Atypical integrative element with strand-biased circularization activity assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis The exchange of antimicrobial resistance (AMR) genes between aquaculture and terrestrial microbial populations has emerged as a serious public health concern. However, the nature of the mobile genetic elements in marine bacteria is poorly documented. To gain insight into the genetic mechanisms underlying AMR gene transfer from marine bacteria, we mated a multidrug-resistant Vibrio alfacsensis strain with an Escherichia coli strain, and then determined the complete genome sequences of the donor and transconjugant strains. Sequence analysis revealed a multidrug resistance conjugative plasmid in the donor strain, which was integrated into the chromosome of the recipient. The plasmid backbone in the transconjugant chromosome was flanked by two copies of a 7.1 kb unclassifiable integrative element harboring a {beta}-lactamase gene. The 7.1 kb element (named SE-6945) and previously reported Tn6283 share four coding sequences, two of which encode the catalytic R-H-R-Y motif of tyrosine recombinases. Polymerase chain reaction and sequencing experiments revealed that SE-6945 generates a circular copy of its one specific strand without generating an empty donor site, in contrast to the movement of integron gene cassette or ICE/IMEs discovered to date. Copy numbers and location of SE-6945 in the chromosome affected antibiotic resistance levels of the transconjugants. SE-6945/Tn6283-related integrative elements are collectively termed SEs (strand-biased circularizing integrative elements). SEs were identified in the genomes of other Vibrio species. Overall, these results suggest that in addition to conjugative plasmids, SEs are involved in the spread of AMR genes among marine bacteria.
Number and proportion of Plasmodium falciparum gametocytes vary from acute infection to chronic carriage despite unaltered sexual commitment rate In areas of seasonal malaria, resuming transmission every wet season relies on persistent asexual parasites during dry season that maintain the ability to produce gametocytes until the mosquito population resurges, following the start of annual rains. Although human asymptomatic P. falciparum reservoirs in the dry season are widely recognized, the longitudinal dynamics of parasite sexual commitment and gametocytogenesis are unclear. Here, we compared the density and proportion of P. falciparum gametocytes in blood of asymptomatic subjects during the dry season versus subjects with symptomatic malaria in the wet season. Blood concentrations of phospholipids potentially involved in gametocytogenesis and parasite transcriptomes were also compared. While blood densities of gametocytes and asexual parasites were lower during the dry season, we found that gametocytes were proportionally higher during the dry versus wet season. Levels of parasite transcripts involved in sexual commitment were unaltered throughout the year, and phospholipid content in the plasma throughout the year did not affect sexual commitment in vitro. Our experimental data and mathematical modelling support that gametocyte density and proportion diverge as infections progress from recently transmitted to chronic carriage, without significant alterations in the rate of sexual commitment over time.
A biologically accurate model of directional hearing in the parasitoid fly Ormia ochracea This manuscript was compiled on October 5, 2021 Although most binaural organisms localize sound sources using neurological structures to amplify the sounds they hear, some animals use mechanically coupled hearing organs instead. One of these animals, the parasitoid fly Ormia ochracea, has astoundingly accurate sound localization abilities and can locate objects in the azimuthal plane with a precision of 2{degrees}, equal to that of humans. This is accomplished despite an intertympanal distance of only 0.5 mm, which is less than 1/100th of the wavelength of the sound emitted by the crickets that it parasitizes. In 1995, Miles et al. developed a model of hearing mechanics in O. ochracea, which works well for incoming sound angles of less than {+/-}30{degrees}, but suffers from reduced accuracy (up to 60% error) at higher angles. Even with this limitation, it has served as the basis for multiple bio-inspired microphone designs for decades. Here, we present critical improvements to the classic O. ochracea hearing model based on information from 3D reconstructions of O. ochraceas tympana. The 3D images reveal that the tympanal organ has curved lateral faces in addition to the flat front-facing prosternal membranes represented in the Miles model. To mimic these faces, we incorporated spatially-varying spring and damper coefficients that respond asymmetrically to incident sound waves, making a new quasi-two-dimensional (q2D) model. The q2D model has high accuracy (average errors of less than 10%) for the entire range of incoming sound angles. This improved biomechanical hearing model can inform the development of new technologies and may help to play a key role in developing improved hearing aids.

Significance StatementThe ability to identify the location of sound sources is critical to organismal survival and for technologies that minimize unwanted background noise, such as directional microphones for hearing aids. Because of its exceptional auditory system, the parasitoid fly Ormia ochracea has served as an important model for binaural hearing and a source of bioinspiration for building tiny directional microphones with outsized sound localization abilities. Here, we performed 3D imaging of the flys tympanal organs and used the morphological information to improve the current model for hearing in O. ochracea. This model greatly expands the range of biological accuracy from {+/-}30{degrees} to all incoming sound angles, providing a new avenue for studies of binaural hearing and further inspiration for fly-inspired technologies. Therapeutic resistance in acute myeloid leukemia cells is mediated by a novel ATM/mTOR pathway regulating oxidative phosphorylation While leukemic cells are susceptible to various therapeutic insults, residence in the bone marrow microenvironment typically confers protection from a wide range of drugs. Thus, understanding the unique molecular changes elicited by the marrow is of critical importance towards improving therapeutic outcomes. In the present study, we demonstrate that aberrant activation of oxidative phosphorylation serves to induce therapeutic resistance in FLT3 mutant human AML cells challenged with FLT3 inhibitor drugs. Importantly, our findings show that AML cells are protected from apoptosis following FLT3 inhibition due to marrow-mediated activation of ATM, which in turn up-regulates oxidative phosphorylation via mTOR signaling. mTOR is required for the bone marrow stroma-dependent maintenance of protein translation, with selective polysome enrichment of oxidative phosphorylation transcripts, despite FLT3 inhibition. To investigate the therapeutic significance of this finding, we tested the mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib in primary human AML xenograft models. While marrow resident AML cells were highly resistant to quizartinib alone, the addition of everolimus induced profound reduction in tumor burden and prevented relapse. Taken together, these data provide a novel mechanistic understanding of marrow-based therapeutic resistance, and a promising strategy for improved treatment of FLT3 mutant AML patients. Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose) In Vitro and In Vivo AO_SCPLOWBSTRACTC_SCPLOWADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally-occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts) - optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems. Mechanisms of damage prevention, signalling, and repair impact disease tolerance The insect gut is frequently exposed to pathogenic threats and must not only clear these potential infections, but also tolerate relatively high microbe loads. In contrast to the mechanisms that eliminate pathogens, we currently know less about the mechanisms of disease tolerance. We investigated how well-described mechanisms that either prevent, signal, control, or repair damage during infection contribute to the phenotype of disease tolerance. We established enteric infections with the bacterial pathogen Pseudomonas entomophila in transgenic lines of Drosophila melanogaster fruit flies affecting dcy (a major component of the peritrophic matrix), upd3 (a cytokine-like molecule), irc (a negative regulator of reactive oxygen species) and egfr1 (epithelial growth factor receptor). Flies lacking dcy experienced the highest susceptibility, while loss of function of either irc or upd3 reduced tolerance in both sexes. The disruption of egfr1 resulted in a severe loss in tolerance in male flies but had no substantial effect on the ability of female flies to tolerate P. entomophila infection, despite carrying greater microbe loads than males. Together, our findings provide evidence for the role of damage limitation mechanisms in disease tolerance and highlight how sex differences in these mechanisms could generate sexual dimorphism in disease tolerance. 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. Robustness in regulatory networks of Epithelial Mesenchymal Plasticity as a function of positive and negative feedback loops Epithelial-Mesenchymal plasticity (EMP) is a key arm of cancer metastasis and is observed across many contexts. Cells undergoing EMP can reversibly switch between three classes of phenotypes: Epithelial (E), Mesenchymal (M), and Hybrid E/M. While a large number of multistable regulatory networks have been identified to be driving EMP in various contexts, the exact mechanisms and design principles that enable robustness in driving EMP across contexts are not yet fully understood. Here we investigated dynamic and structural robustness in EMP networks with regards to phenotypic distribution and plasticity. We use two different approaches to simulate these networks: a computationally inexpensive, parameter-independent continuous state space boolean model, and an ODE-based parameter-agnostic framework (RACIPE), both of which yield similar phenotypic distributions. Using perturbations to network topology and by varying network parameters, we show that multistable EMP networks are structurally and dynamically more robust as compared to their randomized counterparts, thereby highlighting their topological hallmarks. These features of robustness are governed by a balance of positive and negative feedback loops embedded in these networks. Using a combination of the number of negative and positive feedback loops weighted by their lengths and sign, we identified a metric that can explain the structural and dynamical robustness of these networks. This metric enabled us to compare networks across multiple sizes, and the network principles thus obtained can be used to identify fragilities in large networks without simulating their dynamics. Our analysis highlights a network topology-based approach to quantify robustness in multistable EMP networks.

Significance StatementEpithelial-Mesenchymal plasticity (EMP) is a key arm of cancer metastasis. Despite extensive intra- and inter-tumor heterogeneity, the characteristics of EMP have been observed to be robust across multiple contexts. We hypothesize that topology of EMP regulatory networks contributes towards this robustness. Here, we measure the robustness of EMP in the form of its phenotypic heterogeneity and multistability and show that EMP networks are more robust to dynamical (change in kinetic parameters) and structural (change in network topology) perturbations as compared to their random network counterparts. Furthermore, we propose a network topology-based metric using the nature and length of feedback loops that explains the observed robustness. Our metric hence serves to quantify robustness in multistable EMP networks without simulating their dynamics. Different contribution of the monkey prefrontal and premotor dorsal cortex in decision making during a transitive inference task Several studies have reported similar neural modulations between brain areas of the frontal cortex, such as the dorsolateral prefrontal (DLPFC) and the premotor dorsal (PMd) cortex, in tasks requiring encoding of the abstract rules for selecting the proper action. Here we compared the neuronal modulation of the DLPFC and PMd of monkeys trained to choose the higher rank from a pair of abstract images (target item), selected from an arbitrarily rank-ordered set (A>B>C>D>E>F) in the context of a transitive inference task. Once acquired by trial-and-error, the ordinal relationship between pairs of adjacent images (i.e., A>B; B>C; C>D; D>E; E>F), monkeys were tested in indicating the ordinal relation between items of the list not paired during learning. During these decisions, we observed that the choice accuracy increased and the reaction time decreased as the rank difference between the compared items enhanced. This result is in line with the hypothesis that after learning, the monkeys built an abstract mental representation of the ranked items, where rank comparisons correspond to the items position comparison on this representation. In both brain areas, we observed higher neuronal activity when the target item appeared in a specific location on the screen with respect to the opposite position and that this difference was particularly enhanced at lower degrees of difficulty. By comparing the time evolution of the activity of the two areas, we observed that the neural encoding of target item spatial position occurred earlier in the DLPFC than in the PMd. Metabarcoding faecal samples to investigate spatiotemporal variation in the diet of the endangered Westland petrel (Procellaria westlandica) As top predators, seabirds can be indirectly impacted by climate variability and commercial fishing activities through changes in marine communities. However, high mobility and foraging behaviour enables seabirds to exploit prey distributed patchily in time and space. This capacity to adapt to environmental change can be described through the study of their diet. Traditionally, the diet of seabirds is assessed through the morphological identification of prey remains in regurgitates. This sampling method is invasive for the bird and limited in terms of taxonomic resolution. However, the recent progress in DNA-based approaches is now providing a non-invasive means to more comprehensively and accurately characterize animal diets. Here, we used a non-invasive metabarcoding approach to characterize the diet of the Westland petrel (Procellaria westlandica), which is an endangered burrowing species, endemic to the South Island of New Zealand. We collected 99 fresh faecal samples at two different seasons and in two different sub-colonies. Our aims were to describe the diet of the Westland petrel, investigate seasonal and spatial variation in the petrels diet, and assess potential impacts of the New Zealand fishery industry. We found that amphipods were the most common prey, followed by cephalopods and fish. Our results could be the result of natural foraging behaviour, but also suggest a close link between the composition of prey items and New Zealands commercial fishing activities. In particular, the high abundance of amphipods could be the result of Westland petrels feeding on discarded fisheries waste (fish guts). Our results also showed significant differences in diet between seasons (before hatching vs chick-rearing season) and between sampling sites (two sub-colonies 1.5 km apart), indicating plasticity in the foraging strategy of the Westland petrel. Due to its non-invasive nature, metabarcoding of faecal samples can be applied to large numbers of samples to help describe dietary variation in seabirds and indicate their ecological requirements. In our example, dietary DNA (dDNA) provided valuable information regarding the dietary preferences of an iconic species in New Zealands unique biodiversity. dDNA can thus inform the conservation of endangered or at-risk species that have elusive foraging behaviours. Evolution of phenotypic variance during adaptation to high temperature in Drosophila Shifts in trait means are widely considered as evidence for adaptive responses, but the impact on phenotypic variance remains largely unexplored. Classic quantitative genetics provides a theoretical framework to predict how selection on phenotypic mean affects the variance. In addition to this indirect effect, it is also possible that the variance of the trait is the direct target of selection, but experimentally characterized cases are rare. Here, we studied gene expression variance of Drosophila simulans males before and after 100 generations of adaptation to a novel hot laboratory environment. In each of the two independently evolved populations, the variance of 125 and 97 genes was significantly reduced. We propose that the drastic loss in environmental complexity from nature to the lab may have triggered selection for reduced variance. Our observation that selection could drive changes in the variance of gene expression could have important implications for studies of adaptation processes in natural and experimental populations. Modeling miRNA-driven post-transcriptional regulation by using exon-intron split analysis (EISA) in pigs The contribution of microRNAs (miRNAs) to mRNA regulation has often been explored by post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes of mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Gottingen minipigs. Exon-intron split analysis (EISA) of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of upregulated miRNAs also in the fed state. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, EISA provides a valuable view about the regulation of gene expression, complementary to canonical differential expression analyses. Using total RNA quality metrics for time since deposition estimates in degrading bloodstains Determining the time since deposition (TSD) of bloodstains would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. The physicochemical changes occurring to biomolecules as a bloodstain degrades can be used to approximate the TSD of bloodstains. Our study aims to quantify the timewise degradation trends and temperature dependence found in total RNA from bloodstains without the use of amplification, expanding the scope of the RNA TSD research which has previously targeted mRNA molecules. Whole bovine blood was stored in plastic microcentrifuge tubes at 21{degrees}C or 4{degrees}C and tested over different timepoints spanning one week. Nine RNA metrics were visually assessed and quantified using linear and mixed models; the RNA Integrity Number equivalent (RINe) and the DV200 demonstrated strong negative trends over time and statistical independence. The RINe model fit was high (R2 = 0.60), and while including the biological replicate as a random effect increased the fit for all RNA metrics, no significant differences were found between biological replicates stored at the same temperature for the RINe and DV200 metrics. Importantly, this suggests that these standardized metrics can likely be directly compared between scenarios and individuals, with DV200 having an inflection point at [~]28 hrs. This study provides a novel approach for blood TSD estimates, producing metrics that are not affected by inter-individual variation and improving our understanding of the rapid degradation occurring in bloodstains.

HIGHLIGHTSO_LIAmplification-free analysis of total RNA in degrading bloodstains.
C_LIO_LIShort-term RNA degradation assessment using high-resolution size measurements.
C_LIO_LITotal RNA quality and quantity metrics were assessed across a one-week.
C_LIO_LITotal RNA quality metrics demonstrated the strongest timewise trends.
C_LIO_LIBiological replicates produced similar results for RNA quality metrics.
C_LI The compound of baicalein, wogonin, and oroxylin-A inhibiting EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. ALLOPURINOL BLOCKS THE AORTIC ANEURYSM IN A MOUSE MODEL OF MARFAN SYNDROME VIA REDUCING AORTIC OXIDATIVE STRESS BackgroundThe pathogenesis and progression of aortic aneurysm in Marfan syndrome (MFS) involves dysregulated TGF-{beta} and nitric oxide signaling, altered hemodynamics, and biomechanical forces. Increasing evidence indicates that redox stress participates in MFS aortopathy, though its contribution is not well established. We reported elevated reactive oxygen species (ROS) formation and NADPH oxidase NOX4 upregulation in MFS patients and mice aortic samples. Here we address the contribution of xanthine oxidoreductase (XOR), which is transcribed as dehydrogenase (XDH) and then post-translationally modified to oxidase (XO), catabolizing mainly purines into uric acid and ROS.

Methods and ResultsWe report that in MFS patients, XOR protein levels increased in aortic samples. In MFS mice (Fbn1C1041G/+), XOR mRNA transcripts augmented in the aorta of young but not older MFS mice. Moreover, the enzymatic activity of XO form increased with respect to XDH. The administration of the XOR inhibitor allopurinol (ALO) blocked the progression of aortic root aneurysm in MFS mice. ALO was also protective when administrated before the appearance of aneurysm. MFS-induced elastic fiber fragmentation, fibrotic remodeling, nuclear translocation of pNRF2, and increased 3-nitrotyrosine levels in the aortic tunica media as well as endothelial dysfunction were all prevented by allopurinol. The large overproduction of H2O2 in MFS aorta did not occur in allopurinol-treated mice whose uric acid plasma levels remained unaltered.

ConclusionsThis study strengthens the concept that redox stress is an important determinant of aortic aneurysm formation and progression in MFS and supports a clinical trial for ALO in the pharmacological treatment of MFS aortopathy.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/464182v3_ufig1.gif" ALT="Figure 1">
View larger version (31K):
org.highwire.dtl.DTLVardef@eab02aorg.highwire.dtl.DTLVardef@1d3b39eorg.highwire.dtl.DTLVardef@1b54d0forg.highwire.dtl.DTLVardef@1d61ad0_HPS_FORMAT_FIGEXP  M_FIG C_FIG 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. Simpati: patient classifier identifies signature pathways based on similarity networks for the disease prediction BACKGROUNDPathway-based patient classification is a supervised learning task which implies a model learning pathways as features to predict the classes of patients. The counterpart of enrichment tools for the pathway analysis are fundamental methods for clinicians and biomedical scientists. They allow to find signature cellular functions which help to define and annotate a disease phenotype. They provide results which lead human experts to manually classify patients. It is a paradox that pathwaybased classifiers which natively resolve this objective are not strongly developed. They could simulate the human way of thinking, decipher hidden multivariate relationships between the deregulated pathways and the disease phenotype, and provide more information than a probability value. Instead, there are currently only two classifiers of such kind, they require a nontrivial hyperparameter tuning, are difficult to interpret and lack in providing new insights. There is the need of new classifiers which can provide novel perspectives about pathways, be easy to apply with different biological omics and produce new data enabling a further analysis of the patients.

RESULTSWe propose Simpati, an innovative and interpretable patient classifier based on pathway-specific patient similarity networks. The first classifier to adopt ad-hoc novel algorithms for such graph type. It standardizes the biological high-throughput dataset of patients profiles with a propagation algorithm that considers the interconnected nature of the cells molecules for inferring a new activity score. This allows Simpati to classify with dense, sparse, and non-homogenous omic data. Simpati organizes patients molecules in pathways represented by patient similarity networks for being interpretable, handling missing data and preserving the patient privacy. A network represents patients as nodes and a novel similarity measure determines how much every pair act co-ordinately in a pathway. Simpati detects signature biological processes based on how much the topological properties of the related networks separate the patient classes. In this step, it includes a new cohesive subgroup detection algorithm to handle patients not showing the same pathway activity as the other class members. An unknown patient is then classified by a unique recommender system which considers how much is similar to known patients and distant from being an outlier. Simpati outperforms previously published classifiers on five cancer datasets described with two biological omics, classifies well with sparse data, identifies more relevant pathways associated to the patients disease than the competitors and has the lowest computational requirements.

CONCLUSIONSimpati can serve as generic-purpose pathway-based classifier of patient classes. It provides signature pathways to unveil the altered biological mechanisms of a disease phenotype and to classify patients according to the learnt pathway-specific similarities. The signature condition and patient prediction can be deciphered considering the patient similarity networks which must reveal the members of a patient class more cohesive and similar than the non-members. Simpati divides the pathways in up and downinvolved. Upinvolved when the signaling cascades generated by the altered molecules of the disease patients impact stronger the pathway than the ones of the control class. We provide an R implementation, a graphical user interface and a visualization function for the patient similarity networks. The software is available at: https://github.com/LucaGiudice/Simpati The Temporal Statistics of Musical Rhythm across Western Genres: An Amplitude Modulation Phase Hierarchy Model Statistical learning by the human brain plays a core role in the development of cognitive systems like language and music. Both music and speech have structured inherent rhythms, however the acoustic sources of these rhythms are debated. Theoretically, rhythm structures in both systems may be related to a novel set of acoustic statistics embedded in the amplitude envelope, statistics originally revealed by modelling childrens nursery rhymes. Here we apply similar modelling to explore whether the amplitude modulation (AM) timescales underlying rhythm in music match those in child-directed speech (CDS). Utilising AM-driven phase hierarchy modelling previously applied to infant-directed speech (IDS), adult-directed speech (ADS) and CDS, we test whether the physical stimulus characteristics that yield speech rhythm in IDS and CDS describe rhythm in music. Two models were applied. One utilized a low-dimensional representation of the auditory signal adjusted for known mechanisms of the human cochlear, and the second utilized probabilistic amplitude demodulation, estimating the modulator (envelope) and carriers using Bayesian inference. Both models revealed a similar hierarchically-nested temporal modulation structure across Western musical genres and instruments. Core bands of AM and spectral patterning matched prior analyses of IDS and CDS, and music showed strong phase dependence between slower bands of AMs, again matching IDS and CDS. This phase dependence is critical to the perception of rhythm. Control analyses modelling other natural sounds (wind, rain, storms, rivers) did not show similar temporal modulation structures and phase dependencies. We conclude that acoustic rhythm in language and music has a shared statistical basis. 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. Hidden dynamics of proteasome autoregulation discovered by cryo-EM data-driven deep learning The cellular functions are executed by macromolecular complexes in nonequilibrium dynamic processes, which exhibit a vast diversity of conformational states. Solving conformational continuum of important complexes at atomic level remains a key challenge in the methodology of computational biophysics. Here we introduce a deep-learning framework, named AlphaCryo4D, which enables atomic-level cryogenic electron microscopy reconstructions of nonequilibrium conformational continuum. AlphaCryo4D integrates 3D deep residual learning with manifold embedding of energy landscapes, which simultaneously improves 3D classification accuracy and reconstruction resolution via an energy-based particle-voting algorithm. Applications of this methodology to six experimental datasets demonstrated its generality in breaking resolution barrier of visualizing dynamic components of functional complexes, in choreographing continuous inter-subunit motions and in exploring their  hidden conformational space. Importantly, AlphaCryo4D identified 64 conformers of the substrate-bound human 26S proteasome and reconstituted single-nucleotide-exchange dynamics of proteasomal AAA-ATPase motor during translocation initiation, which together illuminates a grand hierarchical allostery for proteasome autoregulation. The angiotensin antagonist Losartan shifts social reward motivation and punishment feedback sensitivity via modulating midbrain-striato-frontal circuits Social deficits and dysregulations in dopaminergic midbrain-striato-frontal circuits represent transdiagnostic symptoms across psychiatric disorders. Animal models suggest that modulating interactions between the dopamine and renin-angiotensin system with the angiotensin receptor antagonist Losartan (LT) can modulate learning and reward-related processes. We have therefore determined the behavioral and neural effects of LT on social reward and punishment processing in humans. A pre-registered randomized double-blind placebo-controlled between-subject pharmacological design was combined with a social incentive delay fMRI paradigm during which subjects could avoid social punishment or gain social reward. Healthy volunteers received a single-dose of LT (50mg, n=43) or placebo (n=44). Reaction times and emotional ratings served as behavioral outcomes, on the neural level activation, connectivity and social feedback prediction errors were modelled. Relative to placebo, LT switched reaction times and arousal away from prioritizing punishment towards social reward. On the neural level the LT-enhanced motivational salience of social rewards was accompanied by stronger ventral striatum-prefrontal connectivity during reward anticipation and attenuated activity in the ventral tegmental area (VTA) and associated connectivity with the bilateral insula in response to punishment during the outcome phase. Computational modelling further revealed an LT-enhanced social reward prediction error signal in VTA and dorsal striatum. LT shifted motivational and emotional salience away from social punishment towards social reward via modulating distinct core nodes of the midbrain-striato-frontal circuits. The findings document a modulatory role of the renin-angiotensin system in these circuits and associated social processes, suggesting a promising treatment target to alleviate social dysregulations. Identification of the three zinc-binding sites on Tau protein Tau protein has been extensively studied due to its key roles in microtubular cytoskeleton regulation and in the formation of aggregates found in some neurodegenerative diseases. Recently it has been shown that zinc is able to induce tau aggregation by interacting with several binding sites. However, the precise location of these sites and the molecular mechanism of zinc-induced aggregation remain unknown. Here we used Nuclear Magnetic Resonance (NMR) to identify zinc binding sites on hTau40 isoform. These experiments revealed three distinct zinc binding sites on tau, located in the N-terminal part (H14, H32, H94, and H121), the repeat region (H299, C322, H329 and H330) and the C-terminal part (H362, H374, H388 and H407). Further analysis enabled us to show that the C-terminal and the N-terminal sites are independent of each other. Using molecular simulations, we modeled the structure of each site in a complex with zinc. Given the clinical importance of zinc in tau aggregation, our findings pave the way for designing potential therapies for tauopathies.

HighlightsO_LIZinc is known to induce tau aggregation in neurodegenerative diseases
C_LIO_LIZinc binding locations and mechanism are not yet clear
C_LIO_LIUsing NMR we localized 3 zinc binding site on tau
C_LIO_LIBy molecular simulations, we proposed a modeled structure of each site
C_LIO_LIOur findings pave the way for designing potential therapies for tauopathies
C_LI High expression of VRT2 increases the number of rudimentary basal spikelets in wheat Spikelets are the fundamental building blocks of Poaceae inflorescences and their development and branching patterns determine the various inflorescence architectures and grain yield of grasses. In wheat, the central spikelets produce the most and largest grains, while spikelet size gradually decreases acro- and basipetally, giving rise to the characteristic lanceolate shape of wheat spikes. The acropetal gradient correlates with the developmental age of spikelets, however the basal spikelets are developed first and the cause of their small size and rudimentary development is unclear. Here, we adapted G&T-seq, a low-input transcriptomics approach, to characterise gene expression profiles within spatial sections of individual spikes before and after the establishment of the lanceolate shape. We observed larger differences in gene expression profiles between the apical, central and basal sections of a single spike than between any section belonging to consecutive developmental timepoints. We found that SVP MADS-box transcription factors, including VRT-A2, are expressed highest in the basal section of the wheat spike and display the opposite expression gradient to flowering E-class SEP1 genes. Based on multi-year field trials and transgenic lines, we show that higher expression of VRT-A2 in the basal sections of the spike is associated with increased numbers of rudimentary basal spikelets. Our results, supported by computational modelling, suggest that the delayed transition of basal spikelets from vegetative to floral developmental programmes results in the lanceolate shape of wheat spikes. This study highlights the value of spatially resolved transcriptomics to gain new insights into developmental genetics pathways of grass inflorescences.

One sentence summaryLarge transcriptional gradients exist within a wheat spike and are associated with rudimentary basal spikelet development, resulting in the characteristic lanceolate shape of wheat spikes. Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. A general role for MIA3/TANGO1 in secretory pathway organization and function. Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum, an essential process in eukaryotic cells. In vertebrates, the Mia3 gene encodes two major forms of Transport ANd Golgi Organization Protein 1 (TANGO1S and TANGO1L). Here, using genome engineering of human cells, light microscopy, secretion assays, genomics, and proteomics we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function including limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 and SURF4 to the ER-Golgi Intermediate Compartment and dramatic changes to the ultrastructure of the ER-Golgi interface. Disruption of TANGO1 expression also causes significant changes in early secretory pathway gene and protein expression. Disruption of both TANGO1L and TANGO1S expression impairs secretion not only of large proteins, including procollagens, but of all types of secretory cargo including small soluble proteins. Our data support a general role for Mia3/TANGO1 in maintaining both secretory pathway structure and function in vertebrate cells. Prostaglandin EP3 Receptor signaling is required to prevent insulin hypersecretion and metabolic dysfunction in a non-obese mouse model of insulin resistance When homozygous for the LeptinOb mutation (Ob), Black-and-Tan Brachyury (BTBR) mice become morbidly obese and severely insulin resistant, and by 10 weeks of age, frankly diabetic. Previous work has shown Prostaglandin EP3 Receptor (EP3) expression and activity is up-regulated in islets from BTBR-Ob mice as compared to lean controls, actively contributing to their beta-cell dysfunction. In this work, we aimed to test the impact of beta-cell-specific EP3 loss on the BTBR-Ob phenotype by crossing Ptger3 floxed mice with the Rat insulin promoter (RIP)-CreHerr driver strain. Instead, germline recombination of the floxed allele in the founder mouse - an event whose prevalence we identified as directly associated with underlying insulin resistance of the background strain - generated a full-body knockout. Full-body EP3 loss provided no diabetes protection to BTBR-Ob mice, but, unexpectedly, significantly worsened BTBR-lean insulin resistance and glucose tolerance. This in vivo phenotype was not associated with changes in beta-cell fractional area or markers of beta-cell replication ex vivo. Instead, EP3-null BTBR-lean islets had essentially uncontrolled insulin hypersecretion. The selective up-regulation of constitutively-active EP3 splice variants in islets from young, lean BTBR mice as compared to C57BL/6J, where no phenotype of EP3 loss has been observed, provides a potential explanation for the hypersecretion phenotype. In support of this, high islet EP3 expression in Balb/c females vs. Balb/c males was fully consistent with their sexually-dimorphic metabolic phenotype after loss of EP3-coupled Gz protein. Taken together, our findings provide a new dimension to the understanding of EP3 as a critical brake on insulin secretion.

New and NoteworthyIslet Prostaglandin EP3 receptor (EP3) signaling is well-known as up-regulated in the pathophysiological conditions of type 2 diabetes, contributing to beta-cell dysfunction. Unexpected findings in mouse models of non-obese insulin sensitivity and resistance provide a new dimension to our understanding of EP3 as a key modulator of insulin secretion. A previously-unknown relationship between mouse insulin resistance and the penetrance of Rat insulin promoter-driven germline floxed allele recombination is critical to consider when creating beta-cell-specific knockouts.

For Table of Contents Use Only

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/671289v3_ufig1.gif" ALT="Figure 1">
View larger version (37K):
org.highwire.dtl.DTLVardef@15cf1d5org.highwire.dtl.DTLVardef@108f565org.highwire.dtl.DTLVardef@126e479org.highwire.dtl.DTLVardef@61fdf8_HPS_FORMAT_FIGEXP  M_FIG C_FIG