Non-Newtonian Blood Rheology Impacts Left Atrial Stasis in Patient-Specific Simulations The lack of mechanically effective contraction of the left atrium (LA) during atrial fibrillation (AF) disturbs blood flow, increasing the risk of thrombosis and ischemic stroke. Thrombosis is most likely in the left atrial appendage (LAA), a small narrow sac where blood is prone to stagnate. Slow flow promotes the formation of erythrocyte aggregates in the LAA, also known as rouleaux, causing viscosity gradients that are usually disregarded in patient-specific simulations. To evaluate these non-Newtonian effects, we built atrial models derived from 4D computed tomography scans of patients and carried out computational fluid dynamics simulations using the Carreau-Yasuda constitutive relation. We examined six patients, three of whom had AF and LAA thrombosis or a history of transient ischemic attacks (TIAs). We modeled the effects of hematocrit and rouleaux formation kinetics by varying the parameterization of the Carreau-Yasuda relation and modulating non-Newtonian viscosity changes based on residence time. Comparing non-Newtonian and Newtonian simulations indicates that slow flow in the LAA increases blood viscosity, altering secondary swirling flows and intensifying blood stasis. While some of these effects can be subtle when examined using instantaneous metrics like shear rate or kinetic energy, they are manifested in the blood residence time, which accumulates over multiple heartbeats. Our data also reveal that LAA blood stasis worsens when hematocrit increases, offering a potential new mechanism for the clinically reported correlation between hematocrit and stroke incidence. In summary, we submit that hematocrit-dependent non-Newtonian blood rheology should be considered in calculating patient-specific blood stasis indices by computational fluid dynamics.
New Segmentation and Feature Extraction Algorithm for Classification of White Blood Cells in Peripheral Smear Images This article addresses a new method for the classification of white blood cells (WBCs) using image processing techniques and machine learning methods. The proposed method consists of three steps: detecting the nucleus and cytoplasm, extracting features, and classification. At first, a new algorithm is designed to segment the nucleus. For the cytoplasm to be detected, only a part of it located inside the convex hull of the nucleus is involved in the process. This attitude helps us overcome the difficulties of segmenting the cytoplasm. In the second phase, three shapes and four novel color features are devised and extracted. Finally, by using an SVM model, the WBCs are classified. The segmentation algorithm can detect the nucleus with a dice similarity coefficient of 0.9675. The proposed method can categorize WBCs in Raabin-WBC, LISC, and BCCD datasets with accuracies of 94.65 %, 92.21 %, and 94.20 %, respectively. It is worth mentioning that the hyperparameters of the classifier are fixed only with the Raabin-WBC dataset, and these parameters are not readjusted for LISC and BCCD datasets. The obtained results demonstrate that the proposed method is robust, fast, and accurate.
Transformations, Lineage Comparisons, and Analysis of Down to Up Protomer States of Variants of the SARS-CoV-2 Prefusion Spike Protein Including the UK Variant B.1.1.7 Monitoring and strategic response to variants in SARS-CoV-2 represents a considerable challenge in the current pandemic, as well as potentially future viral outbreaks of similar magnitude. In particular mutations and deletions involving the virions prefusion Spike protein have significant potential impact on vaccines and therapeutics that utilize this key structural viral protein in their mitigation strategies. In this study, we have demonstrated how dominant energetic landscape mappings ("glue points") coupled with sequence alignment information can potentially identify or flag key residue mutations and deletions associated with variants. Surprisingly, we also found excellent homology of stabilizing residue glue points across the lineage of {beta} coronavirus Spike proteins, and we have termed this as "sequence homologous glue points". In general, these flagged residue mutations and/or deletions are then computationally studied in detail using all-atom biocomputational molecular dynamics over approximately one microsecond in order to ascertain structural and energetic changes in the Spike protein associated variants. Specifically, we examined both a theoretically-based triple mutant and the so-called UK or B.1.1.7 variant. For the theoretical triple mutant, we demonstrated through Alanine mutations, which help "unglue" key residue-residue interactions, that these three key stabilizing residues could cause the transition of Down to Up protomer states, where the Up protomer state allows binding of the prefusion Spike protein to hACE2 host cell receptors, whereas the Down state is believed inaccessible. Thus, we are able to demonstrate the importance of glue point residue identification in the overall stability of the prefusion Spike protein. For the B.1.1.7 variant, we demonstrated the critical importance of D614G and N5017 on the structure and binding, respectively, of the Spike protein. Notably, we had previously identified D614 as a key glue point in the inter-protomer stabilization of the Spike protein prior to the emergence of its mutation. The mutant D614G is a structure breaking Glycine mutation demonstrating a relatively more distal Down state RBD and a more stable conformation in general. In addition, we demonstrate that the mutation N501Y may significantly increase the Spike protein binding to hACE2 cell receptors through its interaction with Y41 of hACE2 forming a potentially strong hydrophobic residue binding pair. We note that these two key mutations, D614G and N501Y, are also found in the so-called South African (SA; B.1.351) variant of SARS-CoV-2. Future studies along these lines are, therefore, aimed at mapping glue points to residue mutations and deletions of associated prefusion Spike protein variants in order to help identify and analyze possible "variants of interest" and optimize efforts aimed at the mitigation of this current and future virions. Development of a novel PROTAC using the nucleic acid aptamer as a targeting ligand for tumor-selective degradation of nucleolin PROteolysis TArgeting Chimeras (PROTACs) induce targeted protein degradation by hijacking the intracellular ubiquitin proteasome system, thus emerging as a new strategy for drug development. However, most PROTACs generated lack cell-type selectivity and have poorly soluble in water. To address this drawback, we developed a novel PROTAC ZL216 using aptamer AS1411 as a targeting ligand of nucleolin to conjugate with a small molecule ligand of E3 ligase VHL, which shows high aqueous solubility and serum stability. Based on the differential expression of nucleolin on the cell surface, ZL216 could bind to and internalize into breast cancer cells, but not normal breast cells. Furthermore, we revealed that ZL216 promoted the formation of a nucleolin-ZL216-VHL ternary complex in breast cancer cells and potently induced nucleolin degradation in vitro and in vivo. As a result, ZL216 inhibited the proliferation and migration of breast cancer cells. These studies demonstrate that in addition to peptides and small molecule compounds, nuclei acid aptamers can also be used to generate PROTACs, which broadens the toolbox constructing PROTACs and provides a promising strategy for development of tumor-selective PROTACs. 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. Quercetin handles cellular oxidant / antioxidant systems and mitigates immunosenescence hallmarks in human PBMCs: an in vitro study Immunosenescence, oxidative stress, and low vaccine efficacy are important symptoms of aging. The goal of our study was to see if quercetin had anti-aging and stimulating effects on PBMC immune cells in vitro. In the presence of concanavalin a, PBMCs were isolated from healthy elderly and young people and cultured in a complete RPMI-1640 medium supplemented with quercetin. Cell proliferation was assessed using the MTT colorimetric assay after a 48-hour incubation period. Spectrophophotometric assays were used to assess oxidative biomarkers (PC, MDA, and GSH). The ELISA method was used to determine the amount of IL-2 released. A Griess reagent was used to investigate iNOS activity. When compared to young control cells, aged PBMCs had lower proliferation potency, lower IL-2 and NO release, and higher MDA and PC levels. Importantly, quercetin-treated aged PBMCs have a high proliferative response comparable to young cells, restored iNOS activity, and increased levels of GSH antioxidant defences. In comparison to untreated aged PBMCs, treated PBMCs have lower lipo-oxidative damage but higher PC levels. Quercetin may be used as a promising dietary vaccinal adjuvant in the elderly, it has significant effects in reducing immunosenescence hallmarks, as well as mitigating the lipo-oxidative stress in PBMCs cells.

Practical abstractQuercetin is a common dietary polyphenol found in fruits and vegetables that is well known for its antioxidant activity in radical scavenging and anticancer properties characterized by immune system stimulation. In vitro, quercetin boosts immune responses in the elderly and reduces immunosenescence symptoms, according to our findings. As a result, this molecule could be a safe and promising vaccine adjuvant for boosting immunity and reducing aging complications in immune cells as well as reducing oxidative stress in PBMC cells. Neutralization of SARS-CoV-2 Omicron BA.2 by Therapeutic Monoclonal Antibodies Monoclonal antibody therapy for the treatment of SARS-CoV-2 infection has been highly successful in decreasing disease severity; however, the recent emergence of the heavily mutated Omicron variant has posed a challenge to this treatment strategy. The Omicron variant BA.1 has been found to evade neutralization by several of the therapeutic monoclonal antibodies authorized for emergency use, while Vir-7831 and a cocktail consisting of monoclonal antibodies AZD8895+AZD1061 retain significant neutralizing activity. A newly emerged variant, Omicron BA.2, containing some of the BA.1 mutations plus an additional 6 mutations and 3 deletions, 3 of which lie in the receptor binding domain, has been found to be spreading with increased transmissibility. We report here, using spike protein-pseudotyped lentiviruses, decreased neutralization of BA.2 by several therapeutic monoclonal antibodies but that the mixture of AZD8895+AZD1061 retained substantial neutralizing activity against BA.2. Spatial and Texture Analysis of Root System Distribution with Earth Mover's Distance (STARSEED) PurposeRoot system architectures are complex, multidimensional, and challenging to characterize effectively for agronomic and ecological discovery.

MethodsWe propose a new method, Spatial and Texture Analysis of Root SystEm distribution with Earth movers Distance (STARSEED), for comparing root system distributions that incorporates spatial information through a novel application of the Earth Movers Distance (EMD).

ResultsWe illustrate that the approach captures the response of sesame root systems for different genotypes and soil moisture levels. STARSEED provides quantitative and visual insights into changes that occur in root architectures across experimental treatments.

ConclusionSTARSEED can be generalized to other plants and provides insight into root system architecture development and response to varying growth conditions not captured by existing root architecture metrics and models. The code and data for our experiments are publicly available: https://github.com/GatorSense/STARSEED. Microbial biomass, composition, and functions are responsible for the differential removal of trace organic chemicals in biofiltration systems Biofiltration processes help to remove trace organic chemicals (TOrCs) both in wastewater and drinking water treatment systems. However, the detailed TOrCs biotransformation mechanisms as well as the underlying drivers behind the variability of site specific transformation processes remain elusive. In this study, we used laboratory batch incubations to investigate the biotransformation of 51 TOrCs in eight bioactive filter materials of different origins treating a range of waters, from wastewater effluents to drinking water. Microscopy, 16S rRNA amplicon and whole metagenome sequencing for assessing associations between the biotransformation rate constants, microbial composition and genetic potential complemented chemical analysis. We observed strong differences in the mean global removal of TOrCs between the individual sand filters (-1.4% to 58%), which were mirrored in overall biomass, microbial community composition, and enzyme encoding genes. From the six investigated biomass markers, ATP turned out to be a major predictor of the mean global biotransformation rate, while compound specific biotransformations were correlated with the microbial community composition. High biomass ecosystems were indicated in our systems by a dominance of Nitrospirae, but individual TOrC biotransformation was statistically connected to rare taxa (< 2%) such as Hydrogenophaga, or indiviudal functions such as the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase encoding genes. In general, this study provides new insights into so far rarely addressed variability of TOrCs biotransformation. We propose novel biological indicators for the removal performance of TOrCs in biofiltration systems, highlighting the role of living biomass in predicting and normalizing the global transformation, and the role of the microbial community for the individual transformation of TOrCs in engineered and natural systems.

Contribution to the Field StatementTrace organic chemicals (TOrCs) are an emerging problem in the aquatic environment that has attracted global attention over the last decade. Recent research efforts on this topic have increased our knowledge on the transformation of TOrCs and various technologies have been developed to improve their removal. In this study, we investigated a wide range of biotransformation of TOrCs by eight sand filter materials from wastewater and water treatment plants. Biotransformation rate constants were calculated using first-order kinetics to evaluate TOrC removal performance. We reevaluated the role of biomass and could thus explain a greater part of the global TOrC removal performance. The remaining variation in removal rates of individual compounds correlated with the microbiome of the biofilter. Rare biosphere lineages and specific enzyme categories genes were correlated with the removal of certain compounds. In summary, our research identified future indicators for successful biotransformation of TOrCs in biofilter systems. A unified mechanism for innate and learned visual landmark guidance in the insect central complex Insects can navigate efficiently in both novel and familiar environments, and this requires flexiblity in how they are guided by sensory cues. A prominent landmark, for example, can ellicit strong innate behaviours (attraction or menotaxis) but can also be used, after learning, as a specific directional cue as part of a navigation memory. However, the mechanisms that allow both pathways to co-exist, interact or override each other are largely unknown. Here we propose a model for the behavioural integration of innate and learned guidance based on the neuroanatomy of the central complex (CX), adapted to control landmark guided behaviours. We consider a reward signal provided either by an innate attraction to landmarks or a long-term visual memory in the mushroom bodies (MB) that modulates the formation of a local vector memory in the CX. Using an operant strategy for a simulated agent exploring a simple world containing a single visual cue, we show how the generated short-term memory can support both innate and learned steering behaviour. In addition, we show how this architecture is consistent with the observed effects of unilateral MB lesions in ants that cause a reversion to innate behaviour. We suggest the formation of a directional memory in the CX can be interpreted as transforming rewarding (positive or negative) sensory signals into a mapping of the environment that describes the geometrical attractiveness (or repulsion). We discuss how this scheme might represent an ideal way to combine multisensory information gathered during the exploration of an environment and support optimized cue integration.

1 Author summaryIn this paper, we modeled the neural pathway allowing insects to perform landmark guided behaviours using their internal compass. First, we observed the intrinsic property of the connectome, extracted from drosophila online database, between the internal compass neurons and the steering neurons to support an oriented behaviour towards a single landmark. Then, we proposed and evaluated an adaptation of the bees path integration neural circuit, to sustain flexible landmark guidance behaviours such as attraction or menotaxis. We showed the model ability to form a memory during the exploration of the local environment to support both innate or learned navigation behaviour using a single landmark in the environment. In addtion, we demonstrated the transformation of a simple goodness/badness signal, from innate or long-term memory pathways, into an oriented steering signal that could be applied to other sensory pathways. Furthermore, by reproducing lesion experiments in the mushroom bodies of wood ants we highlight the consistency of the model with biological observation. We then discuss the different emergent properties and the potential outcome that this local, and operant, memory supports. The neurogenic fate of the hindbrain boundaries relies on Notch-dependent asymmetric cell divisions The generation of cell diversity in the central nervous system occurs during embryogenesis and requires a precise balance between cell proliferation, commitment to specific fates, and further neuronal differentiation. The cellular and molecular mechanisms regulating this balance in the embryonic brain are still poorly understood. Here we study how the neurogenic capacity in the embryonic hindbrain is spatiotemporally allocated, and how the fate and the growth of the hindbrain boundary cells are regulated. By generating a CRISPR-based knock-in zebrafish transgenic line to specifically label the hindbrain boundaries, we unveiled that boundary cells undergo a functional transition to become neurogenic during hindbrain segmentation concurrently as they maintain the progenitor cell pool. Boundary cells engaged in neurogenesis coinciding with the onset of Notch signaling, which triggered their asymmetrical cell division. Our findings reveal that distinct neurogenic phases take place during hindbrain growth and suggest that boundary cells contribute to refine the final number, identity, and proportion of neurons in the brain.

SUMMARYHindbrain boundary cells undergo a functional transition to become neurogenic concurrently as they are maintained as a progenitor cell pool. This involves a behavioral switch from symmetrically dividing progenitor cells to asymmetrically dividing progenitors, which depends on Notch-activity. Classification of Pacemaker Dynamics in the Mouse Intestine by Field Potential Microimaging The flexible and sophisticated movement of the gastrointestinal (GI) tract implies the involvement of mechanisms other than enteric neural circuits, to coordinate excitation in microregions. We thus performed microimaging of pacemaker dynamics in the small intestine of mice since it contains typical network-forming pacemaker cells. A dialysis membrane-reinforced low-impedance microelectrode array (MEA) enabled field potentials over a wide frequency range to be stably measured in microregions. The pacemaker dynamics were classified into basic patterns despite large variations. In the developmental process, pacemaker activity was categorized as either an  expanding or a  migrating pattern that was initiated in or propagated to the MEA sensing area, respectively. The intercellular current of the volume conductor complicated the waveform of both activities. The existence of  expanding and  migrating patterns was attributable to duplicated pacemaker systems such as intracellular Ca2+ oscillation-activated and voltage-gated mechanisms. Additionally, from the spatio-temporal feature during the period of pacemaker events, the  bumpy/aberrant pattern was defined by aberrant, incoherent propagation, and associated with local impairment of excitability, while the  colliding/converging pattern involved the interaction of multiple activities in the MEA area. Interconversion between the four micro-coordination patterns occurred in the same microregion. 5-Hydroxytryptamine (5-HT) promoted  migrating activity, implying an improvement or restoration of spatial conductivity. These results agree well with the action of 5-HT to change GI movement toward propulsion. In conclusion, our MEA method of microimaging classification enables the quantitative assessment of spatio-temporal electric coordination underlying GI motility, suggesting its application to small model animals. Comparative reconstruction of the predatory feeding structures of the polyphenic nematode Pristionchus pacificus Pristionchus pacificus is a nematode model for the developmental genetics of morphological polyphenism, especially at the level of individual cells. Morphological polyphenism in this species includes an evolutionary novelty, moveable teeth, which have enabled predatory feeding in this species and others in its family (Diplogastridae). From transmission electron micrographs of serial thin sections through an adult hermaphrodite of P. pacificus, we three-dimensionally reconstructed all epithelial and myoepithelial cells and syncytia, corresponding to 74 nuclei, of its face, mouth, and pharynx. We found that the epithelia that produce the predatory morphology of P. pacificus are identical to Caenorhabditis elegans in the number of cell classes and nuclei. However, differences in cell form, spatial relationships, and nucleus position correlate with gross morphological differences from C. elegans and outgroups. Moreover, we identified fine-structural features, especially in the anteriormost pharyngeal muscles, that underlie the conspicuous, left-right asymmetry that characterizes the P. pacificus feeding apparatus. Our reconstruction provides an anatomical map for studying the genetics of polyphenism, feeding behavior, and the development of novel form in a satellite model to C. elegans. Systematic temporal analysis of peripheral blood transcriptomes using TrendCatcher identifies early and persistent neutrophil activation as a hallmark of severe COVID-19 Studying temporal gene expression shifts during disease progression provides important insights into the biological mechanisms that distinguish adaptive and maladaptive responses. Existing tools for the analysis of time course transcriptomic data are not designed to optimally identify distinct temporal patterns when analyzing dynamic differentially expressed genes (DDEGs). Moreover, there is a lack of methods to assess and visualize the temporal progression of biological pathways mapped from time course transcriptomic datasets. In this study, we developed an open-source R package TrendCatcher (https://github.com/jaleesr/TrendCatcher), which applies the smoothing spline ANOVA model and break point searching strategy to identify and visualize distinct dynamic transcriptional gene signatures and biological processes from longitudinal datasets. We used TrendCatcher to perform a systematic temporal analysis of COVID-19 peripheral blood transcriptomes, including bulk RNA-seq and scRNA-seq time course data. TrendCatcher uncovered the early and persistent activation of neutrophils and coagulation pathways as well as impaired type I interferon (IFN-I) signaling in circulating cells as a hallmark of patients who progressed to severe COVID-19, whereas no such patterns were identified in individuals receiving SARS- CoV-2 vaccinations or patients with mild COVID-19. These results underscore the importance of systematic temporal analysis to identify early biomarkers and possible pathogenic therapeutic targets. 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. Circadian-period variation underlies the local adaptation of photoperiodism in the short-day plant Lemna aequinoctialis. Phenotypic variation is the basis for trait adaptation via evolutionary selection.1, 2, 3 However, the driving forces behind the quantitative trait variations remain unclear owing to their complexity at the molecular level.4, 5, 6 This study focused on the natural variation of the free-running period (FRP) of the circadian clock because FRP is a determining factor of the internal clock phase (chronotype), which is responsible for physiological timing during a day.7 Although natural variations in FRP have been widely reported,8, 10, 11 few studies have shown the association between FRP and adaptive temporal traits. As a clock-dependent physiological process, photoperiodism is a typical target of local adaptation.12, 13 Lemna aequinoctialis in Japan is a paddy-field duckweed exhibiting a latitudinal cline of critical day-lengths (CDLs) for short-day flowering.14 To investigate the relationship between FRP and CDL, we collected 72 strains of L. aequinoctialis within a latitudinal range between 31.5{degrees}N to 43.8{degrees}N. We found a significant correlation (P = 7.5E-8) between FRPs and locally adaptive CDLs, confirming that the variation in FRP-dependent chronotypes underlies geographically differentiated photoperiodism. Diel transcriptome analysis revealed that the induction timing of a florigen gene is key for connecting chronotypes to photoperiodism at the molecular level. Based on these results, we propose a fundamental rule concerning the "chronotype effect" in evolution: the variation of FRP functions as a resource for the variation of temporal traits. This study highlights the adaptive significance of FRP variation and provides a reason for the maintenance of FRP variation in natural populations. 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. The impact of housing conditions on mesenchymal stromal/stem cell populations differ between adipose tissue and skeletal muscle BackgroundIn pigs, the ratio between lean mass and fat mass in the carcass determines production efficiency and is strongly influenced by the number and size of cells in tissues. During growth, the increase in the number of cells results from the recruitment of different populations of multipotent mesenchymal stromal/stem cells (MSCs) residing in the tissues. We hypothesized that the impact of hygiene of housing conditions during growth on the proportions of MSCs in adipose tissue and skeletal muscle may differ between pigs with different residual feed intake (RFI), a measure of feed efficiency.

MethodsAt the age of 12 weeks, Large White pigs from two lines divergently selected for low and high RFI were housed in two contrasting hygiene conditions (good vs poor). After six weeks, pigs were slaughtered (n = 30; 5-9/group). Samples of subcutaneous adipose tissue and longissimus skeletal muscle were collected, and cells from the stromal vascular fraction (SVF), which includes mesenchymal stromal/stem cell populations, were isolated from each tissue. Adipose and muscle cell populations from the SVF were phenotyped by flow cytometry using antibodies that targeted different cell surface markers (CD45 to separate hematopoietic cells from MSCs; CD34, CD38, CD56 and CD140a to identify MSC populations with adipogenic and/or myogenic potential).

ResultsAdipose tissue and muscle shared some common MSC populations although MSC diversity was higher in muscle than in adipose tissue. In muscle, the CD45-CD56+CD34-CD140a+ and CD45-CD56+CD34+CD140a+ cell populations were abundant. Of these two cell populations, only the proportions of CD45-CD56+CD34+CD140a+ cells increased (P < 0.05) in pigs housed in poor hygiene as compared with pigs in good hygiene conditions. For the CD45-CD56-CD34- cell population, present in low proportion, there was an interaction between hygiene condition and genetic line (P < 0.05) with a decrease in low RFI pigs housed in poor hygiene conditions. In adipose tissue, the two abundant MSC populations were CD45-CD56-CD34- and CD45-CD56+CD34-. The proportion of CD45-CD56-CD34- cells increased (P < 0.05) whereas the proportion of CD45-CD56+CD34- tended to decrease (P < 0.1) in pigs housed in poor conditions. This study shows that the proportions of some MSC populations were affected by hygiene of housing conditions in a tissue-dependent manner in pigs of both RFI lines. It suggests that MSCs may play a significant role in adipose tissue and skeletal muscle homeostasis and may influence later growth and body composition in growing animals. From GWAS Variant to Function: a Study of ~148,000 Variants for Blood Cell Traits. Genome-wide association studies (GWAS) have identified hundreds of thousands of genetic variants associated with complex diseases and traits. However, most variants are noncoding and not clearly linked to genes, making it challenging to interpret these GWAS signals. We present a systematic variant-to-function study, prioritizing the most likely functional elements of the genome for experimental follow-up, for >148,000 variants identified for hematological traits. Specifically, we developed VAMPIRE: Variant Annotation Method Pointing to Interesting Regulatory Effects, an interactive web application implemented in R Shiny (http://shiny.bios.unc.edu/vampire/). This tool efficiently integrates and displays information from multiple complementary sources, including epigenomic signatures from blood cell relevant tissues or cells, functional and conservation summary scores, variant impact on protein and gene expression, chromatin conformation information, as well as publicly available GWAS and phenome-wide association study (PheWAS) results. Leveraging data generated from independently performed functional validation experiments, we demonstrate that our prioritized variants, genes, or variant-gene links are significantly more likely to be experimentally validated. This study not only has important implications for systematic and efficient revelation of functional mechanisms underlying GWAS variants for hematological traits, but also provides a prototype that can be adapted to many other complex traits, paving the path for efficient variant to function (V2F) analyses. Migrators Within Migrators: Exploring Transposable Element Landscapes, Activity, and Host Gene Associations in the Monarch Butterfly, Danaus plexippus BackgroundLepidoptera (butterflies and moths) are an important model system in ecology and evolution. A high-quality chromosomal genome assembly is available for the monarch butterfly (Danaus plexippus), but it lacks an in-depth transposable element (TE) annotation, presenting an opportunity to explore monarch TE dynamics and the impact of TEs on shaping the monarch genome.

ResultsWe find 6.21% of the monarch genome is comprised of TEs, a reduction of 6.85% compared to the original TE annotation performed on the draft genome assembly. Monarch TE content is low compared to two closely related species with available genomes, Danaus chrysippus (33.97% TE) and Danaus melanippus (11.87% TE). The biggest TE contributions to genome size in the monarch are LINEs and Penelope-like elements, and three newly identified families, r2-hero_dPle (LINE), penelope-1_dPle (Penelope-like), and hase2-1_dPle (SINE), collectively contribute 34.92% of total TE content. We find evidence of recent TE activity, with two novel Tc1 families rapidly expanding over recent timescales (tc1-1_dPle, tc1- 2_dPle). LINE fragments show signatures of genomic deletions indicating a high rate of TE turnover. We investigate associations between TEs and wing colouration and immune genes and identify a three-fold increase in TE content around immune genes compared to other host genes.

ConclusionsWe provide a detailed TE annotation and analysis for the monarch genome, revealing a considerably smaller TE contribution to genome content compared to two closely related Danaus species with available genome assemblies. We identify highly successful novel DNA TE families rapidly expanding over recent timescales, and ongoing signatures of both TE expansion and removal highlight the dynamic nature of repeat content in the monarch genome. Our findings also suggest that insect immune genes are promising candidates for future interrogation of TE-mediated host adaptation. Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies Dementia with Lewy bodies is pathologically defined by the cytoplasmic accumulation of alpha-synuclein within neuronal cells in the brain. Alpha-synuclein is predominately pre-synaptic, but has been reported present in various subcellular compartments in cell and animal models. In particular, nuclear alpha-synuclein is evident in-vitro and in disease models and has been associated with altered DNA integrity, gene transcription, nuclear homeostasis. However, owing to various factors, the presence of alpha-synuclein in the nuclei of human brain cells remains controversial, as does its role in synucleinopathies.

Here, we close this gap and provide a unique demonstration confirming the presence of nuclear alpha-synuclein in post-mortem brain tissue obtained from cases of dementia with Lewy bodies as well as from controls via immunohistochemistry, immunoblot, and label-free mass-spectrometry.

Discrete intra-nuclear alpha-synuclein puncta reactive against phosphorylated serine 129-alpha-synuclein and pan-alpha-synuclein antibodies were observed in cortical neurons and non-neuronal cells in fixed brain sections and in isolated nuclear preparations from Dementia with Lewy bodies cases and matched controls. Subsequent biochemical analysis of subcellular fractionated tissue confirmed alpha-synuclein as present in a nuclear fraction at levels ~ 10-fold lower than in the cytoplasm. Critically, however, an increase in monomeric nuclear alpha-synuclein phosphorylated as serine 129 was observed in cases of dementia with Lewy bodies alongside higher molecular weight pan- and phosphorylation reactive alpha-synuclein species, consistent with the formation of intranuclear phosphorylated alpha-synuclein oligomers. Furthermore, the presence of nuclear alpha-synuclein was confirmed via label free mass spectrometry, as 6 unique alpha-synuclein derived peptide sequences were identified in nuclear fractions (71.4% sequence coverage).

Collectively, our data confirm the presence of nuclear alpha-synuclein in human brain tissue and describe nuclear pathology associated with dementia with Lewy bodies. These findings address a major controversy in the synucleinopathy field by confirming the presence of nuclear alpha-synuclein in autoptic human brain tissue and, for the first time, identify that alpha-synuclein is aggregated into novel and potentially pathological assemblies in the nucleus as part of the disease process associated with dementia with Lewy bodies and thus may contribute to the disease phenotype. Fascicles Split or Merge Every ~560 Microns Within the Human Cervical Vagus Nerve 1Vagus nerve stimulation (VNS) is FDA approved for stroke rehabilitation, epilepsy, and depression; however, the vagus functional anatomy underlying the implant is poorly understood. We used microCT to quantify fascicular structure and neuroanatomy within human cervical vagus nerves. Fascicles split or merged every ~560 m (17.8 {+/-} 6.1 events/cm). The high degree of fascicular splitting and merging in humans may explain the clinical heterogeneity in patient responses. TeamTree analysis: a new approach to evaluate scientific production Advances in science and technology depend on the work of research teams and the publication of results through peer-reviewed articles representing a growing socio-economic resource. Current methods to mine the scientific literature regarding a field of interest focus on content, but the workforce credited by authorship remains largely unexplored, and appropriate measures of scientific production are debated. Here, a new bibliometric approach named TeamTree analysis is introduced that visualizes the development and composition of the workforce driving a field. A new citation-independent measure that scales with the H index estimates impact based on publication record, genealogical ties and collaborative connections. This author-centered approach complements existing tools to mine the scientific literature and to evaluate research across disciplines. Combined MEK and STAT3 inhibition reprograms stromal inflammation to overcome immunotherapy resistance in pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is characterized by immune exclusion, pro-inflammatory polarization of cancer-associated fibroblasts (CAF), and resistance to immune checkpoint inhibition (ICI). We have previously demonstrated that reciprocally activated RAS/MEK/ERK and JAK/STAT3 pathways mediate therapeutic resistance, while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes such resistance in preclinical models. We now show that combined MEKi+STAT3i not only alters stromal architecture but also uncovers stromal plasticity by revealing a substantial attenuation of Il6/Cxcl1-expressing secretory and Lrrc15-expressing myofibroblastic CAF phenotypes with a concomitant enrichment of Ly6a/Cd34-expressing CAF phenotypes exhibiting mesenchymal progenitor-like properties via single-cell RNA sequencing in Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox(PKT) mice. This remodeling of CAF heterogeneity is associated with reprogramming of immunosuppressive myeloid populations and enhanced trafficking of CD8+ T-cells which exhibit a distinct effector transcriptional program. These MEKi+STAT3i-mediated repercussions are in part CAF-dependent, since CRISPR/Cas9 genetic silencing of CAF-restricted Mek1/Stat3 mitigates inflammatory CAF polarization and myeloid infiltration in vivo. Addition of MEKi+STAT3i to PD-1 blockade overcomes ICI resistance by significantly augmenting anti-tumor responses and dramatically improving survival in PKT mice compared with anti-PD-1 monotherapy. The addition of MEKi+STAT3i to PD-1 blockade not only augments the recruitment of activated and memory T-cell populations, but also improves their degranulating capacity and functional cytotoxicity compared to PD-1 blockade alone. Importantly, treatment of a patient with chemotherapy-refractory metastatic PDAC with MEKi (Trametinib), STAT3i (Ruxolitinib), and PD-1 inhibitor (Nivolumab) was well-tolerated and yielded clinical benefit. These data uncover a novel paradigm in which combined MEKi+STAT3i reprograms stromal inflammation and immune tolerance to overcome immunotherapy resistance in PDAC. Regulatory perturbations of ribosome allocation reshape the growth proteome with a trade-off in adaptation capacity Bacteria regulate their cellular resource allocation to enable fast growth-adaptation to a variety of environmental niches. We studied the ribosomal allocation, growth and expression profiles of two sets of fast-growing mutants of Escherichia coli K-12 MG1655 in glucose minimal medium. Mutants with only 3 of the seven copies of ribosomal RNA operons grew faster than the wild-type strain in minimal media and show similar phenotype to previously studied rpoB mutants. Higher growth rates due to increased ribosome content affected resource allocation. Expression profiles of fast-growing mutants shared downregulation of hedging functions and upregulated growth functions. Mutants showed longer diauxic shifts and reduced activity of gluconeogenic promoters during glucose-acetate shifts, suggesting reduced availability of the RNA Polymerase for expressing hedging proteome. These results show that the regulation of ribosomal allocation underlies the growth/hedging phenotypes obtained from laboratory evolution experiments. We show how two different regulatory perturbations (rRNA promoters or rpoB mutations) reshape the proteome for growth with a concomitant fitness cost

HighlightsMutants with only 3 ribosomal operons grow faster than wild-type in minimal medium

 {Delta}4 rrn and rpoB mutants share phenotypic traits

Faster growth of mutants is achieved by increased ribosome content

Fast-growing mutants display reduced hedging expression and adaptation trade-offs

Despite similar ribosomal content in rich medium the mutants present growth defects Bacterial H-NS contacts DNA at the same irregularly spaced sites in both bridged and hemi-sequestered linear filaments Gene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the structures, arrangements of DNA-binding domains (DBDs), and positions of DBD-DNA contacts in linear and bridged filaments are uncertain. To characterize the contacts that silence transcription by RNA polymerase, we combined {middle dot}OH footprinting, molecular dynamics, statistical modeling, and DBD mapping using a chemical nuclease (Fe2+-EDTA) tethered to the DBDs (TEN-map). We find that H-NS DBDs contact DNA at indistinguishable locations in bridged or linear filaments and that the DBDs vary in orientation and position with ~10-bp average spacing. Our results support a hemi-sequestration model of linear-to-bridged H-NS switching in which linear filaments able to inhibit only transcription initiation switch to bridged filaments able to inhibit both initiation and elongation using the same irregularly spaced DNA contact sites.

HighlightsO_LITethered-nuclease mapping (TEN-map) of H-NS DNA-binding domains detects DNA contacts
C_LIO_LIBridged and linear H-NS filaments use the same DNA contact sites
C_LIO_LIH-NS-DNA contacts are unevenly spaced with ~10 bp average separation
C_LIO_LIAT-steps, minor groove width, and electrostatic potential best predict contact sites
C_LI