Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling.

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

The mTORC1 pathway stimulates glutamine metabolism and cell proliferation by repressing SIRT4.

Proliferating mammalian cells use glutamine as a source of nitrogen and as a key anaplerotic source to provide metabolites to the tricarboxylic acid cycle (TCA) for biosynthesis. Recently, mammalian target of rapamycin complex 1 (mTORC1) activation has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutaminolysis has been reported. Here, we show that mTORC1 promotes glutamine anaplerosis by activating glutamate dehydrogenase (GDH). This regulation requires transcriptional repression of SIRT4, the mitochondrial-localized sirtuin that inhibits GDH. Mechanistically, mTORC1 represses SIRT4 by promoting the proteasome-mediated destabilization of cAMP-responsive element binding 2 (CREB2). Thus, a relationship between mTORC1, SIRT4, and cancer is suggested by our findings. Indeed, SIRT4 expression is reduced in human cancer, and its overexpression reduces cell proliferation, transformation, and tumor development. Finally, our data indicate that targeting nutrient metabolism in energy-addicted cancers with high mTORC1 signaling may be an effective therapeutic approach.

Selective whole-genome amplification reveals population genetics of Leishmania braziliensis directly from patient skin biopsies.

In Brazil, Leishmania braziliensis is the main causative agent of the neglected tropical disease, cutaneous leishmaniasis (CL). CL presents on a spectrum of disease severity with a high rate of treatment failure. Yet the parasite factors that contribute to disease presentation and treatment outcome are not well understood, in part because successfully isolating and culturing parasites from patient lesions remains a major technical challenge. Here we describe the development of selective whole genome amplification (SWGA) for Leishmania and show that this method enables culture-independent analysis of parasite genomes obtained directly from primary patient skin samples, allowing us to circumvent artifacts associated with adaptation to culture. We show that SWGA can be applied to multiple Leishmania species residing in different host species, suggesting that this method is broadly useful in both experimental infection models and clinical studies. SWGA carried out directly on skin biopsies collected from patients in Corte de Pedra, Bahia, Brazil, showed extensive genomic diversity. Finally, as a proof-of-concept, we demonstrated that SWGA data can be integrated with published whole genome data from cultured parasite isolates to identify variants unique to specific geographic regions in Brazil where treatment failure rates are known to be high. SWGA provides a relatively simple method to generate Leishmania genomes directly from patient samples, unlocking the potential to link parasite genetics with host clinical phenotypes.

Identification of an Electrogenic 2Cl-/H+ Exchanger, ClC5, as a Chloride-Secreting Transporter Candidate in Kidney Cyst Epithelium in Tuberous Sclerosis.

Kidney cyst expansion in tuberous sclerosis complex (TSC) or polycystic kidney disease (PKD) requires active secretion of chloride (Cl-) into the cyst lumen. In PKD, Cl- secretion is primarily mediated via the cystic fibrosis transmembrane conductance regulator (CFTR) in principal cells. Kidney cystogenesis in TSC is predominantly composed of type A intercalated cells, which do not exhibit noticeable expression of CFTR. The identity of the Cl--secreting molecule(s) in TSC cyst epithelia remains speculative. RNA-sequencing analysis results were used to examine the expression of FOXi1, the chief regulator of acid base transporters in intercalated cells, along with localization of Cl- channel 5 (ClC5), in various models of TSC. Results from Tsc2+/- mice showed that the expansion of kidney cysts corresponded to the induction of Foxi1 and correlated with the appearance of ClC5 and H+-ATPase on the apical membrane of cyst epithelia. In various mouse models of TSC, Foxi1 was robustly induced in the kidney, and ClC5 and H+-ATPase were expressed on the apical membrane of cyst epithelia. Expression of ClC5 was also detected on the apical membrane of cyst epithelia in humans with TSC but was absent in humans with autosomal dominant PKD or in a mouse model of PKD. These results indicate that ClC5 is expressed on the apical membrane of cyst epithelia and is a likely candidate mediating Cl- secretion into the kidney cyst lumen in TSC.

A fast machine-learning-guided primer design pipeline for selective whole genome amplification.

Addressing many of the major outstanding questions in the fields of microbial evolution and pathogenesis will require analyses of populations of microbial genomes. Although population genomic studies provide the analytical resolution to investigate evolutionary and mechanistic processes at fine spatial and temporal scales-precisely the scales at which these processes occur-microbial population genomic research is currently hindered by the practicalities of obtaining sufficient quantities of the relatively pure microbial genomic DNA necessary for next-generation sequencing. Here we present swga2.0, an optimized and parallelized pipeline to design selective whole genome amplification (SWGA) primer sets. Unlike previous methods, swga2.0 incorporates active and machine learning methods to evaluate the amplification efficacy of individual primers and primer sets. Additionally, swga2.0 optimizes primer set search and evaluation strategies, including parallelization at each stage of the pipeline, to dramatically decrease program runtime. Here we describe the swga2.0 pipeline, including the empirical data used to identify primer and primer set characteristics, that improve amplification performance. Additionally, we evaluate the novel swga2.0 pipeline by designing primer sets that successfully amplify Prevotella melaninogenica, an important component of the lung microbiome in cystic fibrosis patients, from samples dominated by human DNA.

Kidney intercalated cells and the transcription factor FOXi1 drive cystogenesis in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is caused by mutations in either TSC1 or TSC2 genes and affects multiple organs, including kidney, lung, and brain. In the kidney, TSC presents with the enlargement of benign tumors (angiomyolipomata) and cysts, which eventually leads to kidney failure. The factors promoting cyst formation and tumor growth in TSC are incompletely understood. Here, we report that mice with principal cell-specific inactivation of Tsc1 develop numerous cortical cysts, which are overwhelmingly composed of hyperproliferating A-intercalated (A-IC) cells. RNA sequencing and confirmatory expression studies demonstrated robust expression of Forkhead Transcription Factor 1 (Foxi1) and its downstream targets, apical H+-ATPase and cytoplasmic carbonic anhydrase 2 (CAII), in cyst epithelia in Tsc1 knockout (KO) mice but not in Pkd1 mutant mice. In addition, the electrogenic 2Cl-/H+ exchanger (CLC-5) is significantly up-regulated and shows remarkable colocalization with H+-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Deletion of Foxi1, which is vital to intercalated cells viability and H+-ATPase expression, completely abrogated the cyst burden in Tsc1 KO mice, as indicated by MRI images and histological analysis in kidneys of Foxi1/Tsc1 double-knockout (dKO) mice. Deletion of CAII, which is critical to H+-ATPase activation, caused significant reduction in cyst burden and increased life expectancy in CAII/Tsc1 dKO mice vs. Tsc1 KO mice. We propose that intercalated cells and their acid/base/electrolyte transport machinery (H+-ATPase/CAII/CLC-5) are critical to cystogenesis, and their inhibition or inactivation is associated with significant protection against cyst generation and/or enlargement in TSC.

Lymphangioleiomyomatosis (LAM) Cell Atlas.

Lymphangioleiomyomatosis (LAM) is a rare lung disease of women, causing cystic remodelling of the lung and progressive respiratory failure. The cellular composition, microenvironment and cellular interactions within the LAM lesion remain unclear. To facilitate data sharing and collaborative LAM research, we performed an integrative analysis of single-cell data compiled from lung, uterus and kidney of patients with LAM from three research centres and developed an LAM Cell Atlas (LCA) Web-Portal. The LCA offers a variety of interactive options for investigators to search, visualise and reanalyse comprehensive single-cell multiomics data sets to reveal dysregulated genetic programmes at transcriptomic, epigenomic and cell-cell connectome levels.

Imbibition Dynamics in a U-Groove Microchannel with Sudden Enlargement.

Imbibition dynamics in a rectangular U-groove that is connected to a sudden enlargement and complicated by the presence of Concus-Finn (CF) filaments is investigated using many-body dissipative particle dynamics. For open-ended sudden enlargement, four flow types are identified and depend on the contact angle θy, the critical angle θf associated with the occurrence of CF filaments, and the critical angle θc associated with the occurrence of main flow. First, for θy > θf and θy > θc, the corner flow is absent, and the main flow stops at the end of the small U-groove. Second, for θc > θy > θf, the corner flow vanishes, but the main flow occurs. Third, for θf > θy > θc, the corner flow takes place in the large U-groove, but the main flow is still absent. Fourth, for θy < θf and θy < θc, both the corner and main flows appear in the large U-groove. Additionally, the flow dynamics is greatly influenced by the length of the large U-groove (le). For closed-ended sudden enlargement, similar findings can be obtained. However, the outcome of the third case is altered for sufficiently small le, and the sudden enlargement can eventually be filled.

Acute Hemodynamics in the Fontan Circulation: Open-Label Study of Vasopressin.

OBJECTIVE: To describe the acute hemodynamic effect of vasopressin on the Fontan circulation, including systemic and pulmonary pressures and resistances, left atrial pressure, and cardiac index. DESIGN: Prospective, open-label, nonrandomized study (NCT04463394). SETTING: Cardiac catheterization laboratory at Lucile Packard Children's Hospital, Stanford. PATIENTS: Patients 3-50 years old with a Fontan circulation who were referred to the cardiac catheterization laboratory for hemodynamic assessment and/or intervention. INTERVENTIONS: A 0.03 U/kg IV (maximum dose 1 unit) bolus of vasopressin was administered over 5 minutes, followed by a maintenance infusion of 0.3 mU/kg/min (maximum dose 0.03 U/min). MEASUREMENTS AND MAIN RESULTS: Comprehensive cardiac catheterization measurements before and after vasopressin administration. Measurements included pulmonary artery, atrial, and systemic arterial pressures, oxygen saturations, and systemic and pulmonary flows and resistances. There were 28 patients studied. Median age was 13.5 (9.1, 17) years, and 16 (57%) patients had a single or dominant right ventricle. Following vasopressin administration, systolic blood pressure and systemic vascular resistance (SVR) increased by 17.5 (13.0, 22.8) mm Hg ( Z value -4.6, p < 0.001) and 3.8 (1.8, 7.5) Wood Units ( Z value -4.6, p < 0.001), respectively. The pulmonary vascular resistance (PVR) decreased by 0.4 ± 0.4 WU ( t statistic 6.2, p < 0.001), and the left atrial pressure increased by 1.0 (0.0, 2.0) mm Hg ( Z value -3.5, p < 0.001). The PVR:SVR decreased by 0.04 ± 0.03 ( t statistic 8.1, p < 0.001). Neither the pulmonary artery pressure (median difference 0.0 [-1.0, 1.0], Z value -0.4, p = 0.69) nor cardiac index (0.1 ± 0.3, t statistic -1.4, p = 0.18) changed significantly. There were no adverse events. CONCLUSIONS: In Fontan patients undergoing cardiac catheterization, vasopressin administration resulted in a significant increase in systolic blood pressure, SVR, and left atrial pressure, decrease in PVR, and no change in cardiac index or pulmonary artery pressure. These findings suggest that in Fontan patients vasopressin may be an option for treating systemic hypotension during sedation or general anesthesia.

Associations Between Perceived Social Support, Perceived Competence, and Physical Activity in Hong Kong Children With Disabilities During the COVID-19 Pandemic.

This study aimed to examine the associations between perceived social support, perceived competence, and physical activity in children with physical and intellectual disabilities during the COVID-19 pandemic. During the third wave of the pandemic in Hong Kong (i.e., July through December 2020), 291 participants age 6-17 years from 27 special schools were included. After controlling for demographic variables, the total variance explained by perceived social support and perceived competence was 24%, F(2, 240) = 12.42, p < .001, with perceived competence having a stronger association with physical activity (ß = 0.29, p < .001) than perceived social support (ß = 0.07, p = .22). This study highlights two key facilitators for shaping physical activity involvement among children with disabilities during the COVID-19 pandemic.

Ca2+/calmodulin kinase II-dependent regulation of ßIV-spectrin modulates cardiac fibroblast gene expression, proliferation, and contractility.

Fibrosis is a pronounced feature of heart disease and the result of dysregulated activation of resident cardiac fibroblasts (CFs). Recent work identified stress-induced degradation of the cytoskeletal protein ßIV-spectrin as an important step in CF activation and cardiac fibrosis. Furthermore, loss of ßIV-spectrin was found to depend on Ca2+/calmodulin-dependent kinase II (CaMKII). Therefore, we sought to determine the mechanism for CaMKII-dependent regulation of ßIV-spectrin and CF activity. Computational screening and MS revealed a critical serine residue (S2250 in mouse and S2254 in human) in ßIV-spectrin phosphorylated by CaMKII. Disruption of ßIV-spectrin/CaMKII interaction or alanine substitution of ßIV-spectrin Ser2250 (ßIV-S2254A) prevented CaMKII-induced degradation, whereas a phosphomimetic construct (ßIV-spectrin with glutamic acid substitution at serine 2254 [ßIV-S2254E]) showed accelerated degradation in the absence of CaMKII. To assess the physiological significance of this phosphorylation event, we expressed exogenous ßIV-S2254A and ßIV-S2254E constructs in ßIV-spectrin-deficient CFs, which have increased proliferation and fibrotic gene expression compared with WT CFs. ßIV-S2254A but not ßIV-S2254E normalized CF proliferation, gene expression, and contractility. Pathophysiological targeting of ßIV-spectrin phosphorylation and subsequent degradation was identified in CFs activated with the profibrotic ligand angiotensin II, resulting in increased proliferation and signal transducer and activation of transcription 3 nuclear accumulation. While therapeutic delivery of exogenous WT ßIV-spectrin partially reversed these trends, ßIV-S2254A completely negated increased CF proliferation and signal transducer and activation of transcription 3 translocation. Moreover, we observed ßIV-spectrin phosphorylation and associated loss in total protein within human heart tissue following heart failure. Together, these data illustrate a considerable role for the ßIV-spectrin/CaMKII interaction in activating profibrotic signaling.

Effects of different modes of exercise on skeletal muscle mass and function and IGF-1 signaling during early aging in mice.

Skeletal muscle mass and function tend to decline with increasing age. Insulin-like growth factor 1 (IGF-1) plays a key role in promoting skeletal muscle growth. Exercise improves skeletal muscle mass and function via the activation of IGF-1 signaling. The aim of this study was to investigate whether different types of exercise can promote muscle hypertrophy, exercise and metabolic capacities, and activate IGF-1 signaling during early aging in mice. We randomly assigned 12 month old male C57/BL6 mice into five groups: control, aerobic exercise, resistance exercise, whole-body vibration and electrical stimulation group. Gastrocnemius muscle mass, myofiber size, levels of IGF-1 signaling, oxidative stress, protein synthesis and degradation, and apoptosis were detected. C2C12 cells were used to explore the mechanism by which exercise exerts its effects. We confirmed that the four modes of exercise increased skeletal muscle mass, exercise capacity, indicators of metabolism and protein synthesis, and inhibited oxidative stress and apoptosis via activation of the IGF-1 pathway. The most effective intervention was resistance exercise. Whole-body vibration promoted muscle hypertrophy better than aerobic exercise. Furthermore, in the in vitro experiment, the importance of IGF-1/IGF-1R-PI3K/Akt signaling for maintaining skeletal muscle mass was confirmed. Aerobic exercise, resistance exercise, whole-body vibration and electrical stimulation increased skeletal muscle mass, exercise capacity, protein synthesis and metabolic enzyme activity, and inhibited protein degradation and apoptosis in mice undergoing early aging via activation of IGF-1 signaling. Of these, whole-body vibration has been shown to be significantly effective and is similar to conventional exercise in promoting muscle hypertrophy.

Atypical vesicles and membranes with monolayer and multilayer structures formed by graft copolymers with diblock side-chains: nonlamellar structures and curvature-enhanced permeability.

Graft copolymers with diblock side-chains Am(-graft-B3Ay)n in a selective solvent have been reported to self-assemble into vesicles, but the structure is expected to differ distinctly from those of lipid bilayers. Surprisingly, the number of alternating hydrophobic A-block and hydrophilic B-block layers in the vesicle can vary from a monolayer to multilayers such as the hepta-layer, subject to the same copolymer concentration. The area density of the copolymer layer is not uniform across the membrane. This structural difference among different layers is attributed to the neighboring environment and the curvature of the layer. Because of the unusual polymer conformations, nonlamellar structures of polymersomes are formed, and they are much more intricate than those of liposomes. In fact, a copolymer can contribute to a single or two hydrophilic layers, and it can provide up to three hydrophobic layers. The influence of the backbone length (m) and side-chain length (y) and the permeation dynamics are also studied. The thickness of hydrophobic layers is found to increase with increasing side-chain length but is not sensitive to the backbone length. Although the permeation time increases with the layer number for planar membranes, the opposite behavior is observed for spherical vesicles owing to the curvature-enhanced permeability associated with Laplace pressure.

mTOR Signaling Network in Cell Biology and Human Disease.

The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates multiple processes, including gene transcription, protein synthesis, ribosome biogenesis, autophagy, cell metabolism, and cell growth [...].

Comprehensive Assessment of Secreted Immuno-Modulatory Cytokines by Serum-Differentiated and Stem-like Glioblastoma Cells Reveals Distinct Differences between Glioblastoma Phenotypes.

Glioblastoma is refractory to therapy and presents a significant oncological challenge. Promising immunotherapies have not shown the promise observed in other aggressive cancers. The reasons for this include the highly immuno-suppressive tumour microenvironment controlled by the glioblastoma cells and heterogeneous phenotype of the glioblastoma cells. Here, we wanted to better understand which glioblastoma phenotypes produced the regulatory cytokines, particularly those that are implicated in shaping the immune microenvironment. In this study, we employed nanoString analysis of the glioblastoma transcriptome, and proteomic analysis (proteome profiler arrays and cytokine profiling) of secreted cytokines by different glioblastoma phenotypes. These phenotypes were cultured to reflect a spectrum of glioblastoma cells present in tumours, by culturing an enhanced stem-like phenotype of glioblastoma cells or a more differentiated phenotype following culture with serum. Extensive secretome profiling reveals that there is considerable heterogeneity in secretion patterns between serum-derived and glioblastoma stem-like cells, as well as between individuals. Generally, however, the serum-derived phenotypes appear to be the primary producers of cytokines associated with immune cell recruitment into the tumour microenvironment. Therefore, these glioblastoma cells have considerable importance in shaping the immune landscape in glioblastoma and represent a valuable therapeutic target that should not be ignored.

BACTERIAL IDENTIFICATION AND ANTIBIOTIC SENSITIVITY FROM THE ABSCESSES OF CAPTIVE TORTOISES-CLINICAL ANTIBIOTIC RECOMMENDATIONS.

Bacterial abscesses are commonly seen in tortoises. The morbidity and the resultant mortality are high. Multifactorial problems, antibiotics misapplication. and antibiotic-resistant bacteria make abscess treatment complicated and ineffective. This study identifies the etiological bacterial species and determines the best antibiotics for abscess treatment in captive tortoises. Sterile swab specimens from 40 tortoises with abscesses were analyzed using the Analytical Profile Index (API) system. Sixty-five bacteria species were identified covering facultative anaerobic gram-negative (n = 30, 46.2%), facultative anaerobic gram-positive (n = 19, 29.2%), and aerobic gram-negative bacteria (n = 16, 24.6%). The antibiotic sensitivity of these bacteria to 30 antibiotics was assessed using the Kirby-Bauer disk-diffusion method. Greater than 80% anaerobic gram-negative bacterial species showed sensitivity to amikacin and ceftazidime. Greater than 80% anaerobic gram-positive bacterial species were sensitive to amoxicillin, ampicillin, carbenicillin, and penicillin. In addition, more than 80% aerobic gram-negative bacterial species were sensitive to ceftazidime, colistin sulphate, amikacin, gentamicin, kanamycin, polymyxin B, and tobramycin. This study provides clinicians significant information for initial antibiotic options, which could elevate the abscess therapy success rate and improve the life quality of tortoises.

Comprehensive analysis of inhibitory checkpoint ligand expression by glioblastoma cells.

Glioblastoma is a highly aggressive brain malignancy commonly refractory to classical and novel chemo-, radio- and immunotherapies, with median survival times of ~15 months following diagnosis. Poor immunological responses exemplified by the downregulation of T-cell activity, and upregulation of immunosuppressive cells within the tumor microenvironment have limited the effectiveness of immunotherapy in glioblastoma to date. Here we show that glioblastoma cells express a large repertoire of inhibitory checkpoint ligands known to control effector T cell responses. Furthermore, flow cytometry analysis reveals that glioblastoma cells with an enhanced stem cell-like phenotype express several investigated ligands at significant levels on their cell surface. This reveals that glioblastoma stem-like cells express suppressive ligands with the potential of suppressing major T cell checkpoint receptors. With this information, it is now essential that we understand the relevance of this extensive repertoire of immune checkpoint ligands and their functional consequence on immune evasion in glioblastoma. This is necessary to develop effective immunotherapeutics and to be able to match treatment to patient, especially in the light of CheckMate 143.

Floating and Diving Loops of ABA Triblock Copolymers in Lipid Bilayers and Stability Enhancement for Asymmetric Membranes.

Hybrid membranes of lipids and AxByAz triblock copolymers can possess better biocompatibility and mechanical stability. In this work, triblock copolymer conformations and stability of asymmetric membranes are explored by dissipative particle dynamics. The triblock copolymers in the membranes exhibit either the bridge or loop conformation. As hydrophobic B-blocks interact attractively with lipid heads, bridge-shaped copolymers are significantly inhibited and loop-shaped copolymers prefer to stay at the interface between hydrophilic and hydrophobic layers. This floating loop has a flattened conformation, consistent with the experimental findings. In contrast, for repulsive interactions between B-blocks and lipid heads, bridge-shaped copolymers are abundant and loop-shaped copolymers tend to plunge into the hydrophobic layer. This diving loop displays a random coil conformation. The asymmetric membrane in which the fractions of loop-shaped copolymers in the upper and lower leaflets are different is thermodynamically unstable. Two approaches are proposed to acquire kinetically stable asymmetric membranes. First, membrane symmetrization is arrested by eliminating bridge-shaped copolymers, which is achieved by B-block/lipid head attraction and B-block/lipid tail repulsion. Second, asymmetric triblock copolymers (x ≠ z) are used to prevent the passage of the long A-block through the hydrophobic layer.

Strengthening mechanism of the mechanical properties of graft copolymers with incompatible pendant groups: nano-clusters and weak cross-linking.

It is known that the adhesive property and mechanical strength of an apolar polymer can be improved by grafting with polar side chains, whereas the underlying mechanism is still elusive. In this work, the equilibrium structure and mechanical moduli of the melt of graft copolymers have been explored by dissipative particle dynamics. Due to the strong immiscibility of the non-polar backbone and polar side chains, nano-clusters of side chains formed and acted as physical crosslinkers. Moreover, non-affinity adsorption of polar side chains in the melt to the wall was observed, revealing an improvement in the adhesion property. Subjecting graft copolymers to cyclic deformation, the storage and loss moduli were acquired, and they grew with increasing grafting density. The melt strength in terms of the crossover frequency ascended with more side chains on the backbone. Our findings reveal that the strengthening of the mechanical properties of graft copolymers can be attributed to the formation of weakly cross-linked structures, thus offering an insight into the molecular design to aid the development of stronger graft copolymers.