonia solanacearum plant pathogenic bacterium species complex Ralstonia solanacearum is a destructive plant pathogenic bacterium and the causative agent of bacterial wilt disease, infecting over 200 plant species worldwide. In addition to chromosomal genes, its virulence is mediated by mobile genetic elements including integrated DNA of bacteriophages, i.e., prophages, which may carry fitness-associated auxiliary genes or modulate host gene expression. Although experimental studies have characterised several prophages that shape R. solanacearum virulence, the global diversity, distribution, and wider functional gene content of R. solanacearum prophages is unknown. In this study, prophages were identified in a diverse collection of 192 R. solanacearum draft genome assemblies originating from six continents. Prophages were identified bioinformatically and their diversity investigated using genetic distance measures, gene content, GC, and total length. Prophage distribution was characterised using metadata on R. solanacearum geographic origin and lineage classification (phylotypes), and their functional gene content was assessed by identifying putative prophage-encoded auxiliary genes. In total, 343 intact prophages were identified, forming ten genetically distinct clusters. These included five prophage clusters belonging to the Inoviridae, Myoviridae, and Siphoviridae phage families, and five uncharacterised clusters, possibly representing novel, previously undescribed phages. The prophages had broad geographical distribution being present across multiple continents. However, they were generally host phylogenetic lineage-specific, and overall, prophage diversity was proportional to the genetic diversity of their hosts. The prophages contained a myriad of auxiliary genes involved in metabolism and virulence of both phage and bacteria. Our results show that while R. solanacearum prophages are highly diverse globally, they make lineage-specific contributions to the R. solanacearum accessory genome, which could have resulted from shared coevolutionary history.
Somatic mutation detection: a critical evaluation through simulations and reanalyses in oaks O_LIMutation, the source of genetic diversity, is the raw material of evolution; however, the mutation process remains understudied, especially in plants. Using both a simulation and reanalysis framework, we set out to test the performance of two types of variant callers, generic ones and those developed for cancer research, to detect de novo somatic mutations.
C_LIO_LIIn an in silico experiment, we generated Illumina-like sequence reads spiked with simulated mutations at different allele frequencies to compare the performance of seven commonly-used variant callers to recall them. More empirically, we then reanalyzed two of the largest datasets available for plants, both developed for identifying within-individual variation in long-lived pedunculate oaks.
C_LIO_LIEven in plants, variant callers developed for cancer research outperform generic callers regarding mutation recall and precision, especially at low allele frequency. Such variants at low allele frequency are typically expected for within-individual de novo plant mutations. Reanalysis of published oak data with the best-performing caller based on our simulations identified up to 7x more somatic mutations than initially reported.
C_LIO_LIOur results advocate the use of cancer research callers to boost de novo mutation research in plants, and to reconcile empirical reports with theoretical expectations.
C_LI The initial response of females towards congeneric males matches the propensity to hybridise in Ophthalmotilapia. AO_SCPLOWBSTRACTC_SCPLOWCichlid radiations often harbour closely related species with overlapping niches and distribution ranges. Such species sometimes hybridise in nature, which raises the question how can they coexist. This also holds for the Tanganyika mouthbrooders Ophthalmotilapia ventralis and O. nasuta. Earlier studies found indications of asymmetrical hybridisation with females of O. ventralis accepting males of O. nasuta, but not the other way around. We hypothesised that this was due to differences in the capacity for species recognition. Given the higher propensity of O. ventralis females towards hybridisation, we expect a reduced ability for species recognition in O. ventralis females, compared to O. nasuta females. We staged two experiments, one focusing on 22 female O. nasuta and one on 21 female O. ventralis. These fish were placed in one half of a tank and briefly exposed to a conspecific or a heterospecific male, a conspecific female, or nothing (control). Female response was evaluated by scoring six tracking parameters and by noting the occurrence of ten discrete behaviours before and during the encounter. Females always responded to the presence of another fish by approaching it. Remarkably, for both O. nasuta and O. ventralis, we did not find a different response between encounters with conspecific males and females. However, in agreement with our hypothesis, O. nasuta females behaved differently towards conspecific or heterospecific males, whereas O. ventralis females did not. When presented with a heterospecific male, O. nasuta females performed a lower number of  ram behaviours. Additionally, they never displayed the  flee behaviour, a component of the species mating repertoire that was seen in all but one of the presentations with a conspecific male. Our findings show that differences in species recognition at first encounter predict to a large degree the outcome of the mating process, even in the absence of mating behaviour. RapidoPGS: A rapid polygenic score calculator for summary GWAS data without a test dataset MotivationPolygenic scores (PGS) aim to genetically predict complex traits at an individual level. PGS are typically trained on genome-wide association summary statistics and require an independent test dataset to tune parameters. More recent methods allow parameters to be tuned on the training data, removing the need for independent test data, but approaches are computationally intensive. Based on fine-mapping principles, we present RapidoPGS, a flexible and fast method to compute PGS requiring summary-level GWAS datasets only, with little computational requirements and no test data required for parameter tuning.

ResultsWe show that RapidoPGS performs slightly less well than two out of three other widely-used PGS methods (LDpred2, PRScs, and SBayesR) for case-control datasets, with median r2 difference: -0.0092, -0.0042, and 0.0064, respectively, but up to 17,000-fold faster with reduced computational requirements. RapidoPGS is implemented in R and can work with user-supplied summary statistics or download them from the GWAS catalog.

Availability and implementationOur method is available with a GPL license as an R package from GitHub. Glypiated FGF directs contact-mediated bidirectional signaling to self-regulate tissue-specific dispersion How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and activates target-specific bidirectional FGF-FGFR signaling through cytonemes. FGF-source and recipient ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through CAM-like FGF-FGFR binding. Contact-mediated FGF-FGFR binding induces bidirectional signaling, which, in turn, promotes ASP and source cells to polarize cytonemes toward each other and reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source cell membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion and signaling. Natural Selection as the Sum Over All Histories. As evolution can be connected to the principle of least action, and if it is depicted in evolution-space versus time then it corresponds to the direction of ultimate causation. As an organism evolves and follows a path of proximate causation, if the vector is closely parallel to that of the Ultimate Causation then the changes will confer desirable attributes which will lead to further development. If, however, the variations do not occur in a direction close to that of the ultimate causation vector the evolved organism will quickly die out. Therefore Natural Selection may be viewed as similar to Feynmans "sum over all histories". This approach is compatible with both Neutral Theory and Selection, as it includes both positive and negative mutations and selection. Therefore, the principle of least action gives a direction, but not a purpose, to evolution. Phytoene synthase 2 in tomato fruits remains functional and contributes to abscisic acid formation In ripening tomato fruits, the leaf-specific carotenoids biosynthesis mediated by phytoene synthase 2 (PSY2) is replaced by a fruit-specific pathway by the expression of two chromoplast-specific genes: phytoene synthase 1 (PSY1) and lycopene-{beta}-cyclase (CYCB). Though both PSY1 and PSY2 genes express in tomato fruits, the functional role of PSY2 is not known. To decipher whether PSY2-mediated carotenogenesis operates in ripening fruits, we blocked the in vivo activity of lycopene-{beta}-cyclases in fruits of several carotenoids and ripening mutants by CPTA (2-(4-Chlorophenylthio)triethylamine hydrochloride), an inhibitor of lycopene-{beta}-cyclases. The CPTA-treatment induced accumulation of lycopene in leaves, immature-green and ripening fruits. Even, in psy1 mutants V7 and r that are deficient in fruit-specific carotenoid biosynthesis, CPTA triggered lycopene accumulation but lowered the abscisic acid level. Differing from fruit-specific carotenogenesis, CPTA-treated V7 and r mutant fruits accumulated lycopene but not phytoene and phytofluene. The lack of phytoene and phytofluene accumulation was reminiscent of PSY2-mediated leaf-like carotenogenesis, where phytoene and phytofluene accumulation is never seen. The lycopene accumulation was associated with the partial transformation of chloroplasts to chromoplasts bearing thread-like crystalline structures. Our study uncovers the operation of a parallel carotenogenesis pathway mediated by PSY2 that provides precursors for abscisic acid biosynthesis in ripening tomato fruits.

Significance statementIt is believed that in ripening tomato fruits phytoene synthase 2 that drives carotenoid biosynthesis in leaves is redundant. Contrary to this, we show that in phytoene synthase 1 mutant fruit that is bereft of lycopene, the chemical inhibition of lycopene {beta}-cyclases triggers lycopene accumulation. Our results uncover that phytoene synthase 2 remains functional in ripening fruits and provides precursors for abscisic acid formation in fruits. Muscle Activation Profile During Perturbed Walking is Modulated According to Body State During an unexpected loss of balance, avoiding a fall requires people to readjust their footing rapidly and effectively. We investigated the activation patterns of the ankle and knee muscles, and muscle fiber type recruitment resulting from unannounced, medio-lateral (i.e., right/left) horizontal-surface walking perturbations in twenty healthy adults (27.00{+/-}2.79 years, 10 females). Surface electromyography (sEMG) total spectral power for specific frequency bands (40-60Hz, 60-150Hz, 150-250Hz, 250-400Hz and 400-1000Hz), from tibialis anterior (TA) and vastus lateralis (VL) muscles were analyzed. Compared to non-perturbed walking, we found a significant increase in the total spectral power of lower-extremity muscles during the first 3 seconds after perturbation. When two feet were on the ground in time of perturbation we found a different muscle fiber type recruitment pattern between VL and TA muscles. This was not significant for perturbations implemented when one foot was on the ground. Our findings suggest that muscle operating frequency is modulated in real time to fit body state and somatosensory input in the context of functional goal requirements such as a rapid change of footing in response to unexpected loss of balance in single and double-support phases of gait.

New & NoteworthyTo study muscle spectral profiles in response to loss of balance, we investigated the dynamics of muscle spectral power changes, across different frequency bands after unannounced mechanical perturbations during walking. We showed increased activation of high-frequency motor units of the lower-limb muscles, subside 3 seconds after perturbation. Differences in power increase of specific frequency bands suggest that muscle activation is modulated in real time to fit body state in the context of functional goal requirements. Comparing genome-based estimates of relatedness for use in pedigree-based conservation management Researchers have long debated which estimator of relatedness best captures the degree of relationship between two individuals. In the genomics era, this debate continues, with relatedness estimates being sensitive to the methods used to generate markers, marker quality, and levels of diversity in sampled individuals. Here, we compare six commonly used genome-based relatedness estimators (kinship genetic distance (KGD), Wang Maximum Likelihood (TrioML), Queller and Goodnight (Rxy), Kinship INference for Genome-wide association studies (KING-robust), and Pairwise Relatedness (RAB), allele-sharing co-ancestry (AS)) across five species bred in captivity-including three birds and two mammals-with varying degrees of reliable pedigree data, using reduced-representation and whole genome resequencing data. Genome-based relatedness estimates varied widely across estimators, sequencing methods, and species, yet the most consistent results for known first order relationships were found using Rxy, RAB, and AS. However, AS was found to be less consistently correlated with known pedigree relatedness than either Rxy or RAB. Our combined results indicate there is not a single genome-based estimator that is ideal across different species and data types. To determine the most appropriate genome-based relatedness estimator for each new dataset, we recommend assessing the relative: (1) correlation of candidate estimators with known relationships in the pedigree and (2) precision of candidate estimators with known first-order relationships. These recommendations are broadly applicable to conservation breeding programs, particularly where genome-based estimates of relatedness can complement and complete poorly pedigreed populations. Given a growing interest in the application of wild pedigrees, our results and are also applicable to in-situ wildlife management. Extracellular Matrix Hydrogels Promote Expression of Muscle-Tendon Junction Proteins Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may help understand how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue resulting in junction specific features. ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue specific pre-hydrogel digests. ECM materials were unique in respect to protein composition and included many types of ECM proteins, not just collagens. After digestion and gelation, ECM hydrogels had similar complex viscosities which were less than type I collagen hydrogels at the same concentration. C2C12 myoblasts and tendon fibroblasts were cultured in tissuespecific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. ECM conditioned media had only a minor effect on upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. In addition, the extracellular matrix protein type XXII collagen had similar expression patterns as paxillin, with smaller effect sizes. Using tissue specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscletendon junctions to study the expression of MTJ specific proteins.

Impact StatementThe muscle-tendon junction is an important feature of muscle-tendon units; however, despite crosstalk between the two tissue types, it is overlooked in current research. Deconstructing the cell-matrix interactions will provide the opportunity to study significant junction specific features and markers that should be included in tissue models of the muscletendon unit, while gaining a deeper understanding of the natural junction. This research aims to inform future methods to engineer a more relevant multi-tissue platform to study the muscletendon unit. Improved radiation expression profiling in blood by sequential application of sensitive and specific gene signatures PurposeCombinations of expressed genes can discriminate radiation-exposed from normal control blood samples by machine learning based signatures (with 8 to 20% misclassification rates). These signatures can quantify therapeutically-relevant as well as accidental radiation exposures. The prodromal symptoms of Acute Radiation Syndrome (ARS) overlap those present in Influenza and Dengue Fever infections. Surprisingly, these human radiation signatures misclassified gene expression profiles of virally infected samples as false positive exposures. The present study investigates these and other confounders, and then mitigates their impact on signature accuracy.

MethodsThis study investigated recall by previous and novel radiation signatures independently derived from multiple Gene Expression Omnibus datasets on common and rare non-malignant blood disorders and blood-borne infections (thromboembolism, S. aureus bacteremia, malaria, sickle cell disease, polycythemia vera, and aplastic anemia). Normalized expression levels of signature genes are used as input to machine learning-based classifiers to predict radiation exposure in other hematological conditions.

ResultsExcept for aplastic anemia, these blood-borne disorders modify the normal baseline expression values of genes present in radiation signatures, leading to false-positive misclassification of radiation exposures in 8 to 54% of individuals. Shared changes, predominantly in DNA damage response and apoptosis-related gene transcripts in radiation and confounding hematological conditions, compromise the utility of these signatures for radiation assessment. These confounding conditions (sickle cell disease, thromboembolism, S. aureus bacteremia, malaria) induce neutrophil extracellular traps, initiated by chromatin decondensation, DNA damage response and fragmentation followed by programmed cell death. Riboviral infections (for example, Influenza or Dengue fever) have been proposed to bind and deplete host RNA binding proteins, inducing R-loops in chromatin. R-loops that collide with incoming replication forks can result in incompletely repaired DNA damage, inducing apoptosis and releasing mature virus. To mitigate the effects of confounders, we evaluated predicted radiation-positive samples with novel gene expression signatures derived from radiation-responsive transcripts encoding secreted blood plasma proteins whose expression levels are unperturbed by these conditions.

ConclusionsThis approach identifies and eliminates misclassified samples with underlying hematological or infectious conditions, leaving only samples with true radiation exposures. Diagnostic accuracy is significantly improved by selecting genes that maximize both sensitivity and specificity in the appropriate tissue using combinations of the best signatures for each of these classes of signatures. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. A novel adhesive complex at the base of intestinal microvilli Intestinal epithelial cells form dense arrays of microvilli at the apical membrane to enhance their functional capacity. Microvilli contain a protocadherin-based intermicrovillar adhesion complex localized at their tips which regulates microvillar length and packaging. Here, we identify a second adhesive complex in microvilli of intestinal epithelial cells. This complex is localized at the basal region of microvilli and consists of the adhesion molecule TMIGD1, the phosphoprotein EBP50 and the F-actin - plasma membrane cross-linking protein ezrin. Ternary complex formation requires unmasking of the EBP50 PDZ domains by ezrin binding and is strongly enhanced upon mutating Ser162 located in PDZ domain 2 of EBP50. Dephosphorylation of EBP50 at S162 is mediated by PP1, a serine/threonine phosphatase localized at the microvillar base and involved in ezrin phosphocycling. Importantly, the binding of EBP50 to TMIGD1 enhances the dynamic turnover of EBP50 at microvilli in a Ser162 phosphorylation-dependent manner. We identify an adhesive complex at the microvillar base and propose a potential mechanism that regulates microvillar dynamics in enterocytes. Positive and negative effects of land abandonment on butterfly communities revealed by a hierarchical sampling design across climatic regions Land abandonment may decrease biodiversity but also provides an opportunity for rewilding. It is therefore necessary to identify areas that may benefit from traditional land management practices and those that may benefit from a lack of human intervention. In this study, we conducted comparative field surveys of butterfly occurrence in abandoned and inhabited settlements in 18 regions of diverse climatic zones in Japan to test the hypotheses that species-specific responses to land abandonment correlate with climatic niches and habitat preferences. Hierarchical models that unified species occurrence and habitat preferences revealed that negative responses to land abandonment were associated with species that have cold climatic niches and utilize open habitats, suggesting that species negatively impacted by land abandonment will decline more due to future climate warming. Maps representing species gains and losses due to land abandonment, which were created from the model estimates, showed similar geographic patterns, but some areas exhibited high species losses relative to gains. Our hierarchical modelling approach was useful for scaling up local-scale effects of land abandonment to a macro-scale assessment, which is crucial to developing spatial conservation strategies in the era of depopulation. TMS with fast and accurate electronic control: measuring the orientation sensitivity of corticomotor pathways BackgroundTranscranial magnetic stimulation (TMS) coils allow only a slow, mechanical adjustment of the stimulating electric field (E-field) orientation in the cerebral tissue. Fast E-field control is needed to synchronize the stimulation with the ongoing brain activity. Also, empirical models that fully describe the relationship between evoked responses and the stimulus orientation and intensity are still missing.

ObjectiveWe aimed to (1) develop a TMS transducer for manipulating the E-field orientation electronically with high accuracy at the neuronally meaningful millisecond-level time scale and (2) devise and validate a physiologically based model describing the orientation selectivity of neuronal excitability.

MethodsWe designed and manufactured a two-coil TMS transducer. The coil windings were computed with a minimum-energy optimization procedure, and the transducer was controlled with our custom-made electronics. The electronic E-field control was verified with a TMS characterizer. The motor evoked potential amplitude and latency of a hand muscle were mapped in 3{degrees} steps of the stimulus orientation in 16 healthy subjects for three stimulation intensities. We fitted a logistic model to the motor response amplitude.

ResultsThe two-coil TMS transducer allows one to manipulate the pulse orientation accurately without manual coil movement. The motor response amplitude followed a logistic function of the stimulus orientation; this dependency was strongly affected by the stimulus intensity.

ConclusionThe developed electronic control of the E-field orientation allows exploring new stimulation paradigms and probing neuronal mechanisms. The presented model helps to disentangle the neuronal mechanisms of brain function and guide future non-invasive stimulation protocols. F1F0 ATP Hydrolysis is a Determinant of Metabolic Rate, a Correlate of Lifespan, and a Weakness of Cancer Dramatic rectal temperature decrease, in mice administered with a drug that specifically inhibits F1F0 ATP hydrolysis, suggests that F1F0 ATP hydrolysis is a major determinant of metabolic rate in vivo. Across twelve investigated species, less F1F0 ATP hydrolysis correlates with greater maximal lifespan. Specific drug inhibition of F1F0 ATP hydrolysis exerts potent anti-cancer activity in vitro. Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. Naming of human diseases on the wrong side of history BackgroundIn the medical sphere, understanding naming conventions strengthen the integrity of naming human diseases remains nominal rather than substantial yet. Since the current nosology-based standard for human diseases could not offer a one-size-fits-all corrective mechanism, many idiomatic but flawed names frequently appear in scientific literature and news outlets at the cost of sociocultural impacts.

ObjectiveWe attempt to examine the ethical oversights of current naming practices and propose heuristic rationales and approaches to determine a pithy name instead of an inopportune nosology.

MethodsFirst, we examined the compiled global online news volumes and emotional tones on some inopportune nosology like German measles, Middle Eastern Respiratory Syndrome, Spanish flu, Hong Kong flu, and Huntingtons disease in the wake of COVID-19. Second, we prototypically scrutinize the lexical dynamics and pathological differentials of German measles and common synonyms by leveraging the capacity of the Google Books Ngram Corpus. Third, we demonstrated the empirical approaches to curate an exclusive substitute for an anachronistic nosology German measles based on deep learning models and post-hoc explanations.

ResultsThe infodemiological study shows that the public informed the offensive names with extremely negative tones in textual and visual narratives. The findings of the historiographical study indicate that many synonyms of German measles did not survive, while German measles became an anachronistic usage, and rubella has taken the dominant place since 1994. The PubMedBERT model could identify rubella as a potential substitution for German measles with the highest semantic similarity. The results of the semantic drift experiments further indicate that rubella tends to survive during the ebb and flow of semantic drift.

ConclusionsOur findings indicate that the nosological evolution of anachronistic names could result in sociocultural impacts without a corrective mechanism. To mitigate such impacts, we introduce some ethical principles for formulating an improved naming scheme. Based on deep learning models and post-hoc explanations, our illustrated experiments could provide hallmark references to the remedial mechanism of naming practices and pertinent credit allocations. Bioactive constituents of Verbena officinalis regulate killing efficiency of primary human natural killer cells by accelerating killing processes BackgroundNatural killer (NK) cells play a key role in eliminating tumorigenic and pathogen-infected cells. Verbena officinalis (V. officinalis) has been used as a medical plant in traditional and modern medicine, exhibiting anti-tumor and anti-inflammation activity, but its role in NK cells remains unclear.

PurposeThe impact of bioactive constituents of V. officinalis on immune responses remains largely elusive. In this work we investigated the potential targets of V. officinalis and focused on killing efficiency and related functions of NK cells regulated by bioactive constituents of V. officinalis.

Study design/methodsWe used primary human NK cells from peripheral blood mononuclear cells. Potential regulatory roles of selected compounds in NK killing function-related genes were first analyzed by network pharmacology approaches. Potential engagement between compounds and NK receptors was examined by molecular docking assay. Killing efficiency of NK cells was determined with real-time killing assay and live-cell imaging. Proliferation was examined by CFSE staining. Expression of cytotoxic proteins was analyzed using flow cytometry. Lytic granule release was quantified by CD107a degranulation assay. Contact time required for killing and determination of serial killers were analyzed using live cell imaging results.

ResultsResults from network pharmacology approaches and molecular docking analyses predicted that the five bioactive compounds (acteoside, apigenin, kaempferol, verbenalin and hastatoside) from V. officinalis can regulated various key genes essential for NK effector functions and can bind to NK inhibitory receptors as well as activating receptors, indicating their potential role in regulating NK killing capacity. Analyses of NK killing efficiency revealed that out of these five compounds verbenalin exhibited an encouraging potency to enhance NK killing efficiency. Further investigation showed that verbenalin did not affect proliferation, expression of cytotoxic proteins, or lytic granule degranulation, but rather reduced contact time required for killing, enhanced total killing events per NK cell, and elevated the fraction of killing competent NK cells.

ConclusionsIn this work, we have identified verbenalin as the most potent bioactive constituent from V. officinalis to enhance NK cell killing efficiency via accelerating killing processes and converting non-killers to killing competent NK cells without affecting the lytic granule pathway. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants. Structural and mechanistic basis of reiterative transcription initiation Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5 sequences non-complementary to the DNA template. Here, using crystallography and cryo-EM to define structures, protein-DNA-photocrosslinking to map positions of RNAP leading and trailing edges relative to DNA, and single-molecule DNA nanomanipulation to assess RNAP-dependent DNA unwinding, we show that RNA extension in reiterative transcription initiation: (1) occurs without DNA scrunching, (2) involves a short, 2-3 bp RNA-DNA hybrid, and (3) generates RNA that exits RNAP through the portal by which scrunched nontemplate-strand DNA exits RNAP in standard transcription initiation. The results establish that, whereas RNA extension in standard transcription initiation proceeds through a scrunching mechanism, RNA extension in reiterative transcription initiation proceeds through a slippage mechanism, with slipping of RNA relative to DNA within a short RNA-DNA hybrid, and with extrusion of RNA from RNAP through an alternative RNA exit. Heterogeneity of circulating epithelial cells in breast cancer at single-cell resolution: identifying tumor and hybrid cells Circulating tumor cells and hybrid cells formed by the fusion of tumor cells with normal cells are leading players in metastasis and have prognostic relevance. Circulating tumor cells and hybrid cells are identified as CD45-negative and CD45-positive epithelial cells. However, such an approach is challenging because epithelial cells are observed in the blood of healthy individuals. In this study, we applied single-cell RNA sequencing to profile CD45-negative and CD45-positive circulating epithelial cells (CECs) in 20 breast cancer patients and one healthy donor. DNA ploidy analysis was used to identify the tumor and hybrid cells among CD45[boxh] and CD45+ CECs in patients, respectively. Functional enrichment analysis was applied to characterize aneuploid and diploid cells. Diploid cells were also annotated to generate cell-type candidates and analyzed for copy-number aberrations (CNAs) to confirm or refute their tumor origin. CD45[boxh] and CD45+ CECs were found in cancer patients (25.5 (range 0-404) and median 6.5 (0-147)) and the healthy donor (8 and 11 cells) and divided into three clusters. Two CD45[boxh] CEC clusters were predominantly aneuploid (97% and 98%), but one cluster contained more diploid (59%) than aneuploid cells. CD45+ CECs were mostly diploid: only clusters 1 and 2 had aneuploid cells (16% and 2%). Diploid CD45[boxh] and CD45+ CECs were annotated as different immune cells and surprisingly harbored many CNAs. Cancer-associated signaling pathways were found only in aneuploid cells of CD45[boxh] CEC cluster 1 and diploid cells of CD45+ CEC cluster 1. Thus, our findings suggest that CECs in breast cancer patients are a highly heterogeneous population comprising aneuploid (tumor and hybrid) and diploid (normal) cells. DNA ploidy analysis is an effective instrument for identifying tumor and hybrid cells among CD45[boxh] and CD45+ CECs, respectively. High Spatial Resolution MALDI Imaging Mass Spectrometry ofFresh-Frozen Bone Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis, osteoporosis, osteomyelitis, and cancer. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) enable the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of native osseous tissue for MALDI IMS has been difficult due to the mineralized composition and heterogenous morphology of the tissue, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs allowing the distribution of endogenous lipids to be linked to specific tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with a microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. Subliming matrix does not induce further bone marrow cracking, and recrystallizing the deposited matrix improves lipid signal. High spatial resolution (10 m) MALDI IMS was employed to characterize lipid distributions in fresh-frozen bone, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment. Aggregation Tool for Genomic Concepts (ATGC): A deep learning framework for sparse genomic measures Deep learning has the ability to extract meaningful features from data given enough training examples. Large scale genomic data are well suited for this class of machine learning algorithms; however, for many of these data the labels are at the level of the sample instead of at the level of the individual genomic measures and features. To leverage the power of deep learning for these types of data we turn to a multiple instance learning framework, and present an easily extensible tool built with TensorFlow and Keras. We show how this tool can be applied to somatic variants (featurizing genomic position, sequence context, and read counts) on a range of artificial tasks (classification, regression, Cox regression). In addition, we confirm the model can achieve high performance on real-world problems, accurately classifying samples according to whether they contain a specific variant (hotspot or tumor suppressor), groups of variants (tumor clonality), or a type of variant (microsatellite instability). Our results suggest this framework will lead to improvements on sample-level tasks that require aggregation of a set of genomic measures. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly.