Spike substitution T813S increases Sarbecovirus fusogenicity by enhancing the usage of TMPRSS2.

SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus' evolution.

CCDC50 promotes tumor growth through regulation of lysosome homeostasis.

The maintenance of lysosome homeostasis is crucial for cell growth. Lysosome-dependent degradation and metabolism sustain tumor cell survival. Here, we demonstrate that CCDC50 serves as a lysophagy receptor, promoting tumor progression and invasion by controlling lysosomal integrity and renewal. CCDC50 monitors lysosomal damage, recognizes galectin-3 and K63-linked polyubiquitination on damaged lysosomes, and specifically targets them for autophagy-dependent degradation. CCDC50 deficiency causes the accumulation of ruptured lysosomes, impaired autophagic flux, and superfluous reactive oxygen species, consequently leading to cell death and tumor suppression. CCDC50 expression is associated with malignancy, progression to metastasis, and poor overall survival in human melanoma. Targeting CCDC50 suppresses tumor growth and lung metastasis, and enhances the effect of BRAFV600E inhibition. Thus, we demonstrate critical roles of CCDC50-mediated clearance of damaged lysosomes in supporting tumor growth, hereby identifying a potential therapeutic target of melanoma.

TRIM21 Ubiquitylates SQSTM1/p62 and Suppresses Protein Sequestration to Regulate Redox Homeostasis.

TRIM21 is a RING finger domain-containing ubiquitin E3 ligase whose expression is elevated in autoimmune disease. While TRIM21 plays an important role in immune activation during pathogen infection, little is known about its inherent cellular function. Here we show that TRIM21 plays an essential role in redox regulation by directly interacting with SQSTM1/p62 and ubiquitylating p62 at lysine 7 (K7) via K63-linkage. As p62 oligomerizes and sequesters client proteins in inclusions, the TRIM21-mediated p62 ubiquitylation abrogates p62 oligomerization and sequestration of proteins including Keap1, a negative regulator of antioxidant response. TRIM21-deficient cells display an enhanced antioxidant response and reduced cell death in response to oxidative stress. Genetic ablation of TRIM21 in mice confers protection from oxidative damages caused by arsenic-induced liver insult and pressure overload heart injury. Therefore, TRIM21 plays an essential role in p62-regulated redox homeostasis and may be a viable target for treating pathological conditions resulting from oxidative damage.

PACT inhibits the replication of SARS-CoV-2 through the blockage of GSK-3ß-N-nsp3 cascade.

The protein activator of protein kinase R (PKR) (PACT) has been shown to play a crucial role in stimulating the host antiviral response through the activation of PKR, retinoic acid-inducible gene I, and melanoma differentiation-associated protein 5. Whether PACT can inhibit viral replication independent of known mechanisms is still unrevealed. In this study, we show that, like many viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks GSK-3ß to facilitate its replication. GSK-3ß-induced phosphorylation on N protein increased the interaction between N protein and nsp3. Thus, GSK-3ß-N-nsp3 cascade promotes viral replication. Although SARS-CoV-2 can sabotage the activation of AKT, the upstream proteins suppressing the activation of GSK-3ß, we found that the host can use PACT, another protein kinase, instead of AKT to decrease the activity of GSK-3ß and the interaction between PACT and GSK-3ß is enhanced upon viral infection. Moreover, PACT inhibited the activity of GSK-3ß independent of its well-studied double-stranded RNA binding and PKR activating ability. In summary, this study identified an unknown function of PACT in inhibiting SARS-CoV-2 replication through the blockage of GSK-3ß-N-nsp3 cascade.

SARS-CoV-2 NSP7 inhibits type I and III IFN production by targeting the RIG-I/MDA5, TRIF, and STING signaling pathways.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a poor inducer of innate antiviral immunity, and the underlying mechanism still needs further investigation. Here, we reported that SARS-CoV-2 NSP7 inhibited the production of type I and III interferons (IFNs) by targeting the RIG-I/MDA5, Toll-like receptor (TLR3)-TRIF, and cGAS-STING signaling pathways. SARS-CoV-2 NSP7 suppressed the expression of IFNs and IFN-stimulated genes induced by poly (I:C) transfection and infection with Sendai virus or SARS-CoV-2 virus-like particles. NSP7 impaired type I and III IFN production activated by components of the cytosolic dsRNA-sensing pathway, including RIG-I, MDA5, and MAVS, but not TBK1, IKKε, and IRF3-5D, an active form of IRF3. In addition, NSP7 also suppressed TRIF- and STING-induced IFN responses. Mechanistically, NSP7 associated with RIG-I and MDA5 prevented the formation of the RIG-I/MDA5-MAVS signalosome and interacted with TRIF and STING to inhibit TRIF-TBK1 and STING-TBK1 complex formation, thus reducing the subsequent IRF3 phosphorylation and nuclear translocation that are essential for IFN induction. In addition, ectopic expression of NSP7 impeded innate immune activation and facilitated virus replication. Taken together, SARS-CoV-2 NSP7 dampens type I and III IFN responses via disruption of the signal transduction of the RIG-I/MDA5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways, thus providing novel insights into the interactions between SARS-CoV-2 and innate antiviral immunity.

Nucleon Energy Correlators for the Color Glass Condensate.

We demonstrate the recently proposed nucleon energy-energy correlator (NEEC) f_{EEC}(x,θ) can unveil the gluon saturation in the small-x regime in eA collisions. The novelty of this probe is that it is fully inclusive just like the deep-inelastic scattering (DIS), with no requirements of jets or hadrons but still provides an evident portal to the small-x dynamics through the shape of the θ distribution. We find that the saturation prediction is significantly different from the expectation of the collinear factorization.

Non-suicidal self-injury and professional psychological help-seeking among Chinese left-behind children: prevalence and influencing factors.

BACKGROUND: Non-suicidal self-injury (NSSI) is a risk factor for suicide. This study aimed to investigate the prevalence of NSSI and professional psychological help-seeking status and influencing factors among left-behind children (LBC) in China. METHODS: We implemented a population-based cross-sectional study in participants aged 10-18 years. Sociodemographic characteristics, NSSI, help-seeking status and coping style were measured by self-reported questionnaires. A total of 16,866 valid questionnaires were collected, including 6096 LBC. Binary logistic regression models were used to analyze the factors influencing NSSI and professional psychological help-seeking. RESULTS: The incidence of NSSI among LBC was 4.6%, significantly higher than that of non-left-behind children (NLBC). This incidence was higher among girls. Moreover, 53.9% of LBC with NSSI did not receive any treatment and only 22.0% sought professional psychological help. LBC often adopt emotion-oriented coping styles, specifically, those with NSSI. LBC with NSSI who seek professional help tend to adopt problem-oriented coping styles. Logistic regression analysis revealed that girls, learning stage, single-parent, remarried families, patience, and emotional venting were risk factors for NSSI in LBC, while problem-solving and social support seeking were protective factors. Moreover, problem-solving was also a predictor for seeking professional psychological help, patience will prevent it. LIMITATIONS: This was an online survey. CONCLUSIONS: The prevalence of NSSI in LBC is high. Gender, grade, family structure, and coping style affect the occurrence of NSSI among LBC. Only a few LBC with NSSI seek professional psychological help, while the coping style will affect the help-seeking behavior.

Inhibition of ALG3 stimulates cancer cell immunogenic ferroptosis to potentiate immunotherapy.

Immune checkpoint blockade therapy has drastically improved the prognosis of certain advanced-stage cancers. However, low response rates and immune-related adverse events remain important limitations. Here, we report that inhibiting ALG3, an a-1,3-mannosyltransferase involved in protein glycosylation in the endoplasmic reticulum (ER), can boost the response of tumors to immune checkpoint blockade therapy. Deleting N-linked glycosylation gene ALG3 in mouse cancer cells substantially attenuates their growth in mice in a manner depending on cytotoxic T cells. Furthermore, ALG3 inhibition or N-linked glycosylation inhibitor tunicamycin treatment synergizes with anti-PD1 therapy in suppressing tumor growth in mouse models of cancer. Mechanistically, we found that inhibiting ALG3 induced deficiencies of post-translational N-linked glycosylation modification and led to excessive lipid accumulation through sterol-regulated element-binding protein (SREBP1)-dependent lipogenesis in cancer cells. N-linked glycosylation deficiency-mediated lipid hyperperoxidation induced immunogenic ferroptosis of cancer cells and promoted a pro-inflammatory microenvironment, which boosted anti-tumor immune responses. In human subjects with cancer, elevated levels of ALG3 expression in tumor tissues are associated with poor patient survival. Taken together, we reveal an unappreciated role of ALG3 in regulating tumor immunogenicity and propose a potential therapeutic strategy for enhancing cancer immunotherapy.

Unsupervised Learning-Guided Accelerated Discovery of Alkaline Anion Exchange Membranes for Fuel Cells.

Without insight into the correlation between the structure and properties, anion exchange membranes (AEMs) for fuel cells are developed usually using the empirical trial and error method or simulation methods. Here, a virtual module compound enumeration screening (V-MCES) approach, which does not require the establishment of expensive training databases and can search the chemical space containing more than 4.2×105 candidates was proposed. The accuracy of the V-MCES model was considerably improved when the model was combined with supervised learning for the feature selection of molecular descriptors. Techniques from V-MCES, correlating the molecular structures of the AEMs with the predicted chemical stability, generated a ranking list of potential high stability AEMs. Under the guidance of V-MCES, highly stable AEMs were synthesized. With understanding of AEM structure and performance by machine learning, AEM science may enter a new era of unprecedented levels of architectural design.

Reverse genetics systems for SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) has caused severe public health crises and heavy economic losses. Limited knowledge about this deadly virus impairs our capacity to set up a toolkit against it. Thus, more studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology are urgently needed. Reverse genetics systems, including viral infectious clones and replicons, are powerful platforms for viral research projects, spanning many aspects such as the rescues of wild-type or mutant viral particles, the investigation of viral replication mechanism, the characterization of viral protein functions, and the studies on viral pathogenesis and antiviral drug development. The operations on viral infectious clones are strictly limited in the Biosafety Level 3 (BSL3) facilities, which are insufficient, especially during the pandemic. In contrast, the operation on the noninfectious replicon can be performed in Biosafety Level 2 (BSL2) facilities, which are widely available. After the outbreak of COVID-19, many reverse genetics systems for SARS-CoV-2, including infectious clones and replicons are developed and given plenty of options for researchers to pick up according to the requirement of their research works. In this review, we summarize the available reverse genetics systems for SARS-CoV-2, by highlighting the features of these systems, and provide a quick guide for researchers, especially those without ample experience in operating viral reverse genetics systems.

ORF8 protein of SARS-CoV-2 reduces male fertility in mice.

As one of the most rapidly evolving proteins of the genus Betacoronavirus, open reading frames (ORF8's) function and potential pathological consequence in vivo are still obscure. In this study, we show that the secretion of ORF8 is dependent on its N-terminal signal peptide sequence and can be inhibited by reactive oxygen species scavenger and endoplasmic reticulum-Golgi transportation inhibitor in cultured cells. To trace the effect of its possible in vivo secretion, we examined the plasma samples of coronavirus disease 2019 (COVID-19) convalescent patients and found that the patients aged from 40 to 60 had higher antibody titers than those under 40. To explore ORF8's in vivo function, we administered the mice with ORF8 via tail-vein injection to simulate the circulating ORF8 in the patient. Although no apparent difference in body weight, food intake, and vitality was detected between vehicle- and ORF8-treated mice, the latter displayed morphological abnormalities of testes and epididymides, as indicated by the loss of the central ductal lumen accompanied by a decreased fertility in 5-week-old male mice. Furthermore, the analysis of gene expression in the testes between vehicle- and ORF8-treated mice identified a decreased expression of Col1a1, the loss of which is known to be associated with mice's infertility. Although whether our observation in mice could be translated to humans remains unclear, our study provides a potential mouse model that can be used to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the human reproductive system.

[Effects of Interleukin-6 Gene Knockout on ß-amyloid Deposition and Cognition in 5×FAD Mouse Model of Alzheimer's Disease].

Objective To explore the effects of interleukin-6 (IL-6) gene knockout on the cognitive function and pathological changes in 5×FAD transgenic mice of Alzheimer's disease.Methods IL-6+/- mice were crossed with 5×FAD mice to establish the 5×FAD;IL-6-/- mouse model,and 3-month-old and 10-month-old mice were selected for experiments.The cognitive function of mice was detected by behavioral tests,and HE staining and ß-amyloid (Aß) immunohistochemical staining were performed to detect the pathological changes of mouse brain tissue.Results The number of 5×FAD;IL-6-/- model mice (3 months old,n=20;10 months old,n=5) and 5×FAD littermate control (3 months old,n=26;10 months old,n=24) conformed to the Mendel's law.Compared with that of the 5×FAD mice at the same age,the discrimination ratio of 3-month-old 5×FAD;IL-6-/- mice increased in the novel object recognition test (q=3.890,P=0.002).Morris water maze test results showed that the 3-month-old 5×FAD;IL-6-/- mice had longer time spent in target quadrant (q=3.797,P=0.012) and more times of crossing platform (q=2.505,P=0.017) than the 5×FAD mice at the same age.The results of immunohistochemical staining showed that IL-6 knockout reduced the Aß deposition in the hippocampus (q=13.490,P=0.002;q=45.680,P<0.001) and cortex (q=16.830,P=0.001;q=14.180,P=0.001) of 5×FAD mice.Conclusion IL-6 gene knockout can significantly improve the spatial memory and reduce the Aß deposition in the brain of 5×FAD mice.

Female reproductive abnormalities in mouse adolescent pregnancy.

There are around 300 million adolescent pregnancies worldwide, accounting for 11% of all births worldwide. Accumulating evidence demonstrates that many adverse perinatal outcomes are associated with adolescent pregnancies. However, how and why these abnormalities occur remain to be defined. In this study, pregnancy at different stages was compared between 25- and 30- day-old and mature female mice. We found that the litter size of adolescent pregnancy is significantly decreased from F1 to F3 generations compared to mature pregnancy. On days 8 and 12 of pregnancy, multiple abnormalities in decidual and placental development appear in F3 adolescent pregnancy. On days 5 and 8, uterine endoplasmic reticulum stress is dysregulated in F3 adolescent pregnancy. Embryo implantation and decidualization are also compromised in adolescent pregnancy. Many genes are abnormally expressed in adolescent estrous uteri. The abnormal endocrine environment and abnormal implantation from uterine immaturity may result in multiple pregnancy failures in adolescent pregnancy. The aim of this study is to shed light on human adolescent pregnancy.

Autophagy mediates degradation of nuclear lamina.

Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3-lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3-lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.

Class IA PI3K p110ß subunit promotes autophagy through Rab5 small GTPase in response to growth factor limitation.

Autophagy is an evolutionarily conserved membrane trafficking process. Induction of autophagy in response to nutrient limitation or cellular stress occurs by similar mechanisms in organisms from yeast to mammals. Unlike yeast, metazoan cells rely more on growth factor signaling for a wide variety of cellular activities including nutrient uptake. How growth factor availability regulates autophagy is poorly understood. Here we show that, upon growth factor limitation, the p110ß catalytic subunit of the class IA phosphoinositide 3-kinases (PI3Ks) dissociates from growth factor receptor complexes and increases its interaction with the small GTPase Rab5. This p110ß-Rab5 association maintains Rab5 in its guanosine triphosphate (GTP)-bound state and enhances the Rab5-Vps34 interaction that promotes autophagy. p110ß mutants that fail to interact with Rab5 are defective in autophagy promotion. Hence, in mammalian cells, p110ß acts as a molecular sensor for growth factor availability and induces autophagy by activating a Rab5-mediated signaling cascade.

A PET imaging study of the brain changes of glucose metabolism in patients with temporal lobe epilepsy and depressive disorder.

BACKGROUND: This study aims to compare the difference of the brain changes of glucose metabolism between temporal lobe epilepsy patients (TLE) with major depressive disorder and temporal TLE without major depressive disorder. METHODS: A total of 24 TLE patients, who met the inclusion criteria of our hospital, were enrolled in this study. They were divided into a TLE with depression group (n = 11) and a TLE without depression group (n = 13), according to the results of the HAMD-24 Scale. Two groups patients were examined using 18F-FDG PET brain imaging. RESULTS: The low metabolic regions of the TLE with depression group were mainly found in the left frontal lobe, temporal lobe and fusiform gyrus, while the high metabolic regions of the TLE with depression group were mainly located in the right frontal lobe, visual joint cortex and superior posterior cingulate cortex. Both of the TLE groups had high metabolic compensation in the non-epileptic area during the interictal period. CONCLUSIONS: There is an uptake difference of 18F-FDG between TLE patients with depression and TLE patients without depression in multiple encephalic regions.

High-dose omega-3 polyunsaturated fatty acid supplementation might be more superior than low-dose for major depressive disorder in early therapy period: a network meta-analysis.

BACKGROUND: The application of n-3 Polyunsaturated Fatty Acids (n-3 PUFAs) supplementation for major depressive disorder (MDD) has been widely discussed in recent years, but its efficacy and application are still controversial. This network meta-analysis was conducted to compare the efficacy of different dosages of n-3 PUFAs on MDD patients in the early period of treatment. METHODS: Randomized controlled trials (RCTs) exploring the efficacy of n-3 PUFA supplementation for patients with MDD were retrieved from the databases of Pubmed, Embase and the Cochrane Library. RCTs comparing the efficacy of n-3 PUFA for adult (≥18 years) MDD patients without comorbidity were eligible for our study. The score of depressive symptoms in early therapy period of the treatment (≤9 weeks) was extracted. Standardized mean deviations (SMDs) of all the sores from the eligible RCTs were synthesized in a pairwise meta-analysis in frequentist framework and a random-effects network meta-analysis in Bayesian framework for the overall and subgroups (high- and low-dose) efficacy of n-3 PUFAs. RESULTS: A total of 910 MDD patients in 10 trials with 3 adjuvant therapy strategies (high-dose n-3 PUFAs, low-dose n-3 PUFAs and placebo) were included. Results of pairwise meta-analysis showed that n-3 PUFAs were superior to placebo (SMD: 1.243 ± 0.596; 95% CI: 0.060 ~ 2.414). Results of the network meta-analysis showed that both the high (SMD: 0.908 ± 0.331; 95% CI: 0.262 ~ 1.581) and the low-dose (SMD: 0.601 ± 0.286; 95% CI: 0.034 ~ 1.18) n-3 PUFAs were superior to placebo, and the efficacy of high-dose n-3 PUFAs is superior to that of low-dose. CONCLUSIONS: High-dose n-3 PUFAs supplementation might be more superior than low-dose in the early therapy period for MDD. More head-to-head clinical trials need to be carried out to provide more direct comparison and enhance the evidence of the efficacy of n-3PUFAs for MDD.

Dexamethasone suppresses osteogenesis of osteoblast via the PI3K/Akt signaling pathway in vitro and in vivo.

The hypofunction of osteoblasts induced by glucocorticoids (GCs) has been identified as a major contributing factor for GC-induced osteoporosis (GIO). However, the biological mechanism underlying the effect of GC in osteoblasts are not fully elucidated. Recent studies implicated an important role of phosphoinositide 3-kinase (PI3K)/protein kinase B(Akt) signaling pathway in the regulation of bone growth. We propose that the PI3K/Akt signaling may be implicated in the process of GC-induced osteogenic inhibition in osteoblasts. In this study, primary osteoblasts were used in vitro and in rats in vivo to evaluate the biological significance of the PI3K/Akt pathway in GC-induced bone loss. In vivo, dexamethasone (Dex)-treated rats had low bone mineral density and decreased expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), and phosphorylated Akt (p-Akt) in bone tissue. In vitro study shows that Dex over the dose of 10-8 M remarkably inhibited cellular osteogenesis, as represented by decreased cell viability, lessened ALP activity, and suppressed osteogenic protein expressions including ALP and OCN. Meanwhile, a dramatic downregulation in the PI3K/Akt pathway phosphorylation was also observed in Dex-treated osteoblasts. These changes were marked rescued by treatment with a PI3K agonist 740Y-P. Moreover, downregulation of ALP and OCN expressions by LY294002 can mimic the suppressive effects of Dex. These data together reveal that the suppressed PI3K/Akt pathway is involved in the regulatory action of Dex on osteogenesis.

Triptolide induces p53-dependent cardiotoxicity through mitochondrial membrane permeabilization in cardiomyocytes.

Triptolide (TP), a major active component of Tripterygium wilfordii Hook f., is widely used in the treatment of inflammation and autoimmune disorders. Its clinical application is limited by severe adverse effects, especially cardiotoxicity. Accumulative evidences indicate that TP induces DNA damage by inhibiting RNA polymerase. Considering the relationship among DNA damage, p53, and the role of p53 in mitochondria-dependent apoptosis, we speculate that TP-induced cardiotoxicity results from p53 activation. In this study, the role of p53 in TP-induced cardiotoxicity was investigated in H9c2 cells, primary cardiomyocytes, and C57BL/6 genetic background p53-/- mice. p53 protein level was elevated by TP in vitro and in acute heart injury models. With TP administration (1.2 mg/kg), p53 deficiency prevented heart histology injury and decreased serum cardiac troponin I (cTn-I) and apoptotic proteins. Mechanistically, immunoblotting and immunofluorescence staining identified that TP-induced toxicity is dependent on p53 nuclear translocation and transactivation of Bcl2 family genes, leading to mitochondrial outer membrane permeabilization (MOMP) and mitochondria dysfunction. Consistently, p53 antagonist PFTα counteracted TP-induced p53 overexpression and regulation of Bcl2 family transcription, which improved mitochondrial membrane integrity and prevented apoptosis. Moreover, Bax antagonist Bax inhibitor peptide (BIP) V5 ameliorated TP-induced apoptosis through suppressing membrane depolarization and ROS accumulation. These results suggest that TP-induced cardiotoxicity is p53-dependent by promoting Bax-induced mitochondria-mediated apoptosis.