Effects of Zinc Combined with Metformin on Zinc Homeostasis, Blood-Epididymal Barrier, and Epididymal Absorption in Male Diabetic Mice.

Diabetes increases the likelihood of germ cell damage, hypogonadism, and male infertility. Diabetes leads to lower zinc (Zn) levels, an important micronutrient for maintaining male fertility, and zinc deficiency can lead to decreased male fertility through multiple mechanisms. The aim of this study was to investigate the effect of combined metformin and zinc administration on epididymis in diabetic mice; 10 of 50 male mice were randomly selected as the control group (group C), and the remaining 40 mice were randomly divided into untreated diabetes group (group D), diabetes + zinc group (group Z), diabetes + metformin group (group M), and diabetes + metformin + zinc group (group ZM) with 10 mice each. Diabetic mice in group Z received oral zinc (10 mg/kg) once daily for 4 weeks; diabetic mice in group M received oral metformin (200 mg/kg) once daily for 4 weeks; diabetic mice in group ZM received oral metformin and zinc once daily for 4 weeks; and groups C and D received the same amount of sterile water by gavage. Overnight fasted mice were sacrificed, and blood samples, mouse epididymides, and sperm were collected for further experiments. In group D, fasting blood glucose and insulin resistance index increased significantly, semen quality, serum insulin, and testosterone decreased, and epididymal structure was disordered. In group D, epididymal tissue zinc, free zinc ions in the caput, and cauda of epididymis and zinc transporter (ZnT2) decreased significantly, while ZIP12, metallothionein (MT), and metal transcription factor (MTF1) increased significantly. In addition, the expressions of blood-epididymal barrier (BEB)-related molecules (including ZO-1 ß-catenin and N-cadherin) and aquaporins (AQPs, including AQP3, AQP9, and AQP11) in the epididymis of mice in group D were significantly decreased. In addition, compared with groups D, Z, and M, in the ZM group, the expression of BEB-related molecules (including ZO-1, ß-catenin, and N-cadherin) and aquaporins (AQP3, AQP9, and AQP11) in epididymis tissue were significantly increased, and sperm motility and serum testosterone were significantly increased. It was concluded that male diabetic mice have a disturbed epididymal structure and decreased semen quality by causing an imbalance in epididymal zinc homeostasis, BEB, and impaired absorptive function. The combination of zinc and metformin is an effective and safe alternative treatment and provides additional benefits over metformin alone.

Biochemical Markers of Zinc Nutrition.

Zinc is an important trace element involved in the biochemical and physiological functions of the organism and is essential in the human body. It has been reported that 17.3% of people around the world are at risk of many diseases due to zinc deficiency, which has already affected people's healthy lives. Currently, mild zinc deficiency is difficult to diagnose early due to the lack of typical clinical manifestations, so finding zinc biomarkers is crucial for people's health. The present article reviews the main representative zinc biomarkers, such as body fluid zinc levels, zinc-dependent proteins, tissue zinc, and zinc-containing enzymes, to provide a reference for actively promoting the study of zinc nutritional status and early clinical diagnosis.

Clinical Application of Matrix-Assisted Laser-Resolved Ionization Time-Of-Flight Mass Spectrometry for Rapid Detection of Blood-Borne Pathogens.

Objective: To evaluate the performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical pathogenic microorganisms. Methods: Blood culture-positive specimens were collected from inpatients in our hospital from March to December 2022 and identified using VITEK 2XL (biochemical), VITEK MS (colony), VITEK MS (bacterial membrane) and VITEK MS (separating gel) methods, respectively, to compare the compliance rate and identification values of the four methods. Results: A total of 280 strains were included in the analysis, including 155 (55.36%) Gram-negative and 125 (44.64%) Gram-positive strains. 279 (99.64%) of the 280 strains were identified by VITEK 2XL (biochemical), including 154 (99.35%) Gram-negative and 125 (100%) Gram-positive strains. VITEK MS (colony) identified 278 (99.29%) strains, including 153 (98.71%) Gram-negative and 125 (100%) Gram-positive. 261 (93.21%) strains were identified in VITEK MS (bacterial membrane), including 148 (95.48%) Gram-negative and 113 (90.40%) Gram-positive strains. VITEK MS (separating gel) identified 232 (82.86%) strains, including 136 (87.74%) Gram-negative and 96 (76.80%) Gram-positive strains. Conclusion: MALDI-TOF MS findings are highly consistent with traditional culture identification methods in terms of identification accuracy, and the VITEK MS (bacterial membrane) and VITEK MS (separating gel) identification methods significantly reduce the turnaround time for identification in the laboratory.

A Rapid and Sensitive Detection of HIV-1 with a One-Pot Two-Stage Reverse Transcription Recombinase Aided Real-Time PCR Assay.

Human immunodeficiency virus 1 (HIV-1) attacks the immune system, making people susceptible to various diseases, thus increasing their risk of death. Comprehensive detection of major HIV-1 strains circulating in China is vital for effective HIV-1 infection prevention and treatment. HIV-1 nucleic acid detection is considered effective for HIV-1 diagnosis since traditional immunological testing may fail to detect HIV-1 infection during the window period. This work demonstrates a one-pot two-stage amplification assay (RT-RAP), a combination of reverse transcription recombinase (RT- RAA), and quantitative real-time polymerase chain reaction (qRT-PCR). The turn-around time of the assay is only 50 min and can be performed with commonly available laboratory equipment, the qPCR devices. The RT-RAP assay could detect approximately 5 and 14 copies/reaction of HIV-1 DNA and RNA using recombinant plasmids and standard reference strains, respectively. Additionally, we found that the clinical performance of RT-RAP (detected 169 samples out of 170 specimens) was consistent with that of qRT-PCR. The sensitivity and specificity of RT-RAP were 100.00% (99/99) and 98.59% (70/71), respectively, while its positive and negative predictive values were 99.00% (99/100) and 100.00% (70/70), respectively. The total coincidence rate of the RT-RAP was 99.41% (169/170), with a kappa value of 0.988 (p < 0.05). We demonstrated that RT-RAP could rapidly detect the common HIV-1 subtypes commonly circulating in China with comparable sensitivity and specificity to qRT-PCR.

Simultaneous detection of 9 respiratory pathogens using a newly developed multiplex real-time PCR panel based on an automatic molecular detection and analysis system.

Timely identification of respiratory pathogens guides specific treatment, reduces hospital costs and minimizes the excessive use of antibiotics. A new multiplex real-time PCR panel was developed based on an automatic molecular detection and analysis system (AutoMolec system), consisting of three separate internally controlled assays. Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, human metapneumovirus, influenza B virus, respiratory syncytial virus and human parainfluenza virus 1-3 may be directly detected in original samples. The system's clinical performance was evaluated by comparison with an approved commercial kit, using 517 clinical samples. The limit of detection of the AutoMolec mRT-PCR panel ranged from 4 × 10-4 ∼3.3 TCID50/mL and no cross-reaction with common respiratory pathogens was observed. The AutoMolec mRT-PCR panel had 99.09% sensitivity and 100.0% specificity and overall detection consistency was 99.61%, making it comparable to that of the commercial kit. Therefore, the AutoMolec mRT-PCR panel has great potential for routine screening of respiratory infection in China.

Development of a duplex recombinase-aided amplification assay for direct detection of Mycoplasma pneumoniae and Chlamydia trachomatis in clinical samples.

BACKGROUND: Pneumonia caused by Mycoplasma pneumoniae is common in the elderly and children, and pneumonia caused by Chlamydia trachomatis is prevalent in newborns. This study aimed to establish a rapid, sensitive, and simple method for the direct detection of M. pneumoniae and C. trachomatis in clinical samples without DNA extraction. METHODS: We established a duplex recombinase-aided amplification (RAA) assay with the RNAseP gene as an internal control for detecting the P1 gene of M. pneumoniae and the ORF8 gene of C. trachomatis, respectively. The results were obtained at 39 °C within 15-20 min. A total of 130 clinical samples suspected of M. pneumoniae or C. trachomatis infection were collected and tested by duplex RAA and PCR. DNA extracted via a commercial kit or treated with a nucleic acid-releasing agent was used and compared, respectively. Standard recombinant plasmids were used to test the sensitivity of the duplex RAA assay. In addition, other similar common pathogens were used to verify the specificity of the duplex RAA assay. RESULTS: The sensitivity of the duplex RAA assay for detecting M. pneumoniae and C. trachomatis was 10 copies/µL using recombinant plasmids. Compared with PCR, the sensitivity and specificity of duplex RAA assays for M. pneumoniae and C. trachomatis was 100% using clinical DNA samples extracted using a commercial kit and a nucleic acid-releasing agent, and the Kappa value was 1. CONCLUSION: The advantages of this duplex RAA assay include high sensitivity and specificity, short duration, and simple extraction steps, with potential for use in the on-site detection of M. pneumoniae and C. trachomatis in resource-limited settings.

Detection of low-load Epstein-Barr virus in blood samples by enriched recombinase aided amplification assay.

Epstein-Barr virus (EBV), a common human γ-herpesvirus, infects more than 90% of adults worldwide. The purpose of this study was to establish a novel EBV detection method by combining the recombinase aided amplification (RAA) assay with an initial enrichment step that utilizes magnetic beads coated with a recombinant human mannan-binding lectin (rhMBL, M1 protein). An M1 protein-protein A magnetic bead complex (M1 beads) was prepared and used to achieve separation and enrichment of EBV from blood. After nucleic acid extraction, DNA was amplified by RAA. Using 388 whole blood samples and 1 serum sample, we explored the specificity, sensitivity and applicability of the newly developed detection method and compared it with commercial quantitative real-time polymerase chain reaction (qPCR) following M1 bead enrichment, traditional qPCR and traditional RAA. After enrichment, the positivity rate of EBV was increased from 15.94% to 17.74% by RAA (P < 0.05) and from 7.20% to 15.17% by qPCR (P < 0.05). The viral loads after enrichment were increased by 1.13 to 23.19-fold (P < 0.05). Our data demonstrates that an RAA assay incorporating M1 bead enrichment is a promising tool for detecting low EBV viral loads in blood samples that will facilitate an early response to EBV infection.

Establishment and evaluation of a quadruple quantitative real-time PCR assay for simultaneous detection of human coronavirus subtypes.

BACKGROUND: The newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four seasonal human coronaviruses (HCoVs) (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1) still circulate worldwide. The early clinical symptoms of SARS-CoV-2 and seasonal HCoV infections are similar, so rapid and accurate identification of the subtypes of HCoVs is crucial for early diagnosis, early treatment, prevention and control of these infections. However, current multiplex molecular diagnostic techniques for HCoV subtypes including SARS-CoV-2 are limited. METHODS: We designed primers and probes specific for the S and N genes of SARS-CoV-2, the N gene of severe acute respiratory syndrome coronavirus (SARS-CoV), and the ORF1ab gene of four seasonal HCoVs, as well as the human B2M gene product. We developed and optimized a quadruple quantitative real-time PCR assay (qq-PCR) for simultaneous detection of SARS-CoV-2, SARS-CoV and four seasonal HCoVs. This assay was further tested for specificity and sensitivity, and validated using 184 clinical samples. RESULTS: The limit of detection of the qq-PCR assay was in the range 2.5 × 101 to 6.5 × 101 copies/µL for each gene and no cross-reactivity with other common respiratory viruses was observed. The intra-assay and inter-assay coefficients of variation were 0.5-2%. The qq-PCR assay had a 91.9% sensitivity and 100.0% specificity for SARS-CoV-2 and a 95.7% sensitivity and 100% specificity for seasonal HCoVs, using the approved commercial kits as the reference. Compared to the commercial kits, total detection consistency was 98.4% (181/184) for SARS-CoV-2 and 98.6% (142/144) for seasonal HCoVs. CONCLUSION: With the advantages of sensitivity, specificity, rapid detection, cost-effectiveness, and convenience, this qq-PCR assay has potential for clinical use for rapid discrimination between SARS-CoV-2, SARS-CoV and seasonal HCoVs.

SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) prevents cardiac remodeling after myocardial infarction through ERK/SMAD signaling pathway.

In this study, we aimed to investigate the role of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) in cardiac remodeling after myocardial infarction (MI) and explore the underlying molecular mechanism. MI model was established by ligation of the left anterior descending coronary artery. C57/BL6J mice were randomly administered with 3.0 mg/kg/day PHPS1 (PHPS1-treated group) or normal saline (model group) by intraperitoneal injection. After 4 weeks of infusion, the effects of PHPS1 on cardiac remodeling were evaluated. Echocardiography results showed that PHPS1 treatment aggravated the MI-induced deterioration of cardiac function, with worse cardiac function parameters. PHPS1 treatment significantly increased the infarcted area, as well as the fibrotic area and the expression of collagen I and collagen III. Western blots and immunofluorescence staining showed that PHPS1 treatment up-regulated the expression of p-GRK2, p-SMAD2/3 and p-ERK1/2, while U0126 reversed the effect of PHPS1. The present study indicated that PHPS1 treatment contributed to myocardial fibrosis and infarction by activating ERK/SMAD signaling pathway, suggesting that SHP-2 may be a promising treatment target for cardiac remodeling after MI.

Specific inhibition of SHP2 suppressed abdominal aortic aneurysm formation in mice by augmenting the immunosuppressive function of MDSCs.

AIMS: To address the roles of SHP2 in regulating angiotensin II (Ang II) induced abdominal aortic aneurysm (AAA) and the potential molecular mechanisms. MAIN METHODS: AAA model was established in apolipoprotein E-deficient (apoE-/-) mice infused with Ang II. Suprarenal aortic luminal diameters were ultrasonically measured to determine the presentation of AAA in mice. The inflammatory and immunosuppressive factors in serum were detected by ELISA. AAA lesion size, positive macrophages and elastic laminae degradation were examined by histological analysis. Myeloid-derived suppressor cells (MDSCs) were measured by flow cytometry after magnetic bead sorting. Bioinformatics analysis was applied to screen the crucial genes related the progression of AAA. KEY FINDINGS: Treatment with PHPS1 (SHP2 inhibitor) significantly decreased the vascular diameter of AAA. Histological analysis showed that PHPS1 obviously reduced the Masson positive area, macrophages positive area, as well as the damage rate of elastic laminae. Moreover, PHPS1 suppressed the expression of INF-γ, TNF-α and MMPs, as well as elevated IL-10 and arginase-1 expression. Additionally, PHPS1 enhanced the expression of granulocytic MDSCs (G-MDSCs). By consulting with bioinformatics, STAT3 was selected. In G-MDSCs, PHPS1 stimulation obviously increased the phosphorylation level of STAT3, as well as elevated the protein expression of C/EBPß and arginase-1. However, the above phenomena can be blocked after Stattic (STAT3 inhibitor) treatment. SIGNIFICANCE: SHP2 may affect the AAA progression by interfering with expansion and function of MDSCs to regulate the body immunity, which might afford a novel direction for the treatment of patients with AAA.

Comprehensive analysis on expression and biological role of miR-224 in hepatocellular carcinoma / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To analyze the expression level of miR-224 in cancer tissues and plasma of hepatocellular carcinoma (HCC) patients, and its correlation with clinicopathological characteristics, diagnosis and prognosis of HCC patients, and to further analyze its mechanism of action in the occurrence and development of liver cancer through bioinformatics analysis and in vitro experiments. Methods: The expression level of miR-224 in HCC tissues and normal tissues was analyzed using large sample data from Gene Expression Omnibus (GEO). qPCR method was used to verify the expression level of miR-224 in the tumor tissues and corresponding adjacent tissues that surgically resected from 80 HCC patients in Hebei Provincial People ’s Hospital from January 2017 to January 2020; in addition, the miR-224 level was also examined in plasma samples from 30 HCC patients. The Kaplan-Meier plotter database was used to analyze the correlation between the miR-224 expression and the overall survival time of HCC patients. The biological processes and signal pathways involving miR-224 were analyzed using bioinformatics tools. Hepatocellular carcinoma HepG2 cells were transfected with miR-224 inhibitor, and then Clone formation experiment, Transwell chamber experiment, qPCR and WB methods were used to detect the effect of miR-224 knockdown on the proliferation and invasion of HepG2 cells and the expression level of EMT-related molecules. Results: The results of GEO database analysis showed that the expression level of miR-224 in HCC tissues was significantly higher than that in normal tissues. The results of clinical specimen verification showed that the expression level of miR-224 in the tumor tissues and plasma of HCC patients was significantly higher than that in the corresponding adjacent tissues and plasma from healthy controls (all P<0.01). The expression level of miR-224 was significantly correlated with the TNM stage, lymph node metastasis status and tumor size of HCC patients (P<0.05 or P<0.01). ROC analysis indicated that miR-224 showed a prominent diagnostic value in liver cancer, and the increased expression level of miR-224 was significantly related to the poor prognosis of HCC patients (P<0.05). Functional enrichment analysis revealed that miR-224 was mainly involved in the mTOR signaling pathway, AGE-RAGE signaling pathway, Rap1 signaling pathway, Ras signaling pathway, ErbB signaling pathway, HIF-1 signaling pathway and p53 signaling pathway and other signaling pathways related to tumor occurrence and development. Knockdown of miR-224 could significantly inhibit the colony formation and invasion of HepG2 cells and affect the expression of EMT-related markers (P<0.05 or P<0.01). Conclusion: miR-224 is highly expressed in HCC tissues and plasma and is significantly related to the poor prognosis of HCC patients. Knockdown of miR-224 expression can inhibit the colony formation, invasion and EMT process of liver cancer HepG2 cells.

A highly sensitive one-tube nested quantitative real-time PCR assay for specific detection of Bordetella pertussis using the LNA technique.

OBJECTIVES: Bordetella pertussis is a highly contagious respiratory agent and is the causative pathogen of pertussis, which primarily affects children. Current diagnostic techniques for this pathogen have a variety of limitations including a long culture time, low bacterial load, and lack of specificity. METHODS: This article reports the development of a one-tube nested quantitative real-time PCR assay using the locked nucleic acid (LNA) technique (LNA-OTN-q-PCR), targeting the BP485 gene and using a simple inexpensive extraction method. A total of 130 clinical samples from patients with clinically suspected pertussis, collected from the Children's Hospital of Hebei, China, were tested by LNA-OTN-q-PCR assay. RT-PCR and two-step semi-nested PCR assays were performed in parallel for comparison. RESULTS: Only strains of B. pertussis were identified as positive, whereas all of the remaining strains were appropriately identified as negative by the LNA-OTN-q-PCR assay. A single copy per reaction can be detected by the LNA-OTN-q-PCR assay. Additionally, the sensitivity of this method was 100 times that of the RT-PCR assay (100 copies per reaction). Sixty-three of the 130 clinical samples were detected positive by LNA-OTN-q-PCR assay; in contrast, RT-PCR was able to detect only 41 positive samples. Following this, all 63 samples were positively identified by two-step semi-nested PCR. Compared with the two-step semi-nested PCR assay, both the specificity and sensitivity of the LNA-OTN-q-PCR assay using purified DNA and crude extract were 100%. CONCLUSIONS: This assay was able to detect B. pertussis infection with high sensitivity and specificity. This test shows great potential as a promising technique to detect B. pertussis in both clinical laboratories and public health settings.

Potential injurious effects of the fine particulate PM2.5 on the progression of atherosclerosis in apoE-deficient mice by activating platelets and leukocytes.

INTRODUCTION: Exposure to the fine particulate matter PM2.5 is strongly associated with atherosclerotic diseases, creating considerable public concern. Nevertheless, the mechanisms have not been fully elucidated. We exposed atherosclerosis-prone apoE-deficient mice to PM2.5 to begin investigating these mechanisms. MATERIAL AND METHODS: Thirty-two 8-week-old male apoE-/- mice were divided to two groups fed with high-fat diet: a control group instilled with 0.9% saline, and an experimental group instilled with PM2.5 (30 mg/kg/day) for 8 weeks. We measured PM2.5 in whole blood by the ICP-MS method, and lipids and inflammatory factors by standard methods. The whole descending arteries were stained with oil red O; Aortic roots were stained with Movat, Sirius Red and immunohistochemical stains for pathological analysis; Brachiocephalic arteries for scanning electron microscopy, the descending arteries for Q-PCR. Echocardiography was used to evaluate cardiac function. RESULTS: In PM2.5 group, we observed elevated heavy metal components, consistent with higher amounts of platelets in total blood. The PM2.5 group also had elevated serum inflammatory factor levels. Finally, the PM2.5 group showed larger atherosclerotic plaques (p = 0.0231), higher numbers of lesion macrophages (p = 0.0183), greater injury to endothelial layers with greater adherence of platelets and leukocytes, elevated inflammatory factor levels, the NAD(P)H oxidase subunits p22phox and p47phox (p = 0.0079 and p = 0.0294), the M1/M2 associated markers IL-6, TNF-α (p = 0.0291, p = 0.0286), iNOS, IL-12 (p = 0.0122 and p = 0.0280) and arginase-1, and CD206 (p = 0.0216 and p = 0.0317). CONCLUSIONS: PM2.5 exposure activated circulating leukocytes, platelets and associated inflammatory factors, contributing to the progression of atherosclerosis in apoE-/- mice.

Relationship between XspI Site Polymorphisms of LDL-R Gene and Serum IL-2 and IL-10 in Patients with Hypercholesterolemia.

BACKGROUND: Relationship has been identified in sporadic reports between polymorphisms and hypercholesterolemia. However, the relationship between inflammatory cytokines and polymorphism of low-density lipoprotein receptor (LDL-R) gene in hypercholesterolemia is unclear. This study aimed to explore the relationship and significance between polymorphisms of LDL-R gene and serum Interleukin-2 (IL-2), IL-10 in patients with hypercholesterolemia. METHODS: PCR-RFLP and direct DNA sequencing assay were employed to determine polymorphism of LDL-R gene in 900 patients with hypercholesterolemia and 400 healthy cases. ELISA was applied to assay serum concentration of IL-2 and IL-10. Blood lipid indexes were tested in all cases. RESULTS: Compared with the healthy controls, level of IL-2 increased significantly, while IL-10 decreased significantly (P < 0.05). Correlation analysis showed that IL-2 was positively correlated with total cholesterol (TC), LDL-c, and genotype (r = 0.542, 0.410, 0.598, P < 0.05) and negatively correlated with HDL-c (r = -0.352, P < 0.05). Negative relationship also was found between TC, LDL-c, genotype, and IL-10 (r = -0.452, -0.390, -0.613, P < 0.05), and positive correlation between HDL-c and IL-10 (r = 0.398, P < 0.05). Multiple linear regression showed that genotypes and TC were independent factors affecting the levels of IL-2 and IL-10 (P < 0.05). CONCLUSION: IL-2 and IL-10 were related to gene polymorphisms of LDL-R, which might be involved in the development and progress of hypercholesterolemia.

[Correlations between tumor infiltrating Th17 cells and clinicopathological parameters in patients with colorectal cancer].

OBJECTIVE: To observe the correlations between the percentage of tumor infiltrating Th17 cells and clinicopathological parameters in patients with colorectal cancer (CRC). METHODS: Tumor and normal mucosal tissues were collected from 28 CRC patients. The proportions of Th17 cells in lymphocytes were detected by flow cytometry. The supernatant levels of IL-17A in cultured normal and tumor tissues were measured by ELISA. The correlations between the proportions of tumor infiltrating Th17 cells as well as the tumor supernatant levels of IL-17A and clinicopathological parameters in CRC patients were further analyzed. RESULTS: There was no significant difference in the expression of Th17 cells between normal tissues (2.24 ± 0.24)% and tumor tissues (2.47 ± 0.34)% (P>0.05). Compared with normal tissues, the supernatant levels of IL-17A in tumor tissues were significantly up-regulated [(257.74±31.36) pg/mL vs (163.53±12.62)pg/mL, P<0.05]. The proportions of tumor infiltrating Th17 cells and the tumor supernatant levels of IL-17A had no associations with age, gender, tumor location, tumor size and growth characteristics (P>0.05), but they were correlated with tumor differentiation, lymph node metastasis and TNM stage (P<0.05). No associations between the proportions of infiltrating Th17 cells as well as the supernatant levels of IL-17A in the normal tissues and all clinicopathological parameters were found (P>0.05). CONCLUSION: Th17 cells may be involved in the immunopathogenesis of the development of CRC.