Hyperbranched, Functional Polyethoxysiloxanes: Tunable Molecular Building Blocks.

Functional silanes are multifaceted cross-linkers, compatibilizers, coupling agents, and surface modifiers. Herein, we present organofunctional polysiloxane building blocks that offer great versatility in terms of molecular weight, degree of condensation, and the choice and loading of organic substituent groups. The organofunctional polyethoxysilanes (funPEOS) are prepared in a one-pot, two-step process: synthesis of the PEOS carrier/substrate, followed by grafting a functional silane "shell", both based on condensation with acetic anhydride. The reaction was optimized at the lab scale and scaled up to a 7 L reactor. The acetylation, condensation, and hyperbranched structure of the carrier were confirmed by 29Si NMR, while 29Si-29Si 2D INADEQUATE NMR provides strong evidence for the grafting of functional silanes onto the carrier (Q-T coupling). IR, 1H, and 13C NMR spectroscopy demonstrate that the functional groups remain intact. The molar mass can be tailored by stoichiometric control of the acetic anhydride to silane monomer ratio (M n 3500-20,000 g/mol). The compounds are stable organic liquids with a long shelf life. Selected applications are presented: scratch-resistant coatings with water contact angles of ∼90°, stable water emulsions, and surfactant-free, mesoporous silica foams.

Truncated variants of MAGEL2 are involved in the etiologies of the Schaaf-Yang and Prader-Willi syndromes.

The neurodevelopmental disorders Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) both arise from genomic alterations within human chromosome 15q11-q13. A deletion of the SNORD116 cluster, encoding small nucleolar RNAs, or frameshift mutations within MAGEL2 result in closely related phenotypes in individuals with PWS or SYS, respectively. By investigation of their subcellular localization, we observed that in contrast to a predominant cytoplasmic localization of wild-type (WT) MAGEL2, a truncated MAGEL2 mutant was evenly distributed between the cytoplasm and the nucleus. To elucidate regulatory pathways that may underlie both diseases, we identified protein interaction partners for WT or mutant MAGEL2, in particular the survival motor neuron protein (SMN), involved in spinal muscular atrophy, and the fragile-X-messenger ribonucleoprotein (FMRP), involved in autism spectrum disorders. The interactome of the non-coding RNA SNORD116 was also investigated by RNA-CoIP. We show that WT and truncated MAGEL2 were both involved in RNA metabolism, while regulation of transcription was mainly observed for WT MAGEL2. Hence, we investigated the influence of MAGEL2 mutations on the expression of genes from the PWS locus, including the SNORD116 cluster. Thereby, we provide evidence for MAGEL2 mutants decreasing the expression of SNORD116, SNORD115, and SNORD109A, as well as protein-coding genes MKRN3 and SNRPN, thus bridging the gap between PWS and SYS.

P80 natural essence spray and lozenges provide respiratory protection against Influenza A, B, and SARS-CoV-2.

Seasonally circulating viruses, such as Influenza, as well as newly emerging viruses and variants thereof, and waning immunity urge the need for safe, easy-to-use and inexpensive drugs to protect from these challenges. To prevent transmission of these viruses and subsequent excessive inflammatory reactions on mucous membranes, we tested the efficacy of the natural essence P80 as spray and in form of lozenges against respiratory infections caused by SARS-CoV-2 variants of concern (VoCs), influenza A (H3N2) and influenza B (Victoria). P80 natural essence, a Dimocarpus longan extract, shielded highly differentiated human airway epithelia from SARS-CoV-2 wildtype and Omicron variant as well as Influenza A and B infection and dampened inflammation by down-modulating pro-inflammatory cytokine and anaphylatoxin secretion. A single application of P80 natural essence spray maintained tissue integrity long-term. This also significantly reduced the release of infectious viral particles and the secretion of IP10, MCP1, RANTES and C3a, all of which mediate the migration of immune cells to the sites of infection. Even P80 lozenges dissolved in distilled water or non-neutralizing saliva efficiently prevented SARS-CoV-2 and Influenza-induced tissue destruction. Consequently, our in vitro data suggest that P80 natural essence can act as antiviral prophylactic, both in form of nasal or oral spray and in form of lozenges, independent of circulating respiratory challenges.

Plasma proteome signature of canine acute haemorrhagic diarrhoea syndrome (AHDS).

Acute haemorrhagic diarrhoea is a common complaint in dogs. In addition to causes like intestinal parasites, dietary indiscretion, intestinal foreign bodies, canine parvovirus infection, or hypoadrenocorticism, acute haemorrhagic diarrhoea syndrome (AHDS) is an important and sometimes life-threatening differential diagnosis. There is some evidence supporting the link between Clostridium perfringens toxins and AHDS. These toxins may be partially responsible for the epithelial cell injury, but the pathogenesis of AHDS is still not fully understood. Recent studies have suggested that severe damage to the intestinal mucosa and associated barrier dysfunction can trigger chronic gastrointestinal illnesses. Besides bloodwork and classical markers for AHDS such as protein loss and intestinal bacterial dysbiosis, we focused mainly on the plasma-proteome to identify systemic pathological alterations during this disease and searched for potential biomarkers to improve the diagnosis. To accomplish the goals, we used liquid chromatography-mass spectrometry. We compared the proteomic profiles of 20 dogs with AHDS to 20 age-, breed-, and sex-matched control dogs. All dogs were examined, and several blood work parameters were determined and compared, including plasma biochemistry and cell counts. We identified and quantified (relative quantification) 207 plasmatic proteins, from which dozens showed significantly altered levels in AHDS. Serpina3, Lipopolysaccharide-binding protein, several Ig-like domain-containing proteins, Glyceraldehyde-3-phosphate dehydrogenase and Serum amyloid A were more abundant in plasma from AHDS affected dogs. In contrast, other proteins such as Paraoxonase, Selenoprotein, Amine oxidases, and Apolipoprotein C-IV were significantly less abundant. Many of the identified and quantified proteins are known to be associated with inflammation. Other proteins like Serpina3 and RPLP1 have a relevant role in oncogenesis. Some proteins and their roles have not yet been described in dogs with diarrhoea. Our study opens new avenues that could contribute to the understanding of the aetiology and pathophysiology of AHDS.

Short-term glycemic variability in non-diabetic, non-obese dogs assessed by common glycemic variability indices.

Glycemic variability (GV) refers to swings in blood glucose levels and is an emerging measure of glycemic control in clinical practice. It is associated with micro- and macrovascular complications and poor clinical outcomes in diabetic humans. Although an integral part of patient assessment in human patients, it is to a large extent neglected in insulin-treated diabetic dogs. This prospective pilot study was performed to describe canine within-day GV in non-diabetic dogs with the aim to provide a basis for the interpretation of daily glucose profiles, and to promote GV as an accessible tool for future studies in veterinary medicine. Interstitial glucose concentrations of ten non-diabetic, non-obese beagles were continuously measured over a 48-h period using a flash glucose monitoring system. GV was assessed using the common indices MAGE (mean amplitude of glycemic excursion), GVP (Glycemic variability percentage) and CV (coefficient of variation). A total of 2260 sensor measurements were obtained, ranging from 3.7 mmol/L (67 mg/dL) to 8.5 mmol/L (153 mg/dL). Glucose profiles suggested a meal-dependent circadian rhythmicity with small but significant surges during the feeding periods. No differences in GV indices were observed between day and night periods (p > 0.05). The MAGE (mmol/L), GVP (%) and CV (%) were 0.86 (± 0.19), 7.37 (± 1.65), 6.72 (± 0.89) on day one, and 0.83 (± 0.18), 6.95 (± 1.52), 6.72 (± 1.53) on day two, respectively. The results of this study suggest that GV is low in non-diabetic dogs and that glucose concentrations are kept within narrow ranges.

Myocardial strain assessment in the human fetus by cardiac MRI using Doppler ultrasound gating and feature tracking.

OBJECTIVES: Assessment of myocardial strain by feature tracking magnetic resonance imaging (FT-MRI) in human fetuses with and without congenital heart disease (CHD) using cardiac Doppler ultrasound (DUS) gating. METHODS: A total of 43 human fetuses (gestational age 28-41 weeks) underwent dynamic cardiac MRI at 3 T. Cine balanced steady-state free-precession imaging was performed using fetal cardiac DUS gating. FT-MRI was analyzed using dedicated post-processing software. Endo- and epicardial contours were manually delineated from fetal cardiac 4-chamber views, followed by automated propagation to calculate global longitudinal strain (GLS) of the left (LV) and right ventricle (RV), LV radial strain, and LV strain rate. RESULTS: Strain assessment was successful in 38/43 fetuses (88%); 23 of them had postnatally confirmed diagnosis of CHD (e.g., coarctation, transposition of great arteries) and 15 were heart healthy. Five fetuses were excluded due to reduced image quality. In fetuses with CHD compared to healthy controls, median LV GLS (- 13.2% vs. - 18.9%; p < 0.007), RV GLS (- 7.9% vs. - 16.2%; p < 0.006), and LV strain rate (1.4 s-1 vs. 1.6 s-1; p < 0.003) were significantly higher (i.e., less negative). LV radial strain was without a statistically significant difference (20.7% vs. 22.6%; p = 0.1). Bivariate discriminant analysis for LV GLS and RV GLS revealed a sensitivity of 67% and specificity of 93% to differentiate between fetuses with CHD and healthy fetuses. CONCLUSION: Myocardial strain was successfully assessed in the human fetus, performing dynamic fetal cardiac MRI with DUS gating. Our study indicates that strain parameters may allow for differentiation between fetuses with and without CHD. CLINICAL RELEVANCE STATEMENT: Myocardial strain analysis by cardiac MRI with Doppler ultrasound gating and feature tracking may provide a new diagnostic approach for evaluation of fetal cardiac function in congenital heart disease. KEY POINTS: • MRI myocardial strain analysis has not been performed in human fetuses so far. • Myocardial strain was assessed in human fetuses using cardiac MRI with Doppler ultrasound gating. • MRI myocardial strain may provide a new diagnostic approach to evaluate fetal cardiac function.

Functional and spatial proteomics profiling reveals intra- and intercellular signaling crosstalk in colorectal cancer.

Precision oncology approaches for patients with colorectal cancer (CRC) continue to lag behind other solid cancers. Functional precision oncology-a strategy that is based on perturbing primary tumor cells from cancer patients-could provide a road forward to personalize treatment. We extend this paradigm to measuring proteome activity landscapes by acquiring quantitative phosphoproteomic data from patient-derived organoids (PDOs). We show that kinase inhibitors induce inhibitor- and patient-specific off-target effects and pathway crosstalk. Reconstruction of the kinase networks revealed that the signaling rewiring is modestly affected by mutations. We show non-genetic heterogeneity of the PDOs and upregulation of stemness and differentiation genes by kinase inhibitors. Using imaging mass-cytometry-based profiling of the primary tumors, we characterize the tumor microenvironment (TME) and determine spatial heterocellular crosstalk and tumor-immune cell interactions. Collectively, we provide a framework for inferring tumor cell intrinsic signaling and external signaling from the TME to inform precision (immuno-) oncology in CRC.

Omicron subvariants illustrate reduced respiratory tissue penetration, cell damage and inflammatory responses in human airway epithelia.

Introduction: To explore whether the reported lower pathogenicity in infected individuals of variant of concern (VoC) Omicron and its current subvariants compared to VoC Delta may be related to fundamental differences in the initial virus-tissue interaction, we assessed their ability to penetrate, replicate and cause damage in a human 3D respiratory model. Methods: For this, we used TEER measurements, real-time PCR, LDH, cytokine and complex confocal imaging analyses. Results and discussion: We observed that Delta readily penetrated deep into the respiratory epithelium and this was associated with major tissue destruction, high LDH activity, high viral loads and pronounced innate immune activation as observed by intrinsic C3 activation and IL-6 release at infection sites. In contrast, Omicron subvariants BA.5, BQ.1.1 and BF7 remained superficially in the mucosal layer resulting merely in outward-directed destruction of cells, maintenance of epithelial integrity, minimal LDH activity and low basolateral release of virus at infection sites, as well as significantly smaller areas of complement activation and lower IL-6 secretion. Interestingly, also within Omicron subvariants differences were observed with newer Omicron subvariants BQ.1.1 and BF.7 illustrating significantly reduced viral loads, IL-6 release and LDH activity compared to BA.5. Our data indicate that earliest interaction events after SARS-CoV-2 transmission may have a role in shaping disease severity.

Association between clinical history in the radiographic request and diagnostic accuracy of thorax radiographs in dogs: A retrospective case-control study.

BACKGROUND: The effect of clinical history on the interpretation of radiographs has been widely researched in human medicine. There is, however, no data on this topic in veterinary medicine. HYPOTHESIS/OBJECTIVES: Diagnostic accuracy would improve when history was supplied. ANIMALS: Thirty client-owned dogs with abnormal findings on thoracic radiographs and confirmation of the disease, and 30 healthy client-owned controls were drawn retrospectively. METHODS: Retrospective case-control study. Sixty radiographic studies of the thorax were randomized and interpreted by 6 radiologists; first, with no access to the clinical information; and a second time with access to all pertinent clinical information and signalment. RESULTS: A significant increase in diagnostic accuracy was noted when clinical information was provided (64.4% without and 75.2% with clinical information; P = .002). There was no significant difference in agreement between radiologists when comparing no clinical information and with clinical information (Kappa 0.313 and 0.300, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: The addition of pertinent clinical information to the radiographic request significantly improves the diagnostic accuracy of thorax radiographs of dogs and is recommended as standard practice.

The negative adipogenesis regulator Dlk1 is transcriptionally regulated by Ifrd1 (TIS7) and translationally by its orthologue Ifrd2 (SKMc15).

Delta-like homolog 1 (Dlk1), an inhibitor of adipogenesis, controls the cell fate of adipocyte progenitors. Experimental data presented here identify two independent regulatory mechanisms, transcriptional and translational, by which Ifrd1 (TIS7) and its orthologue Ifrd2 (SKMc15) regulate Dlk1 levels. Mice deficient in both Ifrd1 and Ifrd2 (dKO) had severely reduced adipose tissue and were resistant to high-fat diet-induced obesity. Wnt signaling, a negative regulator of adipocyte differentiation, was significantly upregulated in dKO mice. Elevated levels of the Wnt/ß-catenin target protein Dlk1 inhibited the expression of adipogenesis regulators Pparg and Cebpa, and fatty acid transporter Cd36. Although both Ifrd1 and Ifrd2 contributed to this phenotype, they utilized two different mechanisms. Ifrd1 acted by controlling Wnt signaling and thereby transcriptional regulation of Dlk1. On the other hand, distinctive experimental evidence showed that Ifrd2 acts as a general translational inhibitor significantly affecting Dlk1 protein levels. Novel mechanisms of Dlk1 regulation in adipocyte differentiation involving Ifrd1 and Ifrd2 are based on experimental data presented here.

Cardiac T1 mapping enables risk prediction of LV dysfunction after surgery for aortic regurgitation.

Background: To assess whether cardiac T1 mapping for detecting myocardial fibrosis enables preoperative identification of patients at risk for early left ventricular dysfunction after surgery of aortic regurgitation. Methods: 1.5 Tesla cardiac magnetic resonance imaging was performed in 40 consecutive aortic regurgitation patients before aortic valve surgery. Native and post-contrast T1 mapping was performed using a modified Look-Locker inversion-recovery sequence. Serial echocardiography was performed at baseline and 8 ± 5 days after aortic valve surgery to quantify LV dysfunction. Receiver operating characteristic analysis was performed to determine the diagnostic accuracy of native T1 mapping and extracellular volume for predicting postoperative LV ejection fraction decrease >-10% after aortic valve surgery. Results: Native T1 was significantly increased in patients with a postoperatively decreased LVEF (n = 15) vs. patients with a preserved postoperative LV ejection fraction (n = 25) (i.e., 1,071 ± 67 ms vs. 1,019 ± 33 ms, p = .001). Extracellular volume was not significantly different between patients with preserved vs. decreased postoperative LV ejection fraction. With a cutoff-of value of 1,053 ms, native T1 yielded an area under the curve (AUC) of .820 (95% CI: .683-.958) for differentiating between patients with preserved vs. reduced LV ejection fraction with 70% sensitivity and 84% specificity. Conclusion: Increased preoperative native T1 is associated with a significantly higher risk of systolic LV dysfunction early after aortic valve surgery in aortic regurgitation patients. Native T1 could be a promising tool to optimize the timing of aortic valve surgery in patients with aortic regurgitation to prevent early postoperative LV dysfunction.

SLC5A1 Variants in Turkish Patients with Congenital Glucose-Galactose Malabsorption.

Congenital glucose-galactose malabsorption is a rare autosomal recessive disorder caused by mutations in SLC5A1 encoding the apical sodium/glucose cotransporter SGLT1. We present clinical and molecular data from eleven affected individuals with congenital glucose-galactose malabsorption from four unrelated, consanguineous Turkish families. Early recognition and timely management by eliminating glucose and galactose from the diet are fundamental for affected individuals to survive and develop normally. We identified novel SLC5A1 missense variants, p.Gly43Arg and p.Ala92Val, which were linked to disease in two families. Stable expression in CaCo-2 cells showed that the p.Ala92Val variant did not reach the plasma membrane, but was retained in the endoplasmic reticulum. The p.Gly43Arg variant, however, displayed processing and plasma membrane localization comparable to wild-type SGLT1. Glycine-43 displays nearly invariant conservation in the relevant structural family of cotransporters and exchangers, and localizes to SGLT1 transmembrane domain TM0. p.Gly43Arg represents the first disease-associated variant in TM0; however, the role of TM0 in the SGLT1 function has not been established. In summary, we are expanding the mutational spectrum of this rare disorder.

The developmental trajectory of object recognition robustness: Children are like small adults but unlike big deep neural networks.

In laboratory object recognition tasks based on undistorted photographs, both adult humans and deep neural networks (DNNs) perform close to ceiling. Unlike adults', whose object recognition performance is robust against a wide range of image distortions, DNNs trained on standard ImageNet (1.3M images) perform poorly on distorted images. However, the last 2 years have seen impressive gains in DNN distortion robustness, predominantly achieved through ever-increasing large-scale datasets-orders of magnitude larger than ImageNet. Although this simple brute-force approach is very effective in achieving human-level robustness in DNNs, it raises the question of whether human robustness, too, is simply due to extensive experience with (distorted) visual input during childhood and beyond. Here we investigate this question by comparing the core object recognition performance of 146 children (aged 4-15 years) against adults and against DNNs. We find, first, that already 4- to 6-year-olds show remarkable robustness to image distortions and outperform DNNs trained on ImageNet. Second, we estimated the number of images children had been exposed to during their lifetime. Compared with various DNNs, children's high robustness requires relatively little data. Third, when recognizing objects, children-like adults but unlike DNNs-rely heavily on shape but not on texture cues. Together our results suggest that the remarkable robustness to distortions emerges early in the developmental trajectory of human object recognition and is unlikely the result of a mere accumulation of experience with distorted visual input. Even though current DNNs match human performance regarding robustness, they seem to rely on different and more data-hungry strategies to do so.

RagD auto-activating mutations impair MiT/TFE activity in kidney tubulopathy and cardiomyopathy syndrome.

Heterozygous mutations in the gene encoding RagD GTPase were shown to cause a novel autosomal dominant condition characterized by kidney tubulopathy and cardiomyopathy. We previously demonstrated that RagD, and its paralogue RagC, mediate a non-canonical mTORC1 signaling pathway that inhibits the activity of TFEB and TFE3, transcription factors of the MiT/TFE family and master regulators of lysosomal biogenesis and autophagy. Here we show that RagD mutations causing kidney tubulopathy and cardiomyopathy are "auto- activating", even in the absence of Folliculin, the GAP responsible for RagC/D activation, and cause constitutive phosphorylation of TFEB and TFE3 by mTORC1, without affecting the phosphorylation of "canonical" mTORC1 substrates, such as S6K. By using HeLa and HK-2 cell lines, human induced pluripotent stem cell-derived cardiomyocytes and patient-derived primary fibroblasts, we show that RRAGD auto-activating mutations lead to inhibition of TFEB and TFE3 nuclear translocation and transcriptional activity, which impairs the response to lysosomal and mitochondrial injury. These data suggest that inhibition of MiT/TFE factors plays a key role in kidney tubulopathy and cardiomyopathy syndrome.

Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification.

BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

The lysosomal LAMTOR / Ragulator complex is essential for nutrient homeostasis in brown adipose tissue.

OBJECTIVE: In brown adipose tissue (iBAT), the balance between lipid/glucose uptake and lipolysis is tightly regulated by insulin signaling. Downstream of the insulin receptor, PDK1 and mTORC2 phosphorylate AKT, which activates glucose uptake and lysosomal mTORC1 signaling. The latter requires the late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) complex, which serves to translate the nutrient status of the cell to the respective kinase. However, the role of LAMTOR in metabolically active iBAT has been elusive. METHODS: Using an AdipoqCRE-transgenic mouse line, we deleted LAMTOR2 (and thereby the entire LAMTOR complex) in adipose tissue (LT2 AKO). To examine the metabolic consequences, we performed metabolic and biochemical studies in iBAT isolated from mice housed at different temperatures (30 °C, room temperature and 5 °C), after insulin treatment, or in fasted and refed condition. For mechanistic studies, mouse embryonic fibroblasts (MEFs) lacking LAMTOR 2 were analyzed. RESULTS: Deletion of the LAMTOR complex in mouse adipocytes resulted in insulin-independent AKT hyperphosphorylation in iBAT, causing increased glucose and fatty acid uptake, which led to massively enlarged lipid droplets. As LAMTOR2 was essential for the upregulation of de novo lipogenesis, LAMTOR2 deficiency triggered exogenous glucose storage as glycogen in iBAT. These effects are cell autonomous, since AKT hyperphosphorylation was abrogated by PI3K inhibition or by deletion of the mTORC2 component Rictor in LAMTOR2-deficient MEFs. CONCLUSIONS: We identified a homeostatic circuit for the maintenance of iBAT metabolism that links the LAMTOR-mTORC1 pathway to PI3K-mTORC2-AKT signaling downstream of the insulin receptor.

GlyPerA™ effectively shields airway epithelia from SARS-CoV-2 infection and inflammatory events.

New SARS-CoV-2 variants of concern (VOCs) and waning immunity illustrate that quick and easy-to-use agents are needed to prevent infection. To protect from viral transmission and subsequent inflammatory reactions, we applied GlyperA™, a novel antimicrobial formulation that can be used as mouth gargling solution or as nasal spray, to highly differentiated human airway epithelia prior infection with Omicron VOCs BA.1 and BA.2. This formulation fully protected polarized human epithelium cultured in air-liquid interphase (ALI) from SARS-CoV-2-mediated tissue destruction and infection upon single application up to two days post infection. Moreover, inflammatory reactions induced by the Omicron VOCs were significantly lowered in tissue equivalents either pre-treated with the GlyperA™ solution, or even when added simultaneously. Thus, the GlyperA™ formulation significantly shielded epithelial integrity, successfully blocked infection with Omicron and release of viral particles, and decreased intracellular complement C3 activation within human airway epithelial cell cultures. Crucially, our in vitro data imply that GlyperA™ may be a simple tool to prevent from SARS-CoV-2 infection independent on the circulating variant via both, mouth and nose.

Structure of the lysosomal mTORC1-TFEB-Rag-Ragulator megacomplex.

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy1. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1)2-5 is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN6,7. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the 'megacomplex'. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.

Cognitive reappraisal, emotional expression and mindfulness in adaptation to bereavement: a longitudinal study.

BACKGROUND AND OBJECTIVES: Maladaptive emotion regulation strategies increase prolonged grief and depressive symptoms following bereavement. However, less is known about the role of adaptive emotion regulation strategies in adaptation to loss. Therefore, we examined the concurrent and longitudinal associations of three putative adaptive emotion regulation strategies (cognitive reappraisal, emotional expression, and mindfulness) with prolonged grief and depression symptoms. DESIGN: A two-wave longitudinal survey. METHODS: A sample of 397 bereaved Dutch adults (89% female, mean age 54 years) completed validated questionnaires to assess trait cognitive reappraisal, emotional expression, mindfulness and prolonged grief and depression symptoms at baseline (T1) and 344 participants completed symptom measures again six months later (T2). RESULTS: Zero-order correlations demonstrated that mindfulness, cognitive reappraisal and emotional expression relate negatively to T1 and T2 prolonged grief and depression symptoms. In multiple regression analyses, controlling for relevant background variables, all emotion regulation strategies related negatively to T1 prolonged grief and depression symptoms. In multiple regression analyses, controlling for T1 symptoms and background variables, mindfulness predicted lower T2 depression symptoms. CONCLUSIONS: Adaptive emotion regulation strategies relate negatively to post-loss psychopathology symptoms, yet only mindfulness longitudinally predicts lower depression symptoms. Dispositional mindfulness may be a protective factor in psychological adaptation to bereavement.

A CRISPR screen in intestinal epithelial cells identifies novel factors for polarity and apical transport.

Epithelial polarization and polarized cargo transport are highly coordinated and interdependent processes. In our search for novel regulators of epithelial polarization and protein secretion, we used a genome-wide CRISPR/Cas9 screen and combined it with an assay based on fluorescence-activated cell sorting (FACS) to measure the secretion of the apical brush-border hydrolase dipeptidyl peptidase 4 (DPP4). In this way, we performed the first CRISPR screen to date in human polarized epithelial cells. Using high-resolution microscopy, we detected polarization defects and mislocalization of DPP4 to late endosomes/lysosomes after knockout of TM9SF4, anoctamin 8, and ARHGAP33, confirming the identification of novel factors for epithelial polarization and apical cargo secretion. Thus, we provide a powerful tool suitable for studying polarization and cargo secretion in epithelial cells. In addition, we provide a dataset that serves as a resource for the study of novel mechanisms for epithelial polarization and polarized transport and facilitates the investigation of novel congenital diseases associated with these processes.