Increasing triacylglycerol formation and lipid storage by unsaturated lipids protects renal proximal tubules in diabetes In diabetic patients, dyslipidemia frequently contributes to organ damage such as diabetic kidney disease (DKD). DKD is associated with excessive renal deposition of triacylglycerol (TAG) in lipid droplets (LD). Yet, it is unclear whether LDs play a protective or damaging role and how this might be influenced by dietary patterns. By using a diabetes mouse model, we find here that high fat diet enriched in the unsaturated oleic acid (OA) caused more lipid storage in LDs in renal proximal tubular cells (PTC) but less tubular damage than a corresponding butter diet with the saturated palmitic acid (PA). Mechanistically, we identify endoplasmic reticulum (ER) stress as the main cause of PA-induced PTC injury. ER stress is caused by elevated cellular levels of saturated TAG precursors and to higher membrane order in the ER. The resulting cell death is preceded by a transcriptional rewiring of phospholipid metabolism. Simultaneous addition of OA rescues the cytotoxic effects by normalizing membrane order and by increasing the total TAG amount. The latter also stimulates the formation of LDs that in turn can release unsaturated lipids upon demand by lipolysis. Our study thus clarifies mechanisms underlying PA-induced cell stress in PTCs and emphasizes the importance of olive oil for the prevention of DKD.
PTEN regulates adipocyte progenitor growth, differentiation and replicative aging The tumor suppressor phosphatase and tensin homolog (PTEN) negatively regulates the insulin signaling pathway. Germline PTEN pathogenic variants cause PTEN Hamartoma Tumor Syndrome (PHTS), associated with lipoma development in children. It remains unclear which mechanisms trigger this aberrant adipose tissue growth. Adipocyte progenitor cells (APCs) lose their capacity to differentiate into adipocytes during continuous culture, while APCs from PHTS patients lipomas retain their adipogenic potential over a prolonged period. To investigate the role of PTEN in adipose tissue development we performed functional assays and RNA sequencing of control and PTEN knockdown APCs. Reduction of PTEN levels using siRNA or CRISPR lead to an enhanced proliferation and differentiation of APCs. FOXO1 was downregulated on the mRNA level while inactivation through phosphorylation increased. FOXO1 phosphorylation initiates the expression of the lipogenesis activating transcription factor SREBP1. SREBP1 levels were higher after PTEN knockdown and may account for the enhanced adipogenesis. To validate this we overexpressed constitutively active FOXO1 in PTEN CRISPR cells and found reduced adipogenesis, accompanied by a SREBP1 downregulation. We observed that PTEN levels were upregulated during long term culture of wild type APCs. PTEN CRISPR cells showed less senescence compared to controls and the senescence marker CDKN1A (p21) was downregulated in PTEN knockdown cells. Cellular senescence was the most significantly enriched pathway found in RNA sequencing of PTEN knockdown vs. control cells. These results provide evidence that PTEN is involved in the regulation of APCs proliferation, differentiation and senescence, thereby contributing to aberrant adipose tissue growth in PHTS patients.
Expression and subcellular localization of USH1C/harmonin in the human retina provides insights into pathophysiology and therapy of Usher syndrome Usher syndrome (USH) is the most common form of hereditary deafness-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retina uncovered harmonin a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Muller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones, and outer segments of rods as well as at adhesive junctions of MGCs and photoreceptors in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in MGCs and photoreceptors and rhodopsin in photoreceptors. Subcellular expression and colocalization of harmonin correlates with the clinical phenotype observed in USH1C patients. In addition, primary cilia defects in USH1C patient-derived fibroblasts could be reverted by delivery of harmonin a1 transcript isoform. Our data provide novel insights into photoreceptor cell biology, USH1C pathophysiology and for developing gene therapy treatment. Assessing the impact of grassland management on landscape multifunctionality Land-use intensification has contrasting effects on different ecosystem services, often leading to land-use conflicts. While multiple studies have demonstrated how landscape-scale strategies can minimise the trade-off between agricultural production and biodiversity conservation, little is known about which land-use strategies maximise the landscape-level supply of multiple ecosystem services (landscape multifunctionality), a common goal of stakeholder communities.

We combine comprehensive data collected from 150 German grassland sites with a simulation approach to identify landscape compositions, with differing proportions of low-, medium-, and high-intensity grasslands, that minimise trade-offs between the six main grassland ecosystem services prioritised by local stakeholders: biodiversity conservation, aesthetic value, productivity, carbon storage, foraging, and regional identity. Results are made accessible through an online tool that provides information on which compositions best meet any combination of user-defined priorities (https://neyret.shinyapps.io/landscape_composition_for_multifunctionality/).

Results show that an optimal landscape composition can be identified for any pattern of ecosystem service priorities. However, multifunctionality was similar and low for all landscape compositions in cases where there are strong trade-offs between services (e.g. aesthetic value and fodder production), where many services were prioritised, and where drivers other than land use played an important role. We also found that if moderate service levels are deemed acceptable, then strategies in which both high and low intensity grasslands are present can deliver landscape multifunctionality. The tool presented can aid informed decision-making by predicting the impact of future changes in landscape composition, and by allowing for the relative roles of stakeholder priorities and biophysical trade-offs to be understood by scientists and practitioners alike.

HighlightsO_LIAn online tool identifies optimal landscape compositions for desired ecosystem services
C_LIO_LIWhen the desired services are synergic, the optimum is their common best landscape composition
C_LIO_LIWhen the desired services trade-off, a mix of grassland intensity is most multifunctional
C_LIO_LISuch tools could support decision-making processes and aid conflict resolution
C_LI

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/208199v5_ufig1.gif" ALT="Figure 1">
View larger version (27K):
org.highwire.dtl.DTLVardef@3273f6org.highwire.dtl.DTLVardef@5b3a5aorg.highwire.dtl.DTLVardef@153f526org.highwire.dtl.DTLVardef@103cbea_HPS_FORMAT_FIGEXP  M_FIG C_FIG On-pathway multivalency of a broadly neutralizing evolution drug candidate A promising approach to prevent antibiotic resistance is design of multivalent antibiotics that constrain bacterial evolvability by blocking multiple escape routes. Previously we discovered that an "evolution drug" lead designed to broadly neutralize bacterial dihydrofolate reductase (DHFR) and its trimethoprim escape variants is multivalent in E. coli cells. In this work we identified the second target of this new lead. We analysed antibiotic induced global metabolic perturbations and deployed metabolic-network analysis and structural similarity search of the putative targets and narrowed down the search to a few enzymes. Further, combining in vivo and in vitro analyses, we identified HPPK (folK), an essential protein in bacterial folate-metabolism, as the non-DHFR target of the lead. We observed that compounds with interaction specificity against WT do not develop multivalency against related proteins. This multivalent-design strategy is effective in blocking the emergence of drug-resistant phenotypes.

HighlightsO_LIAn evolution drug lead designed to block bacterial evolvability manifested behaviours suggesting an intra-cellular multivalent activity.
C_LIO_LIA multi-layered metabolomic and structure guided approach unravelled the second intra-cellular target of the evolution-drug lead, showing folate-pathway inhibitions at two metabolic points.
C_LIO_LIThe on-pathway anti-folate activity is exhibited by inhibiting two essential proteins viz. DHFR and its upstream protein HPPK.
C_LIO_LIMultivalent drug development schemes present a promising anti-evolution strategy to address the problem of "drug-resistance" by inhibiting more than one metabolic protein.
C_LI Intermittent KHz-frequency electrical stimulation selectively engages small unmyelinated vagal afferents Afferent and efferent vagal fibers mediate bidirectional communication between the brain and visceral organs. Small, unmyelinated C-afferents constitute the majority of vagal fibers, play critical roles in numerous interoceptive circuits and autonomic reflexes in health and disease and may contribute to the efficacy and safety of vagus nerve stimulation (VNS). Selective engagement of C-afferents with electrical stimuli has not been feasible, due to the default fiber recruitment order: larger fibers first, smaller fibers last. Here, we determine and optimize an electrical stimulus that selectively engages vagal C-afferents. Intermittent KHz-frequency electrical stimulation (KES) activates motor and, preferentially, sensory vagal neurons in the brainstem. During KES, asynchronous activity of C-afferents increases, while that of larger fibers remains largely unchanged. In parallel, KES effectively blocks excitability of larger fibers while moderately suppressing excitability of C-afferents. By compiling selectivity indices in individual animals, we find that optimal KES parameters for C-afferents are >5KHz frequency and 7-10 times engagement threshold (xT) intensity in rats, 15-25xT in mice. These effects can be explained in computational models by how sodium channel responses to KES are shaped by axonal size and myelin. Our results indicate that selective engagement of vagal C-afferents is attainable by intermittent KES. Regulation of the PKD2 channel function by TACAN Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in membrane receptor PKD1 or cation channel PKD2. TACAN (also named TMEM120A), recently reported as an ion channel in neuron cells for mechano and pain sensing, is also distributed in diverse non-neuronal tissues such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes we found that TACAN inhibited the channel activity of PKD2 gain-of-function mutant F604P. The first and last transmembrane domains of TACAN were found to interact with the PKD2 C-and N-terminal portions, respectively. We showed that the TACAN N-terminus acted as a blocking peptide and that TACAN inhibits the PKD2 function through the PKD2/TACAN binding. By patch clamping in mammalian cells, we found that TACAN inhibits both the single channel conductance and open probability of PKD2 and mutant F604P. PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. Furthermore, we also found that TACAN aggravates PKD2-dependent tail curvature and pronephric cysts in larval zebrafish, in support of the in vitro inhibitory effects of TACAN. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechano sensitivity of the PKD2/TACAN channel complex. 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 The obstacles and potential clues of prime editing applications in tomato, a dicot plant Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement. Topology and adenocarcinoma cell localization dataset on the labyrinthin biomarker. The discovery and characterization of tumor associated antigens is increasingly important to advance the field of immuno-oncology. Data on the, 1) topology, 2) amino acid (a.a.) homology analyses and 3) cell surface localization are presented as evidence in support of labyrinthin as a pan-adenocarcinoma marker. Multiple bioinformatics tools were used to collectively predict calcium binding domain(s), N-myristoylation sites, kinase II phosphorylation sites, and transmembrane domain(s) with predicted N- and C-termini orientations. Sequence homology comparisons were found and alignments made for labyrinthin (255 a.a.) vs. the intracellular aspartyl/asparaginyl beta-hydroxylase (ASPH; 758 a.a.) and the ASPH-gene related protein junctate (299 a.a.), which are both type II proteins. The epitopes for commonly used MCA 44-3A6 anti-labyrinthin and FB-50 anti-ASPH/junctate antibodies were present in all three proteins. The selective expression of labyrinthin was investigated by third party fluorescent activated cell sorter (FACS) analyses of MCA 44-3A6 binding to non-permeabilized adenocarcinoma and normal cell line models. Labyrinthin was detected on non-permeabilized A549 human lung adenocarcinoma cells, but not on normal WI-38 human lung fibroblasts nor primary cultures of normal human glandular-related cells. Microscopic images of immunofluorescent labelled MCA 44-3A6 binding to A549 cells at random cell cycle stages complement the FACS results by further showing that labyrinthin persisted on the cell surfaces along with some cell internalization within 20 minutes. The dataset presented herein is related to research article: "Labyrinthin: A distinct pan-adenocarcinoma diagnostic and immunotherapeutic tumor specific antigen" by Babich et al., Heliyon, 2022 [1].

Specifications Table

O_TBL View this table:
org.highwire.dtl.DTLVardef@19222bborg.highwire.dtl.DTLVardef@1264bcdorg.highwire.dtl.DTLVardef@13a8158org.highwire.dtl.DTLVardef@1dcf0org.highwire.dtl.DTLVardef@503a9f_HPS_FORMAT_FIGEXP  M_TBL C_TBL Value of the data O_LIBecause identification and characterization of neoantigens [3, 4, 5] are rare yet increasingly important for diagnostics and therapeutics, the identification of labyrinthin as a pan-adenocarcinoma target is significant for further study by clinicians and cancer researchers.
C_LIO_LIThe data provide a basis to determine to what extent labyrinthin is a distinguishing marker for adenocarcinomas vs. conventional methods.
C_LIO_LIThe results indicate that therapeutic strategies can be employed that take advantage of labyrinthins convenient presence on adenocarcinoma cell surfaces.
C_LIO_LIThe discovery of a calcium binding sequence in labyrinthin provides a basis to research if it is key to explaining the long-known phenomena of elevated calcium and irregular signaling in cancer cells [8, 9].
C_LIO_LIThe data reveal a need to explore distinctions between ASPH and labyrinthin, in particular because ASPH [10, 11] and labyrinthin-based treatments have already been FDA approved for clinical trials.
C_LI 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. Phylogenetic analysis of molecular pathways in Tregs Suppressor Function Treg-mediated suppression of conventional T cells is a fundamental step in regulating the adaptive immune response. It is known that Treg first appeared in vertebrates. However, little is known about the of major suppression pathways mediated by Tregs. We employed artificial intelligence text mining system to highlight the suppression pathways currently known to be utilized by Tregs. Our system identified various pathways mediated by CTLA4, calcium signaling, NfkB and NFAT. We simultaneously employed detailed phylogenetic analysis including multiple sequence alignment, phylogenetic tree building, ancestral sequence reconstruction, neutrality tests and positive selection test to investigate the evolution of Treg mediated pathways. We found that CTLA4 first appeared in vertebrates possibly arising from an IGV containing protein in cartilaginous fish. Conversely, we found that Tregs repurposed ancient pathways such as Calcineurin and CAMP Response Element Modulator. Interestingly, these two pathways were highly conserved between vertebrates and lower invertebrates indicating conservation of function. Taken together, our research indicate that Tregs developed its regulatory systems through evolution in vertebrates as well as reusing conserved ancient regulatory systems that are related to the innate immune system. Identification of CD4+ Sub-population of Resident Cardiac Fibroblasts Linked to Myocardial Fibrosis Infiltration with inflammatory T-cells and accumulation of cardiac myofibroblasts are hallmarks of cardiac fibrosis and maladaptive remodeling. The origin, identity, and functions of the resident cardiac cells involved in this process are, however, unclear. To determine the identity of cells contained in regions exhibiting fibrosis, mass cytometry profiling was performed using resident human ventricular cardiac fibroblasts and right ventricle autopsy tissues from individuals diagnosed with pulmonary hypertension and SUGEN/hypoxia rats. Results showed that a subpopulation of resident myocardial fibroblasts expresses increased levels of CD4+, a helper T-cell surface marker, in addition to mesenchymal markers in humans and rats. Characterization of the resident cardiac fibroblast subpopulation, both structurally and functionally, using transcriptome and secretome analysis of the secreted cytokines, chemokines, proteins, and metabolites, evidenced that IL-1{beta} induces a phenotypic switch of human cardiac fibroblasts from mesenchymal to CD4+ lymphoidal lineage in vitro. RNA sequencing (RNA-seq) analysis of FACS-sorted CD4-expressing cardiac fibroblasts further revealed that the transcriptome of such IL-1{beta}-induced CD4+ fibroblast population exhibited classical lymphoidal and stem cell-like signatures. Lastly, reversal of cell clustering, phosphorylation of MAPK p38 and NF-{kappa}B p65, and phenotypic switching was achieved with the administration of an IL-1R antagonist. In conclusion, we have identified a subpopulation of cardiac fibroblasts which exhibits structural and functional attributes of both mesenchymal and lymphoid cells which is induced by IL-1{beta}-IL-1R-NFkB pathway for differentiation of cardiac fibroblast cells. These data suggest that cardiac fibroblast transdifferentiation during inflammation may form the basis for maladaptive remodeling during myocardial fibrosis. A chemical screen based on an interruption of zebrafish gastrulation identifies the HTR2C inhibitor Pizotifen as a suppressor of EMT-mediated metastasis Metastasis is responsible for approximately 90% of cancer-associated mortality but few models exist that allow for rapid and effective screening of anti-metastasis drugs. Current mouse models of metastasis are too expensive and time consuming to use for rapid and high-throughput screening. Therefore, we created a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. We hypothesized that small chemicals that interrupt zebrafish gastrulation might also suppress metastatic progression of cancer cells and developed a phenotype-based chemical screen to test the hypothesis. The screen used epiboly, the first morphogenetic movement in gastrulation, as a marker and enabled 100 chemicals to be tested in five hours. The screen tested 1280 FDA-approved drugs and identified Pizotifen, an antagonist for serotonin receptor 2C (HTR2C) as an epiboly-interrupting drug. Pharmacologic and genetic inhibition of HTR2C suppressed metastatic progression in a mouse model. Blocking HTR2C with Pizotifen restored epithelial properties to metastatic cells through inhibition of Wnt-signaling. In contrast, HTR2C induced epithelial to mesenchymal transition (EMT) through activation of Wnt-signaling and promoted metastatic dissemination of human cancer cells in a zebrafish xenotransplantation model. Taken together, our concept offers a novel platform for discovery of anti-metastasis drugs. A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component Beyond its role in cellular homeostasis, autophagy plays anti- and pro-microbial roles in host-microbe interactions, both in animals and plants. One prominent role of anti-microbial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in depletion of a bacterial virulence factor and unravels an unprecedented pathogen strategy to counteract defense-related autophagy. 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. Cell surface hydrophobicity determines aggregate assembly type in Pseudomonas aeruginosa Bacteria live in spatially organized aggregates during chronic infections, where they adapt to the host environment, evade immune responses and resist therapeutic interventions. Although it is known that environmental factors such as polymers influence bacterial aggregation, it is not clear how bacterial adaptation during chronic infection impacts the formation and spatial organization of aggregates in the presence of polymers. Here we show that in an in vitro model of cystic fibrosis (CF) containing the polymers eDNA and mucin, O-specific antigen is a major factor in determining the formation of two distinct aggregate assembly types of Pseudomonas aeruginosa due to alterations in cell surface hydrophobicity. Our findings suggest that during chronic infection, interplay between cell surface properties and polymers in the environment may influence the formation and structure of bacterial aggregates, which would shed new light on the fitness costs and benefits of O-antigen production in environments such as CF lungs.

ImportanceDuring chronic infection, several factors contribute to the biogeography of microbial communities. Heterogeneous populations of Pseudomonas aeruginosa form aggregates in cystic fibrosis airways, however, the impact of this population heterogeneity on spatial organization and aggregate assembly is not well understood. In this study we found that changes in O-specific antigen determine the spatial organization of P. aeruginosa cells by altering the relative cell surface hydrophobicity. This finding suggests a role for O-antigen in regulating P. aeruginosa aggregate size and shape in cystic fibrosis airways. Point Mutations in SARS-CoV-2 Variants Induce Long-Range Dynamical Perturbations in Neutralizing Antibodies Monoclonal antibodies are emerging as a viable treatment for the coronavirus disease 19 (COVID-19). However, newly evolved variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can reduce the efficacy of currently available antibodies and can diminish vaccine-induced immunity. Here, we demonstrate that the microscopic dynamics of neutralizing monoclonal antibodies can be profoundly modified by the mutations present in the spike proteins of the SARS-COV-2 variants currently circulating in the world population. The dynamical perturbations within the antibody structure, which alter the thermodynamics of antigen recognition, are diverse and can depend both on the nature of the antibody and on the spatial location of the spike mutation. The correlation between the motion of the antibody and that of the spike receptor binding domain (RBD) can also be changed, modulating binding affinity. Using protein-graph-connectivity networks, we delineated the mutant-induced modifications in the information-flow along allosteric pathway throughout the antibody. Changes in the collective dynamics were spatially distributed both locally and across long-range distances within the antibody. On the receptor side, we identified an anchor-like structural element that prevents the detachment of the antibodies; individual mutations there can significantly affect the antibody binding propensity. Our study provides insight into how virus neutralization by monoclonal antibodies can be impacted by local mutations in the epitope via a change in dynamics. This realization adds a new layer of sophistication to the efforts for rational design of monoclonal antibodies against new variants of SARS-CoV2, taking the allostery in the antibody into consideration. DNA Strand Breaks and Gaps Target Retroviral Binding and Integration Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and reverse transcribed (RT) copy DNA (cDNA). Previous studies demonstrated DNA site recognition limited intasome integration. Using single molecule Forster resonance energy transfer (smFRET), we show Prototype Foamy Virus (PFV) intasomes pause at DNA strand breaks and gaps. The break/gap discontinuities are similar to base excision repair (BER) lesion-processing intermediates, which affect retrovirus integration in vivo. Pausing targeted site-directed integration at the break/gap without inducing intasome conformational alterations. An 8-oxo-guanine lesion normally processes by BER and a G/T mismatch or a +T nucleotide insertion that induce flexibility or a bend in the DNA backbone did not promote intasome pausing or targeted integration. These results suggest that repair intermediates can modulate dynamic intasome-DNA interactions which target retroviral integration. Selective Ablation of 3' RNA ends and Processive RTs Facilitate Direct cDNA Sequencing of Full-length Host Cell and Viral Transcripts Alternative splicing (AS) is necessary for viral proliferation in host cells and a critical regulatory component of viral gene expression. Conventional RNA-seq approaches provide incomplete coverage of AS due to their short read-lengths and are susceptible to biases and artifacts introduced in prevailing library preparation methodologies. Moreover, viral splicing studies are often conducted separately from host cell transcriptome analysis, precluding an assessment of the viral manipulation of host splicing machinery. To address current limitations, we developed a quantitative full-length direct cDNA sequencing strategy to simultaneously profile viral and host cell transcripts. This nanopore-based approach couples processive reverse transcriptases with a novel one-step chemical ablation of 3 RNA ends (termed CASPR) which decreases ribosomal RNA reads and enriches for poly-adenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4kb), while being functionally equivalent to PolyA+ selection for transcript quantification. We used our approach to interrogate host cell and HIV-1 transcript dynamics during viral reactivation and identified novel putative HIV-1 host factors containing exon skipping or novel intron retentions and delineated the HIV-1 transcriptional state associated with these differentially regulated host factors. If Research is Not the Evidence, What is it? The Lack of Research Citations in Health Vlogs Doctor-influencers have the knowledge needed to understand scientific research, and they have the social media popularity to share it with large audiences. In this article, I explore the treatment of evidence in vlogs by Egyptian doctor-influencers: how they present it and what they use instead of it. I answer the following questions: 1) How do doctor-influencers present evidence on social media? 2) If evidence is not the research, then what is it? The data was collected through in-depth interviews with 12 Egyptian doctor-influencers from different specializations; two focus groups; and a critical discourse analysis of 48 of the influencers most popular and most engaging videos. I found that the doctor-influencers cited academic research only in the videos about medical controversies, new treatments, misinformation, or common medical mistakes. In most of their videos, the Egyptian doctor-influencers scarcely referred to medical research. This is because they believe that their audiences would not understand evidence due to their low educational attainment, weak research skills, and the language barrier (as most medical research is published in English rather than Arabic). These findings highlight the challenges of communicating science in societies with a low level of education. In these settings, communicators may need to move beyond mediating research studies on social media and instead focus on simplifying, translating, and connecting evidence-based information to the cultural context. High-throughput variant detection using a color-mixing strategy Many diseases are related to multiple genetic alterations along a single gene. Probing for highly multiple (>10) variants in a single qPCR tube is not possible due to a limited number of fluorescence channels and one variant per channel, so many more tubes are needed. Here, we experimentally validate our novel color-mixing strategy that uses fluorescence combinations as digital color codes to probe multiple variants simultaneously. The color-mixing strategy relies on a simple intra-tube assay that can probe for 15 variants as part of an inter-tube assay that can probe for an exponentially increased number of variants. The color-mixing strategy is achieved using multiplex double-stranded toehold probes modified with fluorophores and quenchers; the probes are designed to be quenched or luminous after binding to wildtype or variant templates. We used the color-mixing strategy to probe for 21 pathogenic mutations in thalassemia and to distinguish between heterozygous and homozygous variants in 6 tubes, with a specificity of 99% and a sensitivity of 94%. To support tuberculosis diagnosis, we used the same strategy to simultaneously probe in Mycobacterium tuberculosis for rifampicin-resistance mutations occurring within one 81-bp region and one 48-bp region in rpoB gene, plus five isoniazid-resistance mutations in inhA and katG genes. The snoGloBe interaction predictor reveals a broad spectrum of C/D snoRNA RNA targets Box C/D small nucleolar RNAs (snoRNAs) are a conserved class of RNA known for their role in guiding ribosomal RNA 2-O-ribose methylation. Recently, C/D snoRNAs were also implicated in regulating the expression of non-ribosomal genes through different modes of binding. Large scale RNA-RNA interaction datasets detect many snoRNAs binding messenger RNA, but are limited by specific experimental conditions. To enable a more comprehensive study of C/D snoRNA interactions, we created snoGloBe, a human C/D snoRNA interaction predictor based on a gradient boosting classifier. SnoGloBe considers the target type, position and sequence of the interactions, enabling it to outperform existing predictors. Interestingly, for specific snoRNAs, snoGloBe identifies strong enrichment of interactions near gene expression regulatory elements including splice sites. Abundance and splicing of predicted targets were altered upon the knockdown of their associated snoRNA. Strikingly, the predicted snoRNA interactions often overlap with the binding sites of functionally related RNA binding proteins, reinforcing their role in gene expression regulation. SnoGloBe is also an excellent tool for discovering viral RNA targets, as shown by its capacity to identify snoRNAs targeting the heavily methylated SARS-CoV-2 RNA. Overall, snoGloBe is capable of identifying experimentally validated binding sites and predicting novel sites with shared regulatory function. Complete neuroanatomy and sensor maps of dragonfly wings for fly-by-feel monitoring of aeroelasticity Can mechanosensors in animal wings allow reconstruction of the wing aeroelastic states? Little is known about how flying animals utilize wing mechanosensation to monitor the dynamic state of their highly deformable wings. Odonata, dragonflies and damselflies, are a basal lineage of flying insects with excellent flight performance, and their wing mechanics have been studied extensively. Here, we present a comprehensive map of the wing sensory system for two Odonata species, including both the external sensor morphologies and internal neuroanatomy. We identified eight morphological classes of sensors; most were mechanosensors innervated by a single neuron. Their innervation patterns and morphologies minimize axon length and allow morphological latency compensation. We further mapped the major veins of another 13 Odonata species across 10 families and identified consistent sensor distribution patterns, with sensor count scaling with wing length. Finally, we constructed a high-fidelity finite element model of a dragonfly wing for structural analysis. Our dynamic loading simulations revealed features of the strain fields that wing sensor arrays could detect to encode different wing deformation states. Taken together, this work marks the first step toward an integrated understanding of fly-by-feel control in animal flight. Flexible control of Pavlovian-instrumental transfer based on expected reward value The Pavlovian-instrumental transfer (PIT) paradigm is widely used to assay the motivational influence of reward-predictive cues, reflected by their ability to invigorate instrumental behavior. Leading theories of incentive learning assume that cues are assigned motivational properties based on the value of the rewards they predict. We outline an alternative account of PIT which recognizes that reward-predictive cues may suppress rather than motivate instrumental behavior under certain conditions. We posit that cues capable of triggering a strong reward expectancy will tend to inhibit instrumental behavior, which is exploratory by nature, in order to facilitate retrieval of the expected reward before it is lost or stolen. According to this view, the motivation to engage in instrumental behavior during a cue should be inversely related to the value of the predicted reward, since there is more to lose by failing to secure a high-value reward than a low-value reward. We tested this hypothesis in rats using a high-conflict PIT protocol which elicits competing instrumental and food-port entry responses. In Experiment 1, cues signaling different reward magnitudes (1, 3, or 9 pellets) differentially influenced these behaviors. Whereas the 1-pellet cue increased instrumental behavior, cues signaling 3 or 9 pellets suppressed instrumental behavior and elicited high levels of food-port activity. Experiment 2 found that reward-predictive cues suppressed instrumental behavior and increased food-port activity in a flexible manner that was disrupted by post-training reward devaluation. We discuss how the PIT task provides a useful tool for studying cognitive control over cue-motivated behavior in rodents. F1F0 ATP Hydrolysis is a Determinant of Metabolic Rate, a Correlate of Lifespan, and a Weakness of Cancer Dramatic rectal temperature decrease, in mice administered with a drug that specifically inhibits F1F0 ATP hydrolysis, suggests that F1F0 ATP hydrolysis is a major determinant of metabolic rate in vivo. Across twelve investigated species, less F1F0 ATP hydrolysis correlates with greater maximal lifespan. Specific drug inhibition of F1F0 ATP hydrolysis exerts potent anti-cancer activity in vitro.