Matrine inhibits inflammatory response induced by TNF-α in human umbilical vein endothelial cells through miR-25-3p-mediated Klf4 pathway / 中国中药杂志

This study aimed to analyze the effect of matrine on tumor necrosis factor-α(TNF-α)-induced inflammatory response in human umbilical vein endothelial cells(HUVECs) and explore whether the underlying mechanism was related to the miR-25-3p-mediated Krüppel-like factor 4(Klf4) pathway. The HUVEC cell inflammation model was induced by TNF-α stimulation. After 24 or 48 hours of incubation with different concentrations of matrine(0.625, 1.25, and 2.5 mmol·L~(-1)), CCK-8 assay was used to detect cell proliferation. After treatment with 2.5 mmol·L~(-1) matrine for 48 h, the expression of TNF-α, interleukin-6(IL-6), interleukin-1β(IL-1β), and Klf4 mRNA and miR-25-3p was detected by real-time fluorescence-based quantitative PCR, and the protein expression of TNF-α, IL-6, IL-1β, and Klf4 was detected by Western blot. The anti-miR-25-3p was transfected into HUVECs, and the effect of anti-miR-25-3p on TNF-α-induced cell proliferation and inflammatory factors was detected by the above method. The cells were further transfected with miR-25-3p and incubated with matrine to detect the changes in proliferation and expression of related inflammatory factors, miR-25-3p, and Klf4. The targeting relationship between miR-25-3p and Klf4 was verified by bioinformatics analysis and dual luciferase reporter gene assay. The results displayed that matrine could inhibit TNF-α-induced HUVEC proliferation, decrease the mRNA and protein expression of TNF-α, IL-6, and IL-1β, increase the mRNA and protein expression of Klf4, and reduce the expression of miR-25-3p. Bioinformatics analysis showed that there were specific complementary binding sites between miR-25-3p and Klf4 sequences. Dual luciferase reporter gene assay confirmed that miR-25-3p negatively regulated Klf4 expression in HUVECs by targeting. The inhibition of miR-25-3p expression can reduce TNF-α-induced cell proliferation and mRNA and protein expression of TNF-α, IL-6, and IL-1β. MiR-25-3p overexpression could reverse the effect of matrine on TNF-α-induced cell proliferation and the mRNA and protein expression of TNF-α, IL-6, IL-1β, and Klf4. This study shows that matrine inhibits the inflammatory response induced by TNF-α in HUVECs through miR-25-3p-mediated Klf4 pathway.

Research progress and clinical application of liposomal drug delivery system / 中国药房

With the advantage of high efficiency， low toxicity and targeting， liposomes have become the research hotspot of new drug preparations at home and abroad. In this paper， the research progress in recent years is reviewed from the aspects of preparation methods， classification and clinical application of liposomes. The results showed that in order to obtain more stable and controllable liposomes， scholars improved and optimized the traditional preparation methods and established novel preparation methods such as supercritical fluid methods， freeze-drying method and double-asymmetric centrifugation method. In order to enhance the efficacy and reduce toxicity， the conventional liposomes were optimized to get novel ones such as environmentally sensitive liposomes， long-circulating liposomes and multifunctional liposomes， which had greatly promoted the clinical application of liposomes. For now， liposomes have been used in many fields， such as anti-cancer agents， antimicrobial and vaccines， but most of the new liposomes are still in the early stage of development.

Construction of iPSC-derived Inhibitory Neural Network Tissue with Synaptic Transmission Potentials / 中山大学学报(医学科学版)

ObjectiveDirected differentiation of human induced pluripotent stem cells （hiPSCs） into spinal cord γ-aminobutyric acid （GABA）-ergic progenitor cells were implanted into an decellularized optical nerve （DON） bioscaffold to construct a hiPSC-derived inhibitory neural network tissue with synaptic activities. This study aimed to provide a novel stem cell-based tissue engineering product for the study and the repair of central nervous system injury. MethodsThe combination of stepwise directional induction and tissue engineering technology was applied in this study. After hiPSCs were directionally induced into human neural progenitor cells （hNPCs） in vitro， they were seeded into a DON for three-dimensional culture， allowing further differentiation into inhibitory GABAergic neurons under the specific neuronal induction environment. Transmission electron microscopy and whole cell patch clamp technique were used to detect whether the hiPSCs differentiated neurons could form synapse-like structures and whether these neurons had spontaneous inhibitory postsynaptic currents， respectively， in order to validate that the hiPSC-derived neurons would form neural networks with synaptic transmission potentials from a structural and functional perspective. ResultsThe inhibitory neurons of GABAergic phenotype were successfully induced from hiPSCs in vitro， and maintained good viability after 28 days of culture. With the transmission electron microscopy， it was observed that many cell junctions were formed between hiPSC-derived neural cells in the three-dimensional materials， some of which presented a synapse- like structure， manifested as the slight thickness of cell membrane and a small number of vesicles within one side of the cell junctions， the typical structure of a presynatic component， and focal thickness of the membrane of the other side of the cell junctions， a typical structure of a postsynaptic component. According to whole-cell patch-clamp recording， the hiPSC-derived neurons had the capability to generate action potentials and spontaneous inhibitory postsynaptic currents were recorded in this biotissue. ConclusionsThe results of this study indicated that hiPSCs can be induced to differentiate into GABAergic progenitor cells in vitro and can successfully construct iPSC-derived inhibitory neural network tissue with synaptic transmission after implanted into a DON for three-dimensional culture. This study would provide a novel neural network tissue for future research and treatment of central nervous system injury by stem cell tissue engineering technology.

Corrigendum: Identification of novel therapeutic candidates against SARS-CoV-2 infections: An application of RNA sequencing toward mRNA based nanotherapeutics.

[This corrects the article DOI: 10.3389/fmicb.2022.901848.].

Identification of Novel Therapeutic Candidates Against SARS-CoV-2 Infections: An Application of RNA Sequencing Toward mRNA Based Nanotherapeutics.

Due to fast transmission and various circulating SARS-CoV-2 variants, a significant increase of coronavirus 2019 infection cases with acute respiratory symptoms has prompted worries about the efficiency of current vaccines. The possible evasion from vaccine immunity urged scientists to identify novel therapeutic targets for developing improved vaccines to manage worldwide COVID-19 infections. Our study sequenced pooled peripheral blood mononuclear cells transcriptomes of SARS-CoV-2 patients with moderate and critical clinical outcomes to identify novel potential host receptors and biomarkers that can assist in developing new translational nanomedicines and vaccine therapies. The dysregulated signatures were associated with humoral immune responses in moderate and critical patients, including B-cell activation, cell cycle perturbations, plasmablast antibody processing, adaptive immune responses, cytokinesis, and interleukin signaling pathway. The comparative and longitudinal analysis of moderate and critically infected groups elucidated diversity in regulatory pathways and biological processes. Several immunoglobin genes (IGLV9-49, IGHV7-4, IGHV3-64, IGHV1-24, IGKV1D-12, and IGKV2-29), ribosomal proteins (RPL29, RPL4P2, RPL5, and RPL14), inflammatory response related cytokines including Tumor Necrosis Factor (TNF, TNFRSF17, and TNFRSF13B), C-C motif chemokine ligands (CCL3, CCL25, CCL4L2, CCL22, and CCL4), C-X-C motif chemokine ligands (CXCL2, CXCL10, and CXCL11) and genes related to cell cycle process and DNA proliferation (MYBL2, CDC20, KIFC1, and UHCL1) were significantly upregulated among SARS-CoV-2 infected patients. 60S Ribosomal protein L29 (RPL29) was a highly expressed gene among all COVID-19 infected groups. Our study suggested that identifying differentially expressed genes (DEGs) based on disease severity and onset can be a powerful approach for identifying potential therapeutic targets to develop effective drug delivery systems against SARS-CoV-2 infections. As a result, potential therapeutic targets, such as the RPL29 protein, can be tested in vivo and in vitro to develop future mRNA-based translational nanomedicines and therapies to combat SARS-CoV-2 infections.

Hetero-bivalent Nanobodies Provide Broad-spectrum Protection against SARS-CoV-2 Variants of Concern including Omicron

Following Delta, Omicron variant triggered a new wave of SARS-CoV-2 infection globally, adaptive evolution of the virus may not stop, the development of broad-spectrum antivirals is still urgent. We previously developed two hetero-bivalent nanobodies with potent neutralization against original WT SARS-CoV-2, termed aRBD-2-5 and aRBD-2-7, by fusing aRBD-2 with aRBD-5 or aRBD-7, respectively. Here, we resolved crystal structures of these nanobodies in complex with RBD, and found the epitope of aRBD-2 differs from that of aRBD-5, aRBD-7. aRBD-2 binds to a conserved epitope which renders its binding activity to all variants of concern (VOCs) including Omicron. Interestingly, although monovalent aRBD-5 and aRBD-7 lost binding to some variants, they effectively improved the overall affinity when transformed into the hetero-bivalent form after being fused with aRBD-2. Consistent with the high binding affinities, aRBD-2-5-Fc and aRBD-2-7-Fc exhibited ultra-potent neutralization to all five VOCs; particularly, aRBD-2-5-Fc neutralized authentic virus of Beta, Delta and Omicron with the IC50of 5.98[~]9.65 ng/mL or 54.3[~]87.6 pM. Importantly, aRBD-2-5-Fc provided in vivo prophylactic protection for mice against WT and mouse-adapted SARS-CoV-2, and provided full protection against Omicron in hamster model when administrated either prophylactically or therapeutically. Taken together, we found a conserved epitope on RBD, and hetero-bivalent nanobodies had increased affinity for VOCs over its monovalent form, and provided potent and broad-spectrum protection both in vitro and in vivo against all tested major variants, and potentially future emerging variants. Our strategy provides a new solution in the development of therapeutic antibodies for COVID-19 caused by newly emergent VOCs.

IgNAR antibody: Structural features, diversity and applications.

In response to the invasion of exogenous microorganisms, one of the defence strategies of the immune system is to produce antibodies. Cartilaginous fish is among those who evolved the earliest humoral immune system that utilizes immunoglobulin-type antibodies. The cartilaginous fish antibodies fall into three categories: IgW, IgM, and IgNAR. The shark Immunoglobulin Novel Antigen Receptor (IgNAR) constitutes disulfide-bonded dimers of two protein chains, similar to the heavy chain of mammalian IgGs. Shark IgNAR is the primary antibody of a shark's adaptive immune system with a serum concentration of 0.1-1.0 mg/mL. Its structure comprises of one variable (V) domain (VNAR) and five constant (C1 -C5) domains in the secretory form. VNARs are classified into several subclasses based on specific properties such as the quantity and position of additional non-canonical cysteine (Cys) residues in the VNAR. The VDJ recombination in IgNAR comprises various fragments; one variable component, three diverse sections, one joining portion, and a solitary arrangement of constant fragments framed in each IgNAR gene cluster. The re-arrangement happens just inside this gene cluster bringing about a VD1D2D3J segment. Therefore, four re-arrangement procedures create the entire VNAR space. IgNAR antibody can serve as an excellent diagnostic, therapeutic, and research tool because it has a smaller size, high specificity for antigen-binding, and perfect stability. The domain characterization, structural features, types, diversity and therapeutic applications of IgNAR molecules are highlighted in this review. It would be helpful for further research on IgNAR antibodies acting as an essential constituent of the adaptive immune system and a potential therapeutic agent.

Clinical efficacy and optimal dose of apatinib combined with chemotherapy in patients with advanced non-small cell lung cancer / 国际肿瘤学杂志

Objective:To explore the clinical efficacy of different doses of apatinib combined with chemotherapy in patients with advanced non-small cell lung cancer (NSCLC) and the adverse reactions.Methods:A total of 69 patients with NSCLC diagnosed in the No. 901 Hospital of the Chinese People′s Liberation Army Joint Logistics Support Force were selected from January 2018 to June 2020, and were divided into chemotherapy alone group (docetaxel+ cisplatin was used), apatinib group A [apatinib (0.25 g)+ docetaxel+ cisplatin was used] and apatinib group B [apatinib (0.50 g)+ docetaxel+ cisplatin was used] according to random number table method, with 23 cases in each group. The objective response rate (ORR), disease control rate (DCR), median overall survival (OS), median progression-free survival (PFS), and incidences of adverse reactions were compared between the three groups of patients.Results:One patients in the apatinib group B withdrew from the study due to acute myocardial infarction. After 4 cycless of treatment, the ORR of the patients in the chemotherapy alone group, apatinib group A and apatinib group B were 17.39% (4/23), 47.83% (11/23) and 54.55% (12/22) respectively, with a statistically significant difference ( χ2=7.41, P=0.024). The ORR of the apatinib group B was higher than that of the chemotherapy alone group, with a statistically significant difference ( χ2=6.77, P=0.009). There were no statistically significant differences in ORR between the apatinib group A and chemotherapy alone group, the apatinib group A and apatinib group B ( χ2=4.85, P=0.028; χ2=0.20, P=0.652). The DCR of the patients in the three groups were 47.83% (11/23), 78.26% (18/23) and 86.36% (19/22) respectively, with a statistically significant difference ( χ2=9.03, P=0.011). The DCR of the apatinib group B was higher than that of the chemotherapy alone group, with a statistically significant difference ( χ2=7.52, P=0.006). There were no statistically significant differences in DCR between the apatinib group A and the chemotherapy alone group, the apatinib group A and apatinib group B ( χ2=4.57, P=0.033; χ2=0.51, P=0.477). The median OS of the patients in the three groups were 6.8, 9.2 and 9.9 months respectively, with a statistically significant different ( χ2=8.91, P=0.022). Compared with the chemotherapy alone group, the median OS of the apatinib group A and apatinib group B were significantly prolonged, with statistically significant differences ( χ2=7.25, P=0.036; χ2=8.60, P=0.029). Compared with the apatinib group A, the median OS of the apatinib group B was prolonged, but there was no statistically significant different ( χ2=1.54, P=0.201). The median PFS of the patients in the three groups were 5.2, 7.7 and 8.2 months respectively, with a statistically significant different ( χ2=8.79, P=0.026). Compared with the chemotherapy alone group, the median PFS of the apatinib group A and apatinib group B were significantly prolonged, with statistically significant differences ( χ2=7.01, P=0.039; χ2=8.36, P=0.031). Compared with the apatinib A group, the median PFS of the apatinib group B was prolonged, but there was no statistically significant different ( χ2=1.68, P=0.186). There were statistically significant differences in the incidences of fatigue [34.78% (8/23) vs. 65.22% (15/23) vs. 72.73% (16/22), χ2=7.50, P=0.024], hypertension [4.35% (1/23) vs. 34.78% (8/23) vs. 68.18% (15/22), χ2=20.07, P<0.001], hand-foot syndrome [4.35% (1/23) vs. 43.48% (10/23) vs. 72.73% (16/22), χ2=22.28, P<0.001] and oral mucositis [8.70% (2/23) vs. 39.13% (9/23) vs. 72.73% (16/22), χ2=19.26, P<0.001] among the three groups. Compared with the chemotherapy alone group, the incidences of hypertension and hand-foot syndrome in the apatinib group A and the incidences of fatigue, hypertension, hand-foot syndrome and oral mucositis in the apatinib group B were increased, with statistically significant differences ( χ2=6.77, P=0.009; χ2=9.68, P=0.002; χ2=6.51, P=0.011; χ2=20.00, P<0.001; χ2=22.37, P<0.001; χ2=19.21, P<0.001). Conclusion:Apatinib (0.50 g) combined with chemotherapy has better short-term efficacy than chemotherapy alone in advanced NSCLC. Apatinib (0.25 g) and apatinib (0.50 g) can prolong the survival of patients, but increasing the treatment dose can not achieve longer survival benefit.

Down-regulation of lncRNA TTTY15 targeting miR-4500 to inhibit the biological characteristics of A172 glioma cells / 中华医学遗传学杂志

OBJECTIVE@#To explore the effect and mechanism of down-regulating lncRNA TTTY15 targeting miR-4500 on the proliferation, apoptosis, migration and invasion of A172 glioma cells.@*METHODS@#The difference in TTTY15 expression between the glioma cells and tissue was determined with a qRT-PCR method. Complementary binding sites of TTTY15 and miR-4500 were predicted with Starbase software, and the targeting relationship was validated with a luciferase reporter system. A172 glioma cells were divided into Control, si-NC (transfected with control siRNA), si-TTTY15 (transfected with TTTY15 siRNA), si-TTTY15+Anti-miR-NC (co-transfected with TTTY15 siRNA and inhibitor control) and si-TTTY15+Anti-miR-4500 (co-transfected with TTTY15 siRNA and miR-4500 inhibitor) groups. Proliferation, apoptosis, migration and invasion, and the expression of Bax, Bcl-2, MMP-2 and MMP-9 proteins of the A172 glioma cells were respectively detected with CCK-8, flow cytometry, Transwell chamber and Western blotting assays.@*RESULTS@#The expression of TTTY15 in glioma cells and glioma tissues have both increased. The expression levels of TTTY15 and miR-4500 in glioma tissues were inversely correlated. TTTY15 and miR-4500 are mutually targeted. Compared with those of the Control and si-NC groups, the glioma cells in the si-TTTY15 group showed increased level of miR-4500, decreased survival rate, increased apoptosis rate, enhanced cell migration and invasion, increased expression of Bax protein, and decreased expression of Bcl-2, MMP-2 and MMP-9 proteins (P<0.05). Compared with those of the si-TTTY15+Anti-miR-NC group, the A172 glioma cells in the si-TTTY15+Anti-miR-4500 group showed decreased level of miR-4500, increased cell survival rate, decreased apoptosis rate, enhanced cell migration and invasion, decreased expression of Bax protein, and increased expression of Bcl-2, MMP-2, and MMP-9 proteins (P<0.05).@*CONCLUSION@#Down-regulating TTTY15 targeting miR-4500 can inhibit the proliferation, migration, invasion and induce apoptosis of the A172 glioma cells.

Research progress of pyruvate kinase type M2 in hepatocellular carcinoma / 中华肝脏病杂志

Primary hepatocellular carcinoma is one of the most common high-grade malignant tumors in the world. Its incidence ranks fifth among malignant tumors in China, and various therapeutic measures have poor curative effect. Pyruvate kinase type M2 is a key enzyme in the glycolytic pathway, and its abnormal expression in liver cancer is closely related to the proliferation, metastasis, diagnosis, treatment, prognosis, as well as drug and radiation resistance. Therefore, multi-pathway targeted regulation of pyruvate kinase type M2 use is expected to become a new direction for the treatment of primary liver cancer.

Anti-tumor Mechanism of Active Ingredients of Glycyrrhizae Radix et Rhizoma and Its Combined Use in Anti-tumor Treatment： A Review / 中国实验方剂学杂志

As a traditional Chinese medicinal material， Glycyrrhizae Radix et Rhizoma has been extensively used in various formulae. According to modern pharmacological research， it has anti-tumor， anti-inflammatory， anti-viral， liver-protecting， anti-heart failure， immunoregulatory， and anti-fibrosis effects. Caused by the interaction of various factors， cancer features complex pathogenesis. It is a global challenge and one of the main causes of death in China. Statistics show that the morbidity and mortality of malignant tumors have been on the rise， particularly for the young， which threaten the health of human beings. At the moment， radiotherapy and chemotherapy are the main countermeasures. Most clinical anti-tumor drugs demonstrate non-selective toxicity. To be specific， they damage normal cells while killing tumor cells， thus injuring vital organs. In addition， long-term medication will reduce the sensitivity of tumor cells. However， traditional Chinese medicine， which is characterized by treatment based on syndrome differentiation， multiple components， and multiple targets， is superior in the treatment of tumor. Studies have shown that the combination of anti-tumor drugs with Chinese medicine can not only enhance the anti-tumor effect but also alleviate toxicity. Therefore， it has been a research hotspot to develop anti-tumor drugs based on traditional Chinese medicine. In recent years， major headway has been made in the research on active ingreddients of Glycyrrhizae Radix et Rhizoma in anti-liver cancer， anti-breast cancer， anti-lung cancer， and anti-colon cancer and the combination with other drugs for anti-tumor. On this basis， we summarized the mechanisms of active ingredients of Glycyrrhizae Radix et Rhizoma in inducing apoptosis， interfering with cell cycle， inducing autophagy， inhibiting glycolysis， regulating immunity， and modulating miRNA and signaling pathways， as well as the combination with other drugs in anti-tumor efficiency， toxicity reduction， and sensitivity enhancement， hoping to lay a theoretical basis for the further development and clinical application of active ingredients of Glycyrrhizae Radix et Rhizoma.

Study on improving the quality of low-dose PET images of children based on generative adversarial networks / 中华核医学与分子影像杂志

Objective:To investigate the value of generative adversarial networks-based PET image reconstruction in improving the quality of low-dose 18F-FDG PET images and lesion detection in pediatric patients. Methods:Retrospective analysis of 61 PET images of children (38 males, 23 females, age (4.0±3.5) years) who underwent 18F-FDG total-body PET/CT imaging in Beijing Friendship Hospital, Capital Medical University from August 2021 to December 2021 was performed. The low-dose images (30 s, 20 s, 10 s) of all children extracted by list mode were input into the generative adversarial networks for deep learning (DL) reconstruction to obtain the corresponding simulated standard full-dose images (DL-30 s, DL-20 s, DL-10 s). The semi-quantitative parameters of the liver blood pool and primary lesion of standard full-dose 120 s, 30 s, 20 s, 10 s, DL-30 s, DL-20 s, and DL-10 s images were measured. The target-to-background ratio (TBR), contrast-to-noise ratio (CNR), and CV were calculated. The 5-point Likert scale was used for subjective scoring of image quality, and the detective abilities for positive lesions of each groups were compared. The sensitivities and positive predictive values of positive lesions detection were calculated. Mann-Whitney U test and Kruskal-Wallis rank sum test and χ2 test were used for data analyses. Results:CNR of the 30 s, 20 s, and 10 s groups were lower than those of DL-30 s, DL-20 s, and DL-10 s groups, respectively ( z values: -3.58, -3.20, -3.65, all P<0.05). Score of DL-10 s group was significantly lower than those of 120 s, DL-30 s and DL-20 s groups (4(3, 4), 5(4, 5), 4(4, 5), 4(4, 5); H=97.70, P<0.001). There were no significant differences in TBR, CNR, CV, SUV max and SUV mean of lesions and liver blood pool in 120 s, DL-30 s, DL-20 s, and DL-10 s groups ( H values: 0.00-6.76, all P>0.05). The sensitivities of positive lesion detection in DL-30 s, DL-20 s, and DL-10 s groups were 97.83%(225/230), 96.96%(223/230), 95.65%(220/230), respectively, and the positive predictive values were 96.57%(225/233), 93.70%(223/238), 84.94%(220/259), respectively. The positive predictive value in DL-10 s group was lower than those in DL-30 s and DL-20 s groups ( χ2=23.51, P<0.001). There were more false-positive and false-negative lesions detected by DL-10 s group than those of DL-30 s and DL-20 s groups in different sites. Conclusion:Based on the generative adversarial networks, the image quality of DL-20 s group is high and can meet the clinical diagnostic requirements.

Mechanism of rebalanced hemostasis in liver cirrhosis and the timing of blood transfusion and anticoagulation / 临床肝胆病杂志

Reconstructed rebalanced hemostasis exists in patients with liver cirrhosis, while such balance is unstable and can be easily broken by stress factors, which may lead to bleeding or thrombosis. There is a lack of effective strategies to prevent and solve the disrupted balance in clinic due to the complex pathogenesis of rebalanced hemostasis, limited testing methods, and insufficient awareness among clinicians. With reference to the articles in recent years, this article summarizes the mechanism of rebalanced hemostasis in liver cirrhosis and the causes of bleeding and thrombosis and discuss the association between blood transfusion and rebalanced hemostasis and the selection of anticoagulant drugs during thrombosis, in order to provide a theoretical basis and new ideas for solving related issues in clinical practice.

Mibefradil reduces hepatic glucose output in HepG2 cells via Ca2+/calmodulin-dependent protein kinase II-dependent Akt/forkhead box O1signaling.

The effects and underlying mechanisms of mibefradil on gluconeogenesis and glycogenesis were investigated using insulin-resistant HepG2 human hepatocellular carcinoma cells and a mouse model of type 2 diabetes mellitus (T2DM). HepG2 cells were divided into one of four groups: control, palmitate (PA)-induced insulin-resistance (0.25 mM), low-concentration mibefradil (0.025 µM), or high-concentration mibefradil (0.05 µM). Glycogen synthesis and glucose consumption were evaluated in these HepG2 cells, and quantitative polymerase chain reaction (qPCR) and western blotting techniques were used to detect expression of forkhead box O1 (FoxO1), phosphoenolpyruvate carboxykinase (PEPCK), and glucose 6-phosphatase (G6Pase). Intracellular calcium concentrations were determined using Fluo-4 AM, and phosphorylation levels of calmodulin-dependent protein kinase II (CaMKII), protein kinase B (Akt) and FoxO1were detected by western blotting. Immunofluorescence was used for the localization and quantification of FoxO1.In vitro results were verified using a mouse model of T2DM. In HepG2 cells and mouse liver tissues, mibefradil decreased PA-induced cytoplasmic calcium levels and CaMKII phosphorylation, but increased the phosphorylation of Akt and FoxO1, thereby contributing to the cytoplasmic localization of FoxO1. Additionally, mibefradil alleviated PA-induced glucose output and insulin resistance through increased glucose consumption and glycogen synthesis, while decreasing the expression of key gluconeogenesis enzymes, including PEPCK and G6Pase. Mibefradil may help to control blood sugar levels by reducing glucose output and insulin resistance, and the mechanism of action may involve the Ca2+-CaMKII-dependent Akt/FoxO1 signaling pathway.

Single-dose AAV-based vaccine induces a high level of neutralizing antibodies and provides long-term protection against SARS-CoV-2 in rhesus macaques

Coronavirus disease 2019 (COVID-19), which is triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, continues to threaten global public health. Developing a vaccine that only requires single immunization but provides long-term protection for the prevention and control of COVID-19 is important. Here, we developed an adeno-associated virus (AAV)-based vaccine expressing a stable receptor-binding domain (SRBD) protein. The vaccine requires only a single shot but provides effective neutralizing antibodies (NAbs) over 598 days in rhesus macaques (Macaca mulatta). Importantly, our results showed that the NAbs were kept in high level and long lasting against authentic wild-type SARS-CoV-2, Beta, Delta and Omicron variants using plaque reduction neutralization test. Of note, although we detected pre-existing AAV2/9 antibodies before immunization, the vaccine still induced high and effective NAbs against COVID-19 in rhesus macaques. AAV-SRBD immune serum also efficiently inhibited the binding of ACE2 with RBD in the SARS-CoV-2 B.1.1.7 (Alpha), B.1.351 (Beta), P.1/P.2 (Gamma), B.1.617.2 (Delta), B.1.617.1/3(Kappa), and C.37 (Lambda) variants. Thus, these data suggest that the vaccine has great potential to prevent the spread of SARS-CoV-2.

Potent in vitro Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain

Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL ([~]0.043 nM) and 3.18 ng/mL ([~]0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnosis reagents for COVID-19. ImportanceTo date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics including SARS-CoV-2 targeting antibodies remains critical. Due to their small size (13-15 kDa), highly solubility and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multi-valent formats, compared to the conventional antibody. Here, we report a serial of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.

Mental health and influencing factors among residents under standardized residency training during the epidemic of COVID-19 / 四川精神卫生

ObjectiveTo analyze the mental health status and the influencing factors among the residents taking standardized residency training during the outbreak and stable period of COVID-19， so as to provide theoretical basis for their mental health education. MethodsOn February 8 to February 10 and April 11 to April 13， 2020， Symptom Checklist 90 （SCL-90）， Beck Srivastava Stress Inventory （BSSI） and Simplified Coping Style Questionnaire （SCSQ） were distributed in online chat groups involving all grades of residents taking standardized residency training in the Second Affiliated Hospital of Kunming Medical University. Through two rounds of questionnaire survey， non-probability sampling method was used to obtain survey samples， and their mental status were analyzed. ResultsA total of 159 valid questionnaires were collected in the first round， and 99 valid questionnaires in the second round. The first survey showed that the total score of SCL-90 was （117.69±37.74） and the detection rate of positive symptoms in SCL-90 was 25.8%， and mainly dominated by obsession， fear and interpersonal sensitivity. In the second survey， the results showed that the total score of SCL-90 was （127.19±51.44）， and the main positive symptoms included obsession， depression and interpersonal sensitivity， with a positive detection rate of 30.3 %. The first survey found that the mental health status had significant differences among residents of different grades （χ2=7.46， P<0.05）， furthermore， the results indicated that lower grade was a risk factor while non-singleton was the protective factor of mental health status （P<0.05）， and SCL-90 total score was positively correlated with score of negative coping styles （r=0.45， P<0.01）. The second survey also classified lower grade as risk factor and non-singleton as protective factor （P<0.05）， and SCL-90 total score was positively correlated with study stress， economic pressure， interpersonal relationship， clinical practice and negative coping styles （r=0.52， 0.46， 0.55， 0.54， P<0.05 or 0.01）. ConclusionResidents under standardized residency training have obvious mental health problems during the outbreak of COVID-19， and the problems become more serious during the stable period of COVID-19.

Effects of different doses of dexmedetomidine on levels of serum claudin-1 and diamine oxidase in patients undergoing laparoscopic radical resection of gynecological malignant tumors / 肿瘤研究与临床

Objective:To explore the effect of different doses of dexmedetomidine (Dex) on levels of tight-junction protein claudin-1 and diamine oxidase (DAO) in patients undergoing laparoscopic radical resection of gynecological malignant tumors.Methods:A total of 60 patients with gynecological malignant tumors who were scheduled to undergo laparoscopic radical resection under general anesthesia from January 2019 to January 2020 in the Second Hospital of Shanxi Medical University were selected, including 43 cases of cervical cancer (stageⅠ-Ⅱ A), 9 cases of ovarian cancer (stageⅠ A-Ⅲ C), and 8 cases of endometrial carcinoma (stageⅠ). Accroding to the random number table method, the patients were divided into control group (group C), low-dose Dex group (group D 1) and high-dose Dex group (group D 2), with 20 cases in each group. Patients in group D 1 were given Dex 0.5 μg·kg -1·h -1 by constant rate intravenous infusion pump after induction until 30 min before the end of operation. Patients in group D 2 were given Dex 1.0 μg·kg -1·h -1 by constant rate intravenous infusion pump after induction until 30 min before the end of operation. Group C adopted the same calculation method and received the same amount of 0.9% sodium chloride solution by infusion pump. At 10 min before induction (T 1), 1 hour after pneumoperitoneum (T 2) and 12 hours after pneumoperitoneum (T 3), 5 ml of brachial vein blood was collected from the patients, and the levels of claudin-1 protein, DAO and blood glucose were measured. Results:At T 1, T 2 and T 3, the expression levels of claudin-1 in group C were (77.05±17.61) pg/ml, (66.76±12.97) pg/ml and (55.93±12.71) pg/ml, and the difference was statistically significant ( F = 10.449, P<0.05); the expression levels of DAO in group C were (4.83±0.93) ng/ml, (5.62±1.01) ng/ml and (5.98±1.21) ng/ml, and the difference was statistically significant ( F = 6.139, P < 0.05); the levels of blood glucose in group C were (4.82±0.66) mmol/L, (7.55±0.94) mmol/L and (6.51±0.54) mmol/L, and the difference was statistically significant ( F = 70.197, P < 0.05). At T 2, the expression level of claudin-1 in group D 1 was (69.12±13.02) pg/ml, which was not significantly different from group C ( t = -0.575, P > 0.05); the expression level of claudin-1 in group D 2 was (76.36±14.89) pg/ml, which was higher than that in group C, and the difference was statistically significant ( t = -2.175, P < 0.05). At T 3, the expression levels of claudin-1 in group D 1 and group D 2 were (66.14±14.36) pg/ml and (73.37±16.93) pg/ml, which were higher than that in group C, and the differences were statistically significant ( t values were -2.380 and -3.682, both P < 0.05). The expression levels of DAO in group D 1 and group D 2 were (5.02±0.84) ng/ml and (4.91±0.93) ng/ml at T 2, and (5.29±0.86) ng/ml and (5.20±0.98) ng/ml at T 3, which were lower than those in group C, and the differences were statistically significant ( t values were 2.051, 2.295, 2.079 and 2.285, all P < 0.05). The levels of blood glucose in group D 1 and group D 2 were (7.10±0.66) mmol/L and (6.77±0.97) mmol/L at T 2, and (5.95±0.94) mmol/L and (5.93±0.74) mmol/L at T 3, which were lower than those in group C, and the differences were statistically significant ( t values were 2.565, 5.374, 2.293 and 2.765, all P < 0.05). Conclusion:Continuous infusion of Dex can inhibit the stress response caused by long-term CO 2 pneumoperitoneum in laparoscopic radical resection of gynecological malignant tumors, and adjust the changes of expression levels of claudin-1 protein and DAO, reduce the damage of intestinal mucosal cells, facilitate the recovery of intestinal function, and the effect of high-dose Dex is better than low-dose Dex.

The Biological Role of miR-340 in Colorectal Cancer Cells and the Regulatory Mechanism of Target Genes / 中国生物化学与分子生物学报

miR-340 can promote the proliferation and invasion of cancer cells, but how miR-340 regulates the occurrence and development of cancer in colon cancer is rarely reported. This study aims to explore the biological function and target gene regulation mechanism of miR-340 in colorectal cancer cells. Firstly, RT-qPCR was used to detect the expression level of miR-340 in different colorectal cancer cell lines, and then miR-340 was overexpressed or inhibited in COLO-205 cells. CCK-8, Transwell migration and invasion assay, and flow cytometry were performed to analyze the cell ability of proliferation, migration and invasion, as well as cell apoptosis and cell cycle. Finally, after bioinformatics prediction of miR-340 target genes, luciferase reporter gene and Western blot experiment were applied to verify those target genes. The results showed that miR-340 was downregulated in COLO-205 cells. Compared with the control group, cell proliferation, migration and invasion were significantly inhibited in the miR-340 overexpression group, but were promoted in the miR-340 suppression group (P<0. 01). The results of flow cytometry showed that the percentage of apoptosis in the miR-340 overexpression group was significantly increased, while the percentage of apoptosis in the miR-340 inhibition group was decreased (P<0. 01). The bioinformatics analysis of the overexpression miR-340 transfection group showed that the 3′UTR of glucose regulated protein 78 kD (GRP78) had a miR-340-5p binding site, and the luciferase activity was significantly reduced in the overexpression miR-340 group (P<0. 01); Western blot results also showed that overexpression of miR-340 can inhibit the expression of GRP78, while inhibiting miR-340 expression, the expression of GRP78 is relieved. In summary, miR-340 can directly target GRP78 to promote the apoptosis of COLO-205 cells and inhibit their proliferation, migration and invasion.

Investigation of radiomics based on 18F-FDG PET/CT in predicting the COG risk stratification of neuroblastoma / 中华核医学与分子影像杂志

Objective:To explore the value of radiomics based on 18F-fluorodeoxyglucose (FDG) PET/CT in predicting the Children′s Oncology Group (COG) risk stratification of neuroblastoma (NB). Methods:From March 2018 to November 2019, the 18F-FDG PET/CT images of 125 NB children (51 males, 74 females, age: 0.5-10.5 years) confirmed pathologically in Beijing Friendship Hospital were retrospectively analyzed. According to the COG classification, patients were divided into high-risk group and non-high-risk group (including low- and intermediate-risk). Imaging radiomics features were extracted from PET and CT images and screened. Logistic regression was used to build the first model based on radiomics features (R_model) and calculate radiomics score (Rad_score), then build the second model (RD_model) based on Rad_score and demographic features and at last build the third model (RDC_modle) based on Rad_score, demographic features and clinical features. The receiver operating characteristic (ROC) curve was used to evaluate the predictive efficacy of these models. Results:The training set contained 94 NB cases (63 high-risk cases, 31 non-high-risk cases), and the validation set contained 31 NB cases (21 high-risk cases, 10 non-high-risk cases). Four radiomics features were obtained by screening, of which two features were based on CT images and the other two features were based on PET images. The area under the curves (AUCs) of the R_model, RD_model and RDC_model in training or validation set were 0.91, 0.94, 0.98 or 0.86, 0.92, 0.95, respectively. The accuracies of the R_model, RD_model and RDC_model in training or validation set were 86%(81/94), 89%(84/94), 93%(87/94) or 84%(26/31), 84%(26/31), 87%(27/31), respectively.Conclusions:Radiomics based on 18F-FDG PET/CT can accurately predict the COG risk stratification of NB. Prediction model of radiomics features combined with demographic and clinical characteristics can further improve the accuracy of predicting NB COG risk stratification, which can help personalized and precise therapy protocol management in NB.