The clinical and immunological characteristics of COVID-19 patients with delayed SARS-CoV-2 virus clearance.

BACKGROUND: The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a great threat to human health. Some severe COVID-19 patients still carried detectable levels of SARS-CoV-2 even after prolonged intensive care unit treatment. However, the immunological features of these COVID-19 patients with delayed virus clearance (CDVC) are still unclear. METHODS: We retrospectively reviewed the clinical and immunological data of 13 CDVC cases, who were admitted into one hospital in Wuhan from February to April 2020. These data were also compared to those of perished (n = 9) and recovered (n = 52) cases. The expression of the exhaustion marker PD-1 on circulating T cells of these patients was measured by flow cytometry. RESULTS: High levels of serum interleukin-6 (IL-6), IL-1ß, IL-8, as well as other inflammatory mediators, were seen in CDVC cases. Severe lymphopenia was observed in CDVC patients with the counts of total lymphocytes (0.9 × 109 /L), CD4+ T cells (0.35 × 109 /L), and CD8+ T cells (0.28 × 109 /L) below their corresponding lower limits of normal range. Similar to the perished group, CDVC cases have higher percentages of CD25+ Foxp3+ regulatory T cells (Treg) in circulation. Moreover, enhanced expression of the exhaustion marker PD-1 on CCR7- CD45RA+ effector, CCR7+ CD45RA- central memory, and CCR7- CD45RA- effector memory CD4+ and CD8+ T cells were also observed in CDVC cases. CONCLUSION: CDVC patients still have SARS-CoV-2 and these cases manifest with severe clinical symptoms due to persistent inflammation. Augmentation of the frequency of circulating Treg, severe lymphopenia, and functional exhaustion of T cells might lead to inefficient clearance of SARS-CoV-2. Therefore, enhancing lymphocyte counts and reversing T-cell exhaustion might be key methods to boost immune responses and eliminate SARS-CoV-2 in CDVC patients.

Anti-PD-1 therapy achieves favorable outcomes in HBV-positive non-liver cancer.

Anti-PD-1 therapy has shown promising outcomes in the treatment of different types of cancer. It is of fundamental interest to analyze the efficacy of anti-PD-1 therapy in cancer patients infected with hepatitis B virus (HBV) since the comorbidity of HBV and cancer is widely documented. We designed a multicenter retrospective study to evaluate the efficacy of anti-PD-1 therapy on non-liver cancer patients infected with HBV. We found anti-PD-1 therapy achieved much better outcomes in HBV+ non-liver cancer patients than their HBV- counterparts. We performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) from esophageal squamous cell carcinoma (ESCC) patients. We found both cytotoxicity score of T cells and MHC score of B cells significantly increased after anti-PD-1 therapy in HBV+ ESCC patients. We also identified CX3CR1high TEFF, a subset of CD8+ TEFF, associated with better clinical outcome in HBV+ ESCC patients. Lastly, we found CD8+ TEFF from HBV+ ESCC patients showing higher fraction of Exhaustionhi T than their HBV- counterpart. In summary, anti-PD-1 therapy on HBV+ non-liver cancer patients is safe and achieves better outcomes than that on HBV- non-liver cancer patients, potentially because HBV+ patients had higher fraction of Exhaustionhi T, which made them more efficiently respond to anti-PD-1 therapy.

A digital twin-driven human-robot collaborative assembly approach in the wake of COVID-19.

In the wake of COVID-19, the production demand of medical equipment is increasing rapidly. This type of products is mainly assembled by hand or fixed program with complex and flexible structure. However, the low efficiency and adaptability in current assembly mode are unable to meet the assembly requirements. So in this paper, a new framework of human-robot collaborative (HRC) assembly based on digital twin (DT) is proposed. The data management system of proposed framework integrates all kinds of data from digital twin spaces. In order to obtain the HRC strategy and action sequence in dynamic environment, the double deep deterministic policy gradient (D-DDPG) is applied as optimization model in DT. During assembly, the performance model is adopted to evaluate the quality of resilience assembly. The proposed framework is finally validated by an alternator assembly case, which proves that DT-based HRC assembly has a significant effect on improving assembly efficiency and safety.

Role of YAP1 gene in proliferation, osteogenic differentiation, and apoptosis of human periodontal ligament stem cells induced by TNF-α.

BACKGROUND: Periodontitis is a chronic inflammatory disease that occurs in periodontal tissues and can cause tooth loosening and loss in severe cases. As the main effector of downstream of Hippo signaling pathway, yes-related protein 1 (YAP1) plays an important role in cell proliferation and differentiation. However, the role of YAP1 in periodontitis has not been reported. METHODS: Cell activity was detected by Cell Counting Kit-8 (CCK-8). YAP1 was overexpressed by cell transfection, and then RT-qPCR and western blot were used to detect the expression of YAP1. The cell proliferation was determined by clone formation assay, and the expression of proliferation-related proteins was determined by western blot. The cell differentiation was detected by ELISA kit of alkaline phosphatase activity (ALP) and alizarin red staining. Finally, western blot was used to detect the expression of differentiation-related protein and Hippo signaling pathway-related proteins. Apoptosis was detected by flow cytometry. RESULTS: With the increase of concentration induced by TNF-α, the cell survival rate of human periodontal ligament stem cells (HPDLSCs) decreased significantly. After the overexpression of YAP1, cell proliferation and proliferation-related protein expression increased. Overexpression of YAP1 can improve the differentiation and the formation of osteoblasts of HPDLSCs induced by TNF-α. The expression of Hippo signaling pathway-related proteins transcriptional coactivators with PDZ binding domains (TAZ), TEA domain family member (TRED) increased and proliferation-related protein P27 decreased, whereas there was no significant change in the expression of MST1. CONCLUSION: TNF-α can inhibit proliferation and osteogenic differentiation of HPDLSCs, which can be ameliorated by the YAP1 gene through the Hippo signaling pathway. Our paper suggested that YAP1 may be a potential therapeutic target for periodontitis.

miR-338-3p regulates osteoclastogenesis via targeting IKKß gene.

This study determined the effects of miR-338-3p on osteoclast (OC) differentiation and activation. The change levels of miR-338-3p in differentiated OCs were investigated by microRNA microarray assay and quantitative real-time PCR analysis. The effects of miR-338-3p on the differentiation and activation of OCs were determined by tartrate-resistant acid phosphatase staining resorption activity assay and Western blot. Target genes of miR-338-3p were identified by target gene prediction and dual-luciferase reporter gene detection assay as well as Western blot. Results showed that miR-338-3p was markedly downregulated in differentiated OCs. miR-338-3p could inhibit the formation and absorption activity of OCs. Western blot showed that miR-338-3p could influence the change levels of OC differentiation-related proteins. Dual-luciferase reporter gene detection assay and Western blot both showed that miR-338-3p directly targeted IKKß gene. In conclusion, miR-338-3p may affect the formation and activity of OCs by targeting the IKKß gene.

3-(4-methoxyl)-1-(2-(4-coumarin)prop)-2-en-1-one inhibits the differentiation of Gaoyou duck embryonic osteoclasts in vitro.

This study determined the influence of 3-(4-methoxyl)-1-(2-(4-coumarin)prop)-2-en-1-one (MCPEO) on the differentiation of Gaoyou duck embryo osteoclasts cultured in vitro. Bone marrow mononuclear cells (BM-MNCs) were harvested from 23-day-old Gaoyou duck embryos and induced by receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in the presence of MCPEO at different concentrations (i.e., 1, 5, 10, 20, and 40 µM). Cell viability measurement, tartrate-resistant acid phosphatase (TRAP) staining, resorption activity assay, and co-staining with Tetramethylrhodamine (TRITC)-conjugated phalloidin and Hoechst 33,258 were conducted. Results indicated that MCPEO influenced the cell viability of the M-CSF + RANKL-induced BM-MNCs in a concentration-dependent manner, reduced the formation of positive multinucleated cells, and restrained the resorption capability of osteoclasts. Microfilament and nuclear staining indicated that MCPEO restricted the differentiation of BM-MNCs into large multinucleated osteoclasts. In short, MCPEO can inhibit the differentiation of BM-MNCs into mature osteoclasts in duck embryos. Therefore, MCPEO is a promising agent for the treatment of poultry osteoporosis.

Effects of Ca2+/calmodulin­dependent protein kinase pathway inhibitor KN93 on osteoclastogenesis.

The aim of the present study was to determine the effects of the Ca2+/calmodulin­dependent protein kinase pathway inhibitor KN93 on osteoclastogenesis. RAW264.7 cells were incubated with macrophage colony­stimulating factor (M­CSF) + receptor activator of nuclear factor kappa­light­chain­enhancer of activated B cells ligand (RANKL) to stimulate osteoclastogenesis and then treated with 10 µM KN93. The methods included tartrate­resistant acid phosphatase (TRAP) staining, bone resorption activity assays, filamentous (F)­actin staining, determination of intracellular calcium ([Ca2+]i) levels, monitoring of osteoclast­specific gene expression levels and measurement of key transcription factors protein levels. The results suggested that KN93 inhibited the formation of TRAP­positive multinucleated cells, shaping of F­actin rings and resorption activity of the cells. In addition, KN93 decreased the concentration of [Ca2+]i, expression levels of osteoclast specific genes and protein levels of critical transcription factors in the M­CSF + RANKL­induced osteoclast model. In summary, KN93 may directly affect the differentiation and activation of osteoclasts, potentially through the Ca2+/calmodulin­dependent protein kinase signaling pathway.

CyberPsychological Computation on Social Community of Ubiquitous Learning.

Under the modern network environment, ubiquitous learning has been a popular way for people to study knowledge, exchange ideas, and share skills in the cyberspace. Existing research findings indicate that the learners' initiative and community cohesion play vital roles in the social communities of ubiquitous learning, and therefore how to stimulate the learners' interest and participation willingness so as to improve their enjoyable experiences in the learning process should be the primary consideration on this issue. This paper aims to explore an effective method to monitor the learners' psychological reactions based on their behavioral features in cyberspace and therefore provide useful references for adjusting the strategies in the learning process. In doing so, this paper firstly analyzes the psychological assessment of the learners' situations as well as their typical behavioral patterns and then discusses the relationship between the learners' psychological reactions and their observable features in cyberspace. Finally, this paper puts forward a CyberPsychological computation method to estimate the learners' psychological states online. Considering the diversity of learners' habitual behaviors in the reactions to their psychological changes, a BP-GA neural network is proposed for the computation based on their personalized behavioral patterns.

Information Dissemination of Public Health Emergency on Social Networks and Intelligent Computation.

Due to the extensive social influence, public health emergency has attracted great attention in today's society. The booming social network is becoming a main information dissemination platform of those events and caused high concerns in emergency management, among which a good prediction of information dissemination in social networks is necessary for estimating the event's social impacts and making a proper strategy. However, information dissemination is largely affected by complex interactive activities and group behaviors in social network; the existing methods and models are limited to achieve a satisfactory prediction result due to the open changeable social connections and uncertain information processing behaviors. ACP (artificial societies, computational experiments, and parallel execution) provides an effective way to simulate the real situation. In order to obtain better information dissemination prediction in social networks, this paper proposes an intelligent computation method under the framework of TDF (Theory-Data-Feedback) based on ACP simulation system which was successfully applied to the analysis of A (H1N1) Flu emergency.

Dual effects of Ginkgo biloba leaf extract on human red blood cells.

Extracts from the leaves of Ginkgo biloba have been used in Chinese medicine for thousands of years. Today, various standardized preparations from G. biloba leaf extract have been developed. G. biloba leaf extract, which contains flavonoids and terpenoids as the major biologically active components, has become one of the most popular and commonly used herbal remedies due to its wide spectrum of beneficial effects on health. In this study, we investigated the effects of G. biloba leaf extract on the properties of human red blood cells in the presence and absence of amyloid peptide (Abeta25-35), peroxide and hypotonic stress. The results suggest that G. biloba leaf extract has a dual action, both protective and disruptive, on red blood cells, depending on whether an exogenous stress is present. G. biloba leaf extract has a protective role on red blood cells against Abeta- and hypotonic pressure-induced haemolysis, peroxide-induced lipoperoxidation, as well as glutathione consumption and methaemoglobin formation. On the other hand, G. biloba leaf extract also exhibited damage to red blood cells by increasing cell fragility, changing cellular morphology and inducing glutathione consumption and methaemoglobin formation, especially when applied at high doses. These anti- and pro-oxidative activities of polyphenolic substances are thought to be involved in the dual function of G. biloba leaf extract. The results of this study suggest that high doses of herbal remedies and dietary supplements can be toxic to cells.

Nicotinamide restores cognition in Alzheimer's disease transgenic mice via a mechanism involving sirtuin inhibition and selective reduction of Thr231-phosphotau.

Memory loss is the signature feature of Alzheimer's disease, and therapies that prevent or delay its onset are urgently needed. Effective preventive strategies likely offer the greatest and most widespread benefits. Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance memory and synaptic plasticity. We evaluated the efficacy of nicotinamide, a competitive inhibitor of the sirtuins or class III NAD(+)-dependent HDACs in 3xTg-AD mice, and found that it restored cognitive deficits associated with pathology. Nicotinamide selectively reduces a specific phospho-species of tau (Thr231) that is associated with microtubule depolymerization, in a manner similar to inhibition of SirT1. Nicotinamide also dramatically increased acetylated alpha-tubulin, a primary substrate of SirT2, and MAP2c, both of which are linked to increased microtubule stability. Reduced phosphoThr231-tau was related to a reduction of monoubiquitin-conjugated tau, suggesting that this posttranslationally modified form of tau may be rapidly degraded. Overexpression of a Thr231-phospho-mimic tau in vitro increased clearance and decreased accumulation of tau compared with wild-type tau. These preclinical findings suggest that oral nicotinamide may represent a safe treatment for AD and other tauopathies, and that phosphorylation of tau at Thr231 may regulate tau stability.

Cep164 is a mediator protein required for the maintenance of genomic stability through modulation of MDC1, RPA, and CHK1.

The activation of the ataxia telangiectasia mutated (ATM) and ATM/Rad3-related (ATR) kinases triggers a diverse cellular response including the initiation of DNA damage-induced cell cycle checkpoints. Mediator of DNA Damage Checkpoint protein, MDC1, and H2AX are chromatin remodeling factors required for the recruitment of DNA repair proteins to the DNA damage sites. We identified a novel mediator protein, Cep164 (KIAA1052), that interacts with both ATR and ATM. Cep164 is phosphorylated upon replication stress, ultraviolet radiation (UV), and ionizing radiation (IR). Ser186 of Cep164 is phosphorylated by ATR/ATM in vitro and in vivo. The phosphorylation of Ser186 is not affected by RPA knockdown but is severely hampered by MDC1 knockdown. siRNA-mediated silencing of Cep164 significantly reduces DNA damage-induced phosphorylation of RPA, H2AX, MDC1, CHK2, and CHK1, but not NBS1. Analyses of Cep164 knockdown cells demonstrate a critical role of Cep164 in G2/M checkpoint and nuclear divisions. These findings reveal that Cep164 is a key player in the DNA damage-activated signaling cascade.

Complementary functions of the Saccharomyces cerevisiae Rad2 family nucleases in Okazaki fragment maturation, mutation avoidance, and chromosome stability.

Rad2 family nucleases, identified by sequence similarity within their catalytic domains, function in multiple pathways of DNA metabolism. Three members of the Saccharomyces cerevisiae Rad2 family, Rad2, Rad27, and exonuclease 1 (Exo1), exhibit both 5' exonuclease and flap endonuclease activities. Deletion of RAD27 results in defective Okazaki fragment maturation, DNA repair, and subsequent defects in mutation avoidance and chromosomal stability. However, strains lacking Rad27 are viable. The expression profile of EXO1 during the cell cycle is similar to that of RAD27 and other genes encoding proteins that function in DNA replication and repair, suggesting Exo1 may function as a back up nuclease for Rad27 in DNA replication. We show that overexpression of EXO1 suppresses multiple rad27 null mutation-associated phenotypes derived from DNA replication defects, including temperature sensitivity, Okazaki fragment accumulation, the rate of minichromosome loss, and an elevated mutation frequency. While generally similar findings were observed with RAD2, overexpression of RAD2, but not EXO1, suppressed the MMS sensitivity of the rad27 null mutant cells. This suggests that Rad2 can uniquely complement Rad27 in base excision repair (BER). Furthermore, Rad2 and Exo1 complemented the mutator phenotypes and cell cycle defects of rad27 mutant strains to differing extents, suggesting distinct in vivo nucleic acid substrates.

Functional alterations of human exonuclease 1 mutants identified in atypical hereditary nonpolyposis colorectal cancer syndrome.

Hereditary Nonpolyposis Colorectal Cancer (HNPCC) is a genetically heterogeneous disorder caused by germ-line mutations in one of several DNA mismatch repair (MMR) genes, most commonly in hMSH2 and hMLH1. Human exonuclease 1 (hExo1) possesses both 5'exonuclease and flap endonuclease activities and plays a role in DNA repair, recombination, and replication. The enzyme interacts with MMR proteins, hMsh2, hMlh1, and hMsh3. Recently, eight missense mutations in hEXO1 were identified in atypical HNPCC patients, who have been screened to be negative for hMSH2, hMLH1, and hMSH6 mutations. To address the question of whether these mutations cause susceptibility to HNPCC, in vitro nuclease activity and protein-protein interaction assays were performed in this study. We found that two mutants, E109K and L410R, lost their exonuclease activities while retaining their capacity to bind to the DNA substrate. Three other mutants, P640S, G759E, and P770L, displayed a reduced capacity to interact with hMsh2. The combination of these three point mutations leads to the binding capacity with hMsh2 to nearly zero. Evidence made available in this study sheds light on the pathogenesis of HNPCC, perhaps initiated by an additional MMR gene, hEXO1.

Suppression of Saccharomyces cerevisiae rad27 null mutant phenotypes by the 5' nuclease domain of Escherichia coli DNA polymerase I.

RNA primer removal from Okazaki fragments during lagging-strand replication and the excision of damaged DNA bases requires the action of structure-specific nucleases, such as the mammalian flap endonuclease 1 (FEN-1). This nuclease contains two conserved motifs enriched with acidic amino acid residues that are important for catalytic function. Similar motifs have been identified in nucleases found in viruses, archebacteria, eubacteria, and in eukaryotes ranging from yeast to humans. Unique among these proteins, the putative FEN-1 homologue in Escherichia coli is contained within the N-terminal region of the DNA polymerase I (PolN). To demonstrate that the cellular functions of FEN-1 reside in PolN, we cloned and expressed the amino terminal domain (323 amino acid residues) of PolI in a Saccharomyces cerevisiae strain lacking the FEN-1 homologue RAD27. Overexpression of PolN suppressed, to varying degrees, phenotypes associated with a rad27 null strain. These include temperature sensitivity, Okazaki fragment processing, a mutator phenotype, a G2/M cell cycle arrest, minichromosome loss, and methyl methane sulfonate sensitivity. We purified Rad27 and PolN proteins in order to determine whether differences in their intrinsic nuclease activities or interaction with proliferating cell nuclear antigen (PCNA) could explain the partial suppression of some phenotypes. We found that the in vitro nuclease activities of Rad27 were more potent than those of PolN and the activity of Rad27, but not PolN, was stimulated by PCNA. We conclude that the N-terminal nuclease domain of E. coli polymerase I encodes a functional homologue of FEN-1.