Targeting Cyclophilin A and CD147 to Inhibit Replication of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and SARS-CoV-2-Induced Inflammation.

Identification and development of effective therapeutics for coronavirus disease 2019 (COVID-19) are still urgently needed. The CD147-spike interaction is involved in the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 invasion process in addition to angiotensin-converting enzyme 2 (ACE2). Cyclophilin A (CyPA), the extracellular ligand of CD147, has been found to play a role in the infection and replication of coronaviruses. In this study, our results show that CyPA inhibitors such as cyclosporine A (CsA) and STG-175 can suppress the intracellular replication of SARS-CoV-2 by inhibiting the binding of CyPA to the SARS-CoV-2 nucleocapsid C-terminal domain (N-CTD), and the IC50 is 0.23 µM and 0.17 µM, respectively. Due to high homology, CsA also had inhibitory effects on SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), and the IC50 is 3.2 µM and 2.8 µM, respectively. Finally, we generated a formulation of phosphatidylserine (PS)-liposome-CsA for pulmonary drug delivery. These findings provide a scientific basis for identifying CyPA as a potential drug target for the treatment of COVID-19 as well as for the development of broad-spectrum inhibitors for coronavirus via targeting CyPA. Highlights: 1) SARS-CoV-2 infects cells via the binding of its S protein and CD147; 2) binding of SARS-CoV-2 N protein and CyPA is essential for viral replication; 3) CD147 and CyPA are potential therapeutic targets for SARS-CoV-2; and 4) CsA is a potential therapeutic strategy by interrupting CD147/CyPA interactions. SIGNIFICANCE STATEMENT: New severe acute respiratory syndrome coronavirus (SARS-CoV)-2 variants and other pathogenic coronaviruses (CoVs) are continually emerging, and new broad-spectrum anti-CoV therapy is urgently needed. We found that binding sites of cyclophilin A/cyclosporin A (CyPA/CsA) overlap with CyPA/N-CTD (nucleocapsid C-terminal domain), which shows the potential to target CyPA during SARS-CoV-2 infection. Here, we provide new evidence for targeting CyPA in the treatment of coronavirus disease 2019 (COVID-19) as well as the potential of developing CyPA inhibitors for broad-spectrum inhibition of CoVs.

WITHDRAWN: Construction and evaluation of risk prediction model for kidney injury in patients treated with PD-1 inhibitors

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Tripterygium glycosides improve abnormal lipid deposition in nephrotic syndrome rat models.

OBJECTIVE: The purpose of this study was to determine the effect of tripterygium glycosides (TGs) on regulating abnormal lipid deposition in nephrotic syndrome (NS) rats. METHODS: Sprague-Dawley (SD) rats were injected with 6 mg/kg doxorubicin to construct nephrotic syndrome models (n = 6 per group), and then administered with TGs (10 mg/kg·d-1), prednisone (6.3 mg/kg·d-1), or pure water for 5 weeks. Biomedical indexes, such as urine protein/creatinine ratio (PCR), blood urea nitrogen (BUN), serum creatinine (Scr), serum albumin (SA), triglycerides (TG), total cholesterol (TC)were investigated to evaluate the renal injury of rats. H&E staining experiment was used to assess the pathological alterations. Oil Red O staining was used to assess the level of renal lipid deposition. Malondialdehyde (MDA) and glutathione (GSH) were measured to assess the extent of oxidative damage to the kidney. TUNEL staining was used to assess the status of apoptosis in the kidney. Western blot analysis was performed to examine the levels of relevant intracellular signaling molecules. RESULTS: After treatment with TGs, those tested biomedical indexes were significantly improved, and the extent of kidney tissue pathological changes and lipid deposition in the kidney was diminished. Treatment with TGs decreased renal oxidative damage and apoptosis. Regarding the molecular mechanism, TGs significantly increased the protein expression levels of Bcl-2 but decreased the levels of CD36, ADFP, Bax, and Cleaved caspase-3. CONCLUSION: TGs alleviates renal injury and lipid deposition induced by doxorubicin, suggesting that it may be a new strategy for reducing renal lipotoxicity in NS.

Development and validation of a prediction model for tuberculous pleural effusion: a large cohort study and external validation.

BACKGROUND: Distinguishing tuberculous pleural effusion (TPE) from non-tuberculosis (TB) benign pleural effusion (BPE) remains to be a challenge in clinical practice. The aim of the present study was to develop and validate a novel nomogram for diagnosing TPE. METHODS: In this retrospective analysis, a total of 909 consecutive patients with TPE and non-TB BPE from Ningbo First Hospital were divided into the training set and the internal validation set at a ratio of 7:3, respectively. The clinical and laboratory features were collected and analyzed by logistic regression analysis. A diagnostic model incorporating selected variables was developed and was externally validated in a cohort of 110 patients from another hospital. RESULTS: Six variables including age, effusion lymphocyte, effusion adenosine deaminase (ADA), effusion lactatedehy drogenase (LDH), effusion LDH/effusion ADA, and serum white blood cell (WBC) were identified as valuable parameters used for developing a nomogram. The nomogram showed a good diagnostic performance in the training set. A novel scoring system was then established based on the nomogram to distinguish TPE from non-TB BPE. The scoring system showed good diagnostic performance in the training set [area under the curve (AUC) (95% confidence interval (CI)), 0.937 (0.917-0.957); sensitivity, 89.0%, and specificity, 89.5%], the internal validation set [AUC (95%CI), 0.934 (0.902-0.966); sensitivity, 88.7%, and specificity, 90.3%], and the external validation set [(AUC (95%CI), 0.941 (0.891-0.991); sensitivity, 93.6%, and specificity, 87.5%)], respectively. CONCLUSIONS: The study developed and validated a novel scoring system based on a nomogram originated from six clinical parameters. The novel scoring system showed a good diagnostic performance in distinguishing TPE from non-TB BPE and can be conveniently used in clinical settings.

Learning Observer-Based Sensor Fault-Tolerant Control of Distributed Generation in an Islanded Microgrid for Bus Voltage Stability Enhancement.

In order to improve robust operating performance and enhance bus voltage stability, a learning observer-based fault-tolerant control strategy is proposed for the distributed generation in islanded microgrid with sensor faults and uncertain disturbances. Firstly, the output feedback control theory and the linear matrix inequality method are used to design closed-loop controller for the voltage source inverter of distributed generation; secondly, a fault-tolerant model and control structure of the distributed generation in an islanded microgrid with sensor faults is analyzed. By employing the fault output signal conversion filter and proportional derivative type learning observer, the online estimation and real-time compensation of the sensor fault signal are realized. Thirdly, the system synthesis of output feedback control and fault-tolerant control is completed. Finally, the multi-scenario sensor fault scheme simulation experiment verifies that the proposed control strategy has strong sensor fault tolerance and adaptability.

Downregulation of lncRNA IGF2-AS-encoded peptide induces trophoblast - cycle arrest.

RESEARCH QUESTION: lncRNA IGF2-AS may be related to early pregnancy loss. Does lncRNA IGF2-AS affect trophoblast cell growth? The aim of the present study was to verify that lncRNA IGF2-AS encodes a polypeptide, IGF2-AS-168aa, and to study its role in the pathogenesis of trophoblasts. DESIGN: A small interfering RNA targeted to the IGF2-AS gene (si-IGF2-AS) was designed and transfected into JEG-3 and JAR cells for in-vitro gene silencing. Quantitative polymerase chain reaction and western blotting were used to determine lncRNA IGF2-AS levels in experimental cells. After IGF2-AS suppression, MTT assay was used to assess cell proliferation and apoptosis was determined by flow cytometry. Target gRNA IGF2-AS-gRNA was designed for knockout conducted the corresponding mRNA. HEK293T cells were transfected with the identified positive clone vectors. Finally, IGF2-AS-168aa was analysed by western blotting after the protein-coding region of the IGF2-AS gene was knocked out by CRISPR/Cas9 gene-editing technology. RESULTS: lncRNA IGF2-AS and IGF2-AS-168aa were significantly downregulated in JEG-3 and JAR cells transfected with si-IGF2-AS (lncRNA IGF2-AS: JAR: NC versus small interfering RNA (siRNA)-1: P = 0.019 NC versus siRNA-2: P = 0.013; JEG-3: NC versus siRNA-1: P = 0.001 NC versus siRNA-2: P = 0.004) (IGF2-AS-168aa: JAR: NC versus siRNA-1: P = 0.030 NC versus siRNA-2: P = 0.018; JEG-3: NC versus siRNA-1: P = 0.004 NC versus siRNA-2: P = 0.001). IGF2-AS gene was incapable of encoding IGF2-AS-168aa after the coding region was successfully knocked out in HEK293T cells. Flow cytometry and the MTT assay revealed that IGF2-AS gene silencing led to cell cycle block in the G1 phase, markedly decreasing cell proliferation and increasing apoptosis. CONCLUSION: The IGF2-AS gene encoded a peptide with a potential function in trophoblast cell cycle arrest.

Pathogenesis of Familial Hyperaldosteronism Type II: New Concepts Involving Anion Channels.

PURPOSE OF REVIEW: The application of advanced genetic techniques has recently begun to unravel the genetic basis for familial primary aldosteronism type 2 (FH-II). RECENT FINDINGS: Whole-exome sequencing in a large family with FH-II revealed a shared rare damaging heterozygous variant in CLCN2 (chr.3: g.184075850C>T, p.Arg172Gln) in three severely affected members. The gene encodes a chloride channel, ClC-2. A cohort of 80 unrelated individuals diagnosed with early-onset primary aldosteronism was also examined for CLCN2 mutations finding three further occurrences of p.Arg172Gln mutations and four single cases of other potentially damaging heterozygous mutations for an overall prevalence of 9.9%. A concurrent report also found a different CLCN2 mutation (p.Gly24Asp) in a single severely affected patient from a cohort of 12 with early-onset PA for a prevalence of 8.3%. Cases of primary aldosteronism associated with CLCN2 mutations appear to be bilateral and respond well to medical treatment. In the adrenal, ClC-2 has been demonstrated to localize predominantly to the zona glomerulosa (ZG), and functional analysis suggests that mutations in ClC-2 predispose ZG cells to depolarization, thus leading to calcium influx via activation of voltage-gated calcium channels and increased aldosterone production. Germline CLCN2 mutations appear to account for a substantial proportion of early-onset primary aldosteronism cases, and genetic testing for mutations in this gene should be considered in appropriate cases.

Roles of Albumin-Binding Proteins in Cancer Progression and Biomimetic Targeted Drug Delivery.

Nutrient transporters have attracted significant attention for their promising application in biomimetic delivery. Due to the active consumption of nutrients, cancer cells generally overexpress nutrient transporters to meet their increased need for energy and materials. For example, albumin-binding proteins (ABPs) are highly overexpressed in malignant cells, stromal cells, and tumor vessel endothelial cells responsible for albumin uptake. ABP (e.g., SPARC) is a promising target for tumor-specific drug delivery, and albumin has been widely used as a biomimetic delivery carrier. Apart from the transportation function, ABPs are closely associated with neoplasia, invasion, and metastasis. Herein, a summary of the roles of ABP in cancer progression and the application of albumin-based biomimetic tumor-targeted delivery through the ABP pathway is presented.

In Primary Aldosteronism, Mineralocorticoids Influence Exosomal Sodium-Chloride Cotransporter Abundance.

Distal tubular sodium retention is a potent driver of hypertension, and the thiazide-sensitive sodium-chloride cotransporter (NCC) has a key role in this process. In humans, factors regulating NCC are unclear, but in animal models, aldosterone is a potent regulator, possibly via effects on plasma potassium. We studied the effects of the mineralocorticoid fludrocortisone on the abundance of NCC and its phosphorylated form (pNCC) as well as WNK lysine deficient protein kinase 4 (WNK4) and STE20/SPS1-related, proline alanine-rich kinase (SPAK) in human urinary exosomes. We isolated exosomes from daily urine samples in 25 patients undergoing fludrocortisone suppression testing (100 µg every 6 hours for 4 days) to diagnose or exclude primary aldosteronism. Over the course of the test, NCC levels increased 3.68-fold (P<0.01) and pNCC levels increased 2.73-fold (P<0.01) relative to baseline. The ratio of pNCC/NCC dropped by 48% (P<0.01). The abundance of WNK4 increased 3.23-fold (P<0.01), but SPAK abundance did not change significantly (P=0.14). Plasma potassium concentration strongly and negatively correlated with pNCC, NCC, and WNK4 abundance (P<0.001 for all). This study shows that, in humans, mineralocorticoid administration is associated with a rapid increase in abundance of NCC and pNCC, possibly via the WNK pathway. These effects may be driven by changes in plasma potassium.

Identification of differentially expressed circular RNAs in human colorectal cancer.

Circular RNA, a class of non-coding RNA, is a new group of RNAs and is related to tumorigenesis. Circular RNAs are suggested to be ideal candidate biomarkers with potential diagnostic and therapeutic implications. However, little is known about their expression in human colorectal cancer. In our study, differentially expressed circular RNAs were detected using circular RNA array in paired tumor and adjacent non-tumorous tissues from six colorectal cancer patients. Expression levels of selected circular RNAs (hsa_circRNA_103809 and hsa_circRNA_104700) were measured by real-time polymerase chain reaction in 170 paired colorectal cancer samples for validation. Statistical analyses were conducted to investigate the association between hsa_circRNA_103809 and hsa_circRNA_104700 expression levels and respective patient clinicopathological features. Receiver operating characteristic curve was constructed to evaluate the diagnostic values. Our results indicated that there were 125 downregulated and 76 upregulated circular RNAs in colorectal cancer tissues compared with normal tissues. We also first demonstrated that the expression levels of hsa_circRNA_103809 ( p < 0.0001) and hsa_circRNA_104700 ( p = 0.0003) were significantly lower in colorectal cancer than in normal tissues. The expression level of hsa_circRNA_103809 was significantly correlated with lymph node metastasis ( p = 0.021) and tumor-node-metastasis stage ( p = 0.011), and the expression level of hsa_circRNA_104700 was significantly correlated with distal metastasis ( p = 0.036). The area under receiver operating characteristic curves of hsa_circRNA_103809 and hsa_circRNA_104700 were 0.699 ( p < 0.0001) and 0.616 ( p < 0.0001), respectively. In conclusion, these results suggest that hsa_circRNA_103809 and hsa_circRNA_104700 may be potentially involved in the development of colorectal cancer and serve as potential biomarkers for the diagnosis of colorectal cancer.

Isolation of a peptide from Ph.D.-C7C phage display library for detection of Cry1Ab.

Traditional ELISA methods of using animal immunity yield antibodies for detection Cry toxin. Not only is this incredibly harmful to the animals, but is also time-intensive. Here we developed a simple method to yield the recognition element. Using a critical selection strategy and immunoassay we confirmed a clone from the Ph.D-C7C phage library, which has displayed the most interesting Cry1Ab-binding characteristics examined in this study (Fig. 1). The current study indicates that isolating peptide is an alternative method for the preparation of a recognition element, and that the developed assay is a potentially useful tool for detecting Cry1Ab.

Prodrug-Like, PEGylated Protein Toxin Trichosanthin for Reversal of Chemoresistance.

Multidrug resistance (MDR) is a main obstacle in cancer chemotherapy. The MDR mechanisms involve P-glycoprotein (P-gp) overexpression, abnormality of apoptosis-related protein, and altered expression of drug-targeting proteins. Therapeutic proteins are emerging as candidates for overcoming cancer MDR because of not only their large molecular size that potentially circumvents the P-gp-mediated drug efflux but also their distinctive bioactivity distinguished from small-molecular drugs. Herein we report trichosanthin, a plant protein toxin, possesses synergistic effect with paclitaxel (PTX) in the PTX-resistance A549/T nonsmall cell lung cancer (NSCLC) cells, by reversing PTX-caused caspase 9 phosphorylation and inducing caspase 3-dependent apoptosis. Moreover, via intein-mediated site-specific protein ligation, a matrix metalloproteinase (MMP)-activatable cell-penetrating trichosanthin delivery system was constructed by modification of a cell-penetrating peptide and MMP-2-sensitive PEGylation to overcome the limitation of in vivo application of trichosanthin, by improving the short half-life and poor tumor targeting, as well as immunogenicity. In a mouse model bearing A549/T tumor, the MMP-activatable trichosanthin was further tested for its application for MDR reversal in combination with PTX liposomes. The delivery system showed synergy effect with PTX-loaded liposome in treating MDR cancer in vivo.

Codelivery of dihydroartemisinin and doxorubicin in mannosylated liposomes for drug-resistant colon cancer therapy.

Multidrug resistance (MDR) is a major hurdle in cancer chemotherapy and makes the treatment benefits unsustainable. Combination therapy is a commonly used method for overcoming MDR. In this study we investigated the anti-MDR effect of dihydroartemisinin (DHA), a derivative of artemisinin, in combination with doxorubicin (Dox) in drug-resistant human colon tumor HCT8/ADR cells. We developed a tumor-targeting codelivery system, in which the two drugs were co-encapsulated into the mannosylated liposomes (Man-liposomes). The Man-liposomes had a mean diameter of 158.8 nm and zeta potential of -15.8 mV. In the HCT8/ADR cells that overexpress the mannose receptors, the Man-liposomes altered the intracellular distribution of Dox, resulting in a high accumulation of Dox in the nuclei and thus displaying the highest cytotoxicity (IC50=0.073 µg/mL) among all the groups. In a subcutaneous HCT8/ADR tumor xenograft model, administration of the Man-liposomes resulted in a tumor inhibition rate of 88.59%, compared to that of 47.46% or 70.54%, respectively, for the treatment with free Dox or free Dox+DHA. The mechanisms underlying the anti-MDR effect of the Man-liposomes involved preferential nuclear accumulation of the therapeutic agents, enhanced cancer cell apoptosis, downregulation of Bcl-xl, and the induction of autophagy.

Isolation of broad-specificity domain antibody from phage library for development of pyrethroid immunoassay.

An antibody to phenoxybenzoic acid (PBA), the conserved chemical region of pyrethroids, was developed using a domain antibody (DAB) library to enable pyrethroid detection in agricultural products. The DAB library, constructed without animal immunization and based on a human VH framework, displayed repertoires on filamentous bacteriophage. After four rounds of panning, we obtained five domain antibodies that are capable of binding to PBA. Antibody A3 has strong identification capability to cypermethrin, ß-cypermethrin, and fenvalerate. The antibody A3 was used to develop an enzyme-linked immunosorbent assay (ELISA). The IC50 values were 2.586, 1.814, and 2.251 µg/ml for cypermethrin, ß-cypermethrin, and fenvalerate, respectively. The assay shows weak competition with flucythrinate but shows no competition with fenpropathrin, deltamethrin, and permethrin. The developed ELISA process was successfully applied to fortified Chinese cabbage samples, with the recoveries of cypermethrin, ß-cypermethrin, and fenvalerate ranging from 84.4 to 112.3%. We developed an immunoassay to detect pyrethroids depending on the domain antibody library, which overcomes the limitation of requiring protein antigen to immunize animals raising antibody.

Design and characterization of human exposure to generated sulfate and soot particles in a pilot chamber study.

UNLABELLED: A number of literatures have documented adverse health effects of exposure to fine particulate matter (PM2.5), and secondary sulfate aerosol and black carbon may contribute to health impacts of PM2.5 exposure. We designed an exposure system to generate sulfate and traffic soot particles, and assessed the feasibility of using it for human exposure assessment in a pilot human exposure study. In the designed exposure system, average mass concentrations of generated sulfate and soot particles were 74.19 µg/m3 and 11.54 µg/m3 in the chamber and did not vary significantly during two-hour human exposure sessions. The size ranges of generated sulfate were largely between 20 to 200 nm, whereas those of generated soot particles were in the size ranges of 50 to 200 nm. Following two-hour exposure to generated sulfate and soot particles, we observed significant increases in fractional exhaled NO (FeNO) in young and health subjects. Building on established human exposure system and health response follow-up methods, future full-scale studies focusing on the effects of mixed particulates and individual PM2.5 components would provide data in understanding the underpinning cardio-respiratory outcomes in relation to air pollution mixture exposure. IMPLICATIONS: Controlled exposure is a useful design to measure the biological responses repeatedly following particulate exposures of target components and set exposure at target levels of health concerns. Our study provides rational and establishes method for future full-scale studies to focus on examining the effects of mixed particulates and individual PM2.5 components.

Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay.

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA-bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I50 and I10 values of purified mAbs were 0.63 and 0.13 µg/ml, respectively, with a dynamic range between 0.19 and 2.04 µg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 µg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.

Lack of association between genetic polymorphisms in three folate-related enzyme genes and male infertility in the Chinese population.

PURPOSE: This study explored the possible association of four single nucleotide polymorphisms (SNPs) of the three folate-related enzyme genes: MTHFR C677T and A1298C, MTR A2756G and MTRR A66G, with male infertility in the Chinese population. METHODS: The polymorphic distributions of the four SNPs (MTHFR C677T and A1298C, MTR A2756G and MTRR A66G) were investigated by the method of SNaPshot in a Chinese cohort including 296 idiopathic infertile males with azoospermia or oligozoospermia and 204 fertile males. RESULTS: We found no evidence for an association between any of these variants (MTHFR C677T and A1298C, MTR A2756G and MTRR A66G) and male infertility. CONCLUSIONS: There is no evidence for an association between male infertility and polymorphism of the three folate-related enzyme genes in the Chinese population.

Synthesis and investigation of the imprinting efficiency of ion imprinted nanoparticles for recognizing copper.

The Cu(2+) imprinted nanoparticles were synthesized using a simple approach including imprinting and crosslinking in aqueous solutions. The morphologies of imprinted polymers were alterable by changing the component ratio and are illustrated using a field emission scanning electron microscope. The obtained nanoparticles were applied as novel polymeric adsorbents with outstanding adsorption capacity and good selectivity to recognize and remove copper ions. The component ratio of PEI, crosslinking agents and template ions was systematically studied with their influence on the morphology and imprinting efficiency of sorbents. The crosslinking degree and the amount of imprinted sites were investigated with different component ratios and were confirmed to be the key factors of the imprinting efficiency. The appropriate dosage ratio of PEI, ECHA and template Cu(2+) was 1 g : 1 g : 2.5 mmol and the imprinting efficiency can be significantly improved. The maximum selectivity coefficients of the nanoparticles for Cu(2+)/Cr(3+) and Cu(2+)/Zn(2+) were 5.21 and 21.00. The results obtained could be extended to other polymers with improvement in imprinting efficiency and further applications.