Knockdown of FOXO4 protects against OGD/R­induced cerebral microvascular endothelial cell injury and regulates the AMPK/Nrf2/HO­1 pathway through transcriptional activation of CTRP6.

Cerebral ischemia is a type of cerebrovascular disease with high disability and mortality rates. The expression of forkhead box protein O4 (FOXO4) in the brain is increased following traumatic brain injury. To the best of our knowledge, however, the role of FOXO4 as well as its mechanism in cerebral ischemia has not been reported so far. For the establishment of an in vitro cellular injury model, human brain microvascular endothelial HCMEC/D3 cells were induced by oxygen-glucose deprivation/reoxygenation (OGD/R). mRNA and protein expressions of FOXO4 and C1q/tumor necrosis factor-related protein 6 (CTRP6) in OGD/R-induced HCMEC/D3 cells were detected by reverse transcription-quantitative (RT-q)PCR and western blotting. The transfection efficacy of small interfering (si)- and overexpression (Ov)-FOXO4 and si-CTRP6 was assessed using RT-qPCR and western blotting. Cell Counting Kit-8 and TUNEL assay were used to assess viability and apoptosis of HCMEC/D3 cells induced by OGD/R, respectively. A FITC-Dextran assay kit was applied to determine endothelial permeability and immunofluorescence assay was used for the measurement of the tight junction protein zonula occludens-1. The levels of oxidative stress markers and inflammatory cytokines were assessed with corresponding assay kits. The binding sites of transcription factor, FOXO4 and CTRP6 promoter were predicted using HDOCK SERVER. Luciferase reporter assay was used to detect the activity of the CTRP6 promoter while chromatin immunoprecipitation assay was used to evaluate the binding ability of the FOXO4 and CTRP6 promoter. Western blotting was used for the detection of apoptosis- and AMPK/Nrf2/heme oxygenase-1 (HO-1) pathway-associated proteins, along with tight junction proteins. The expression of FOXO4 was increased in OGD/R-induced HCMEC/D3 cells. After interfering with FOXO4 in cells, the viability of the OGD/R-induced HCMEC/D3 cells was increased while apoptosis was decreased. Furthermore, FOXO4 interference improved cellular barrier dysfunction but inhibited oxidative stress and the inflammatory response in HCMEC/D3 cells induced by OGD/R. FOXO4 knockdown regulated CTRP6 transcription in HCMEC/D3 cells. Knockdown of FOXO4 regulated expression of CTRP6 and protected OGD/R-induced HCMEC/D3 cell injury via the AMPK/Nrf2/HO-1 pathway. The present study indicated that FOXO4 knockdown activated CTRP6 to protect against cerebral microvascular endothelial cell injury induced by OGD/R via the AMPK/Nrf2/HO-1 pathway.

Transmission of ST45 and ST2407 extended-spectrum ß-lactamase-producing Klebsiella pneumoniae in neonatal intensive care units, associated with contaminated environments.

OBJECTIVES: Given the increasing frequency of infections due to extended-spectrum ß-lactamase (EBSL)-producing Klebsiella pneumoniae in humans over recent decades, infection control against this pathogen is of high importance. METHODS: In this study, the transmission mode of ESBL-producing K. pneumoniae in neonatal intensive care units (NICU) was investigated. We collected K. pneumoniae isolates from patients admitted to the NICU and performed environmental screening of the NICU and nearby obstetrics department. All isolates were analysed using antimicrobial susceptibility testing, whole-genome sequencing, molecular typing, and antimicrobial and virulence determinant screening. The phylogenetic relationships of all the isolates were analysed using core-genome multi-locus sequence type and single-nucleotide polymorphism-based analysis, and their plasmids harbouring antimicrobial resistance genes in ST2407 were compared. RESULTS: Eighteen K. pneumoniae isolates were collected, of which 10 isolates from patients belonged to ST45 and ST2407, and eight isolates from the environment belonged to various other clones. Although 80% and 100% of isolates from patients were ESBL-positive (blaCTX-M-14 and blaCTX-M-55) and possessed siderophores, respectively; fewer environmental isolates harboured antimicrobial resistance and virulence genes. For both ST45 and ST2407 isolates, the phylogenetic assessment revealed a close relationship between clinical and environmental isolates, indicating that bloodstream infections were associated with the contaminated environments. CONCLUSIONS: Based on these results, the environmental prevalence of K. pneumoniae should be considered given its pathogenicity in humans. Early and active infection control measures could decrease the spread of multidrug-resistant K. pneumoniae.

Development and application of quality checklist for the prevention and control of COVID-19 in fever clinic and isolation ward of the general hospital.

To develop a quality control checklist for the prevention and control of coronavirus disease 2019 （COVID-19） in fever clinic and isolation ward of the general hospital and to assess its application. Based on the relevant prevention and control plans and technical guidelines for COVID-19，Delphi method was used to identity items for evaluation，and a quality control checklist for the prevention and control of COVID-19 in the fever clinic and isolation ward was developed in Sir Run Run Shaw Hospital. The checklists included 8 dimensions and 32 items for fever clinic，7 dimensions and 27 items for the isolation ward. The appointed inspectors conducted daily quality control for each shift with this checklist. The expert authority coefficient was 0.88，the mean of the importance of each index in the quality control table was not less than 4.8，and the coefficient of variation was not more than 0.07. During the entire February 2020，8 problems were found and rectified on-the-spot with the application of the checklist. Quality inspection rate was 100% in both isolation wards and fever clinic. The compliance rate and accuracy rate of hand hygiene were 100%; the correct rate of wearing and removing protective equipment increased from 96% to 100%. During the same period，a total of 1915 patients were admitted to the fever clinic，including 191 suspected patients （all were isolated in the hospital，3 were confirmed）. There were no medical staff infected with COVID-19，no cross infection of patients and their families in the hospital. A quality control checklist for the prevention and control of COVID-19 has been developed and applied in the isolation wards and fever clinic，which plays an important role in preventing nosocomial infection.

Elevated microRNA­145­5p increases matrix metalloproteinase­9 by activating the nuclear factor­κB pathway in rheumatoid arthritis.

The present study explored whether miR­145­5p can aggravate the development and progression of rheumatoid arthritis (RA) by regulating the expression of matrix metalloproteinases (MMPs). ELISAs, reverse transcription­quantitative polymerase chain reaction (RT­qPCR), and western blotting were used to examine the expression levels of MMP­1, MMP­3, MMP­9, and MMP­13 in fibroblast­like synoviocytes (FLS) from patients with RA. Levels of MMP­1, MMP­3, MMP­9, and MMP­13 were assessed in the right hind ankles of a murine collagen­induced arthritis (CIA) model by RT­qPCR and immunohistochemical (IHC) analysis. The effects of activation or inhibition of the nuclear factor­κB (NF­κB) pathway on MMPs were evaluated by RT­qPCR and western blotting. Subcellular localization of NF­κB p65 was visualized by confocal microscopy. Overexpression of miR­145­5p increased the expression of MMP­3, MMP­9, and MMP­13 in RA­FLS. Moreover, injection of a miR­145­5p agomir into mice increased MMP­3, MMP­9, and MMP­13, as demonstrated by RT­qPCR and IHC analysis. A chemical inhibitor that selectively targets NF­κB (BAY11­7082) significantly attenuated MMP­9 expression, while it did not influence the levels of MMP­3 and MMP­13. Immunofluorescence analysis revealed that nuclear localization of p65 was significantly enhanced, indicating that miR­145­5p enhances activation of the NF­κB pathway by promoting p65 nuclear translocation. miR­145­5p overexpression also significantly increased phosphorylated p65 levels; however, the levels of IkB­a were reduced in response to this miRNA. Moreover, our results indicated that miR­145­5p aggravated RA progression by activating the NF­κB pathway, which enhanced secretion of MMP­9. In conclusion, modulation of miR­145­5p expression is potentially useful for the treatment of RA inflammation, by regulating the expression of MMPs, and MMP­9 in particular, through inhibition of the NF­κB pathway.

Semaphorin 3A Contributes to Secondary Blood-Brain Barrier Damage After Traumatic Brain Injury.

Semaphorin 3A (SEMA3A) is a member of the Semaphorins family, a class of membrane-associated protein that participates in the construction of nerve networks. SEMA3A has been reported to affect vascular permeability previously, but its influence in traumatic brain injury (TBI) is still unknown. To investigate the effects of SEMA3A, we used a mouse TBI model with a controlled cortical impact (CCI) device and a blood-brain barrier (BBB) injury model in vitro with oxygen-glucose deprivation (OGD). We tested post-TBI changes in SEMA3A, and its related receptors (Nrp-1 and plexin-A1) expression and distribution through western blotting and double-immunofluorescence staining, respectively. Neurological outcomes were evaluated by modified neurological severity scores (mNSSs) and beam-walking test. We examined BBB damage through Evans Blue dye extravasation, brain water content, and western blotting for VE-cadherin and p-VE-cadherin in vivo, and we examined the endothelial cell barrier through hopping probe ion conductance microscopy (HPICM), transwell leakage, and western blotting for VE-cadherin and p-VE-cadherin in vitro. Changes in miR-30b-5p were assessed by RT-PCR. Finally, the neuroprotective function of miR-30b-5p is measured by brain water content, mNSSs and beam-walking test. SEMA3A expression varied following TBI and peaked on the third day which expressed approximate fourfold increase compared with sham group, with the protein concentrated at the lesion boundary. SEMA3A contributed to neurological function deficits and secondary BBB damage in vivo. Our results demonstrated that SEMA3A level following OGD injury almost doubled than control group, and the negative effects of OGD injury can be improved by blocking SEMA3A expression. Furthermore, the expression of miR-30b-5p decreased approximate 40% at the third day and 60% at the seventh day post-CCI. OGD injury also exhibited an effect to approximately decrease 50% of miR-30b-5p expression. Additionally, the expression of SEMA3A post-TBI is regulated by miR-30b-5p, and miR-30b-5p could improve neurological outcomes post-TBI efficiently. Our results demonstrate that SEMA3A is a significant factor in secondary BBB damage after TBI and can be abolished by miR-30b-5p, which represents a potential therapeutic target.

MicroRNA­363­3p inhibits hepatocarcinogenesis by targeting HMGA2 and is associated with liver cancer stage.

The importance of microRNAs (miRNAs) in cancer development has been widely recognized in recent decades. In the present study, the function and mechanism of miRNA­363­3p (miR­363­3p), formerly characterized as a tumor suppressor, in the hepatocarcinogenesis of liver cancer cells was investigated. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was applied to detect the expression of miR­363­3p in liver cancer tissues. Cell proliferation, survival and migration capacities were determined by MTT, colony formation and wound­healing assays, respectively. The targeting of high mobility group AT­hook 2 (HMGA2) mRNA by miR­363­3p was confirmed by bioinformatics analysis, and RT­qPCR, luciferase reporter and western blot assays. The correlation between the expression levels of HMGA2 and miR­363­3p was analyzed. The RT­qPCR results revealed that the levels of miR­363­3p were downregulated in liver cancer tissues. Cellular assays validated that miR­363­3p exerted tumor suppressing functions, including the inhibition of cell proliferation, survival and migration abilities in two liver cancer cell lines. Bioinformatics prediction and subsequent experiments demonstrated that HMGA2 was a direct target of miR­363­3p. Restoration of the expression of HMGA2 in miR­363­3p mimic­transfected cells reversed the tumor suppressing effects caused by miR­363­3p. Finally, there was a significant negative correlation between the expression levels of HMGA2 and miR­363­3p in liver cancer tissues. miR­363­3p was identified as an important tumor suppressor in liver cancer via targeting HMGA2, which may have potential benefits in liver cancer therapy.

Comparison of Three Routine Methods for the Measurement of Serum Bilirubin in a China Laboratory.

BACKGROUND: The reliability of bilirubin measurements is not satisfactory due to the marked variability of methodologies since discovery. METHODS: Imprecision of the three analytical systems (diazo method, vanadate oxidase method, and dry chemistry method) were calculated in accordance with CLSI guidelines EP15-A2. According to Clinical and Laboratory Standards Institute (CLSI) guideline (EP9-A2), 40 analytes were detected to evaluate method performance and comparability among three routine measurements for serum bilirubin in a China laboratory. RESULTS: The within-run precisions and the intermediate imprecision of the three methods all met the CLIA'88 requirement of bilirubin measurement. All three evaluated methods were closely correlated with correlation coefficients ranging from 0.995 to 0.999. The 95% predicted bias confidence intervals between diazo method and vanadate oxidase method for total bilirubin were acceptable. However, for direct bilirubin, the 95% predicted bias confidence intervals was only acceptable at the medium medical decision point. The 95% predicted bias confidence intervals between diazo method and dry medical method for total bilirubin was all acceptable. CONCLUSIONS: All three evaluated routine methods for serum bilirubin in our lab were closely correlated. For measurements of total and direct bilirubin, both dry medical method and vanadate oxidase method had significant differences compared with the diazo method. Further studies are required to reduce the biases among these methods.

miR-145-5p Increases Osteoclast Numbers In Vitro and Aggravates Bone Erosion in Collagen-Induced Arthritis by Targeting Osteoprotegerin.

BACKGROUND Osteoprotegerin (OPG) inhibits bone resorption and binds with strong affinity to receptor activator of NF κB ligand (RANKL), thereby preventing RANKL from binding to its receptor RANK. Osteoclasts have documented effects on bone erosion of rheumatoid arthritis (RA). The aim of this study was to examine the role of miR-145-5p in the regulation of RA osteoclast differentiation and bone erosion. MATERIAL AND METHODS Expression of microRNA-145-5p in human peripheral blood mononuclear cells (PBMC) and synovial tissue was assayed by real-time polymerase chain reaction (RT-PCR). OPG, RANK, and RANKL expression in RAW-264.7 cells was examined by RT-PCR and Western blot analysis. Osteoclast formation was detected by tartrate-resistant acid phosphatase (TRAP) staining. The effect of miR-145-5p on predicted target mRNAs was examined by luciferase reporter assays. Collagen-induced arthritis (CIA) was induced by injecting DBA/1 mice with bovine type II collagen (CII), and miR-145-5p agomir was administered by intravenous injection. Morphological changes in the CIA joint were assessed by micro-computed tomography (CT) and histopathology. RESULTS miR-145-5p levels significantly increased in RA PBMC and synovial tissue compared with normal PBMC and osteoarthritis (OA) tissue. After transfection of RAW-264.7 cells with miR-145-5p, RANK and RANKL expression increased significantly, while OPG expression decreased significantly. TRAP staining results showed osteoclast numbers increased. Micro-CT analysis of the arthritic joints showed that the miR-145-5p agomir caused bone erosion in mice, and histopathological analysis revealed that miR-145-5p agomir aggravates cartilage erosion. CONCLUSIONS Our findings indicate that administration of miR-145-5p aggravates joint erosion in CIA mice. This suggests that miR-145-5p is a potential target for the treatment of RA.

PVT1-derived miR-1207-5p promotes breast cancer cell growth by targeting STAT6.

Accumulating evidence indicates that ectopic expression of non-coding RNAs are responsible for breast cancer progression. Increased non-coding RNA PVT1, the host gene of microRNA-1207-5p (miR-1207-5p), has been associated with breast cancer proliferation. However, how PVT1 functions in breast cancer is still not clear. In this study, we show a PVT1-derived microRNA, miR-1207-5p, that promotes the proliferation of breast cancer cells by directly regulating STAT6. We first confirm the positive correlated expression pattern between PVT1 and miR-1207-5p by observing consistent induced expression by estrogen, and overexpression in breast cancer cell lines and breast cancer patient specimens. Moreover, silence of PVT1 also decreased miR-1207-5p expression. Furthermore, increased miR-1207-5p expression promoted, while decreased miR-1207-5p expression suppressed, cell proliferation, colony formation, and cell cycle progression in breast cancer cell lines. Mechanistically, a novel target of miR-1207-5p, STAT6, was identified by a luciferase reporter assay. Overexpression of miR-1207-5p decreased the levels of STAT6, which activated CDKN1A and CDKN1B to regulate the cell cycle. We also confirmed the reverse correlation of miR-1207-5p and STAT6 expression levels in breast cancer samples. Therefore, our findings reveal that PVT1-derived miR-1207-5p promotes the proliferation of breast cancer cells by targeting STAT6, which in turn controls CDKN1A and CDKN1B expression. These findings suggest miR-1207-5p might be a potential target for breast cancer therapy.

Albuminuria is Suggested as a Potential Health Screening Biomarker for Senior Citizens and General Population with Hypertension or Diabetes in China.

BACKGROUND: To evaluate the possibility of albuminuria as a screening biomarker for seniors or general population with hypertension and diabetes. METHODS: 478 health check-up individuals were enrolled. Albumin to creatinine ratio (ACR) was calculated by testing urinary albumin and creatinine of spot urine sample. Each urine sample was also analyzed by the routine urine test in parallel with ACR. Potential risk factors associated with the presence of albuminuria were analyzed using independent t-test or chi-square test. RESULTS: The total prevalence of albuminuria was 11.9%, and women had a higher positive rate (14.0%) than men (10.6%). Ageing, fasting plasma glucose, and systolic blood pressure were significantly associated with the presence of albuminuria. Individuals are highly probable to have albuminuria when their routine urine test shows positive of urea glucose, red blood cells or urea protein. CONCLUSIONS: Albuminuria should be suggested as a potential health screening biomarker in senior citizens and general population with hypertension and diabetes.

Luminex xMAP combined with Western blot improves HIV diagnostic sensitivity.

Currently, Western blot is used to confirm the initial serodiagnosis of HIV infection by antibody detection. However, a major deficiency of the Western blot relates to a lack of sufficient sensitivity in detecting HIV antibodies. This report describes a simple, sensitive and inexpensive bead-based assay for detection of early HIV infection. A panel of 138 positive specimens including 105 blood donors and 33 MSM with known Western blot results were evaluated using Luminex xMAP at Tianjin Centers for Disease Control and Prevention (CDC). We demonstrate a superior sensitivity of Luminex xMAP compared with Western blot. Of the 87 confirmed HIV positive blood donors, Western blot only confirmed 65 cases with 74.7% (65/87) sensitivity while Luminex xMAP identified 72 cases with 82.8% (72/87) sensitivity (p<0.05). Western blot and Luminex xMAP verified 13 and 19 of 33 MSM specimens, respectively. The sensitivity was 39.4% (13/33) for Western blot and 57.6% (19/33) for Luminex xMAP (p<0.1). Luminex xMAP combined with Western blot improves the diagnostic sensitivity of HIV infection at an early stage, and reduces the chances of missed diagnosis.

[Construction and expression of the recombinant plasmid containing BddhFVIII in HepG2 cells].

OBJECTIVE: To get stable cell line expressing B domain-deleted human FVIII (BDDhFVIII) by constructing the eukaryotic expression plasmid. METHODS: Eukaryotic expression plasmid containing BDDhFVIII was constructed and transfected into HepG2 cells via electroporation. The expression and purification of the target protein was detected by Western blot. RESULTS: Results of enzyme digestion and sequence analysis demonstrated that the gene of BDDhFVIII was correctly inserted into the eukaryotic expression vector pcDNA4/v5-his. Western blot confirmed the successful expression of BDDhFVIII at the protein levels in HepG2 cells. CONCLUSION: The constructed eukaryotic expression vector was able to generate high level expression of human FVIII in HepG2 cells, thus could construct human blood coagulation FVIII stable cell line successfully.

Monitoring of CD95 and CD38 expression in peripheral blood T lymphocytes during active human cytomegalovirus infection after orthotopic liver transplantation.

AIM: The aim of the present study was to quantitatively monitor the response of CD95 molecules expressed on CD3(+) T cells (CD95(+)CD3(+) cells) and CD38 molecules expressed on CD8(+) T cells (CD38(+)CD8(+) cells) to ganciclovir treatment after orthotopic liver transplant (OLT) in recipients with active human cytomegalovirus (HCMV) infection. METHODS: Blood samples were collected from 20 liver transplanted recipients with active HCMV infection and 24 recipients without HCMV infection. CD95(+)CD3(+) cells and CD38(+)CD8(+) cells were quantitatively detected with QuantiBRITE bead methods by dual-color flow cytometry analysis during the post-transplantation period. RESULTS: CD95(+)CD3(+) cells and CD38(+)CD8(+) cells were not significantly different among different ages of healthy adults (P > 0.05). CD95(+)CD3(+) cells and CD38(+)CD8(+) cells were drastically increased in the active HCMV infection group compared with that in the stable group or in the healthy group (P < 0.001), and then they were gradually decreased within the next several weeks after ganciclovir treatment when compared with active HCMV infection recipients (P < 0.001). CONCLUSIONS: The present study showed that CD38(+)CD8(+) T cells can be an appropriate immunological marker for early detection and antiviral therapeutic monitoring of HCMV infection. The evaluation of CD95 molecule levels may be used routinely in clinical practice to assess the level of immunosuppression.

Cytotoxicity of HIV-gp41 segments expressed inE. coli.

The failed attempt to express HIV-gp41 inE. coli led to the investigation of HIV-gp41 segments, which is responsible for the toxicity toE. coli cells. A series of deletion mutants containing different regions ofgp41 gene were constructed and expressed inE. coli BL21(DE3) strain. After IPTG induction, the high mortality of host bacteria was observed in host bacteria carrying the deletion mutants ofgp41 gene except for those transformed with pET-HN2; coordinately, the mRNA transcripts of thegp41 was rapidly decreased; and the release of [3H]uridine increased upon induction. All these data suggested that GP41 protein has a cytotoxic effect onE. coli, and it is the cytotoxicity of thegp41 gene product that contributes to the high mortality when expressed inE. coli.