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Background: ISO 15189:2012 is widely recommended for clinical laboratories. However, it has not been paid enough attention in medical laboratory education and experiments in clinical biochemistry courses. Methods/Approach: The teaching program of clinical biochemistry aims to improve students’ awareness and ability of quality management according to ISO 15189 technical requirements. It is a 4-stage experimental training program to teaching skills: "establish an overall testing process based on the patient's clinical indicator, clarifying principles, improve operational skills, review process and continuous improvement". The program was implemented in our college during the winter semesters of 2019 and 2020. 185 students participate in the program as an experimental group, other 172 students as control group adopt the conventional method. Participants completed an online survey after each class to evaluate the class. Results/Outcomes: The test group had significantly better examination scores not only in experimental operational skills (89.27±7.16 vs. 77.51±4.72, pConclusions: A 4-stage experimental training program based on ISO 15189 technical requirements combined with CBL in clinical biochemistry is an effective and acceptable teaching strategy for the conventional training program.

Thrombospondin-1 (THBS1) derived from platelets and acted as a critical mediator of hemostasis promoting platelet activation in thrombus formation. The biological connection of genetic variants and mRNA expression ofTo evaluate the association of single nucleotide polymorphisms (SNPs) and mRNA expression ofA case-control study consisted of 4,584 IS patients recruited from five hospitals in Jiangsu, China, and 4,663 age-gender-matched controls free of IS. A cohort study enrolled 4,098 participants free of stroke and lasted from 2009 to 2022. Early collected 3158 IS patients aged between 35 and 80 years were followed up an average of 5.86-year to follow up their long-term death outcomes. Two tagSNPs of theThere is no significant difference in genotype and haplotype frequencies of rs2236741 and rs3743125 between IS cases and controls (allTherefore, our study suggested that there is no significant association of

Despite initial response to androgen signaling therapy, most prostate cancer (PCa) patients eventually relapse and remain incurable. ADAR1-mediated A-to-G editing plays oncogenic roles in various tumors. However, the specific function of ADAR1 and the global RNA edited targets governing PCa progression remain underexplored. Here, we demonstrate that highly expressed ADAR1 as a crucial oncogenic target in PCa, and develop a novel small-molecule ADAR1 inhibitor ZYS-1 with significant anti-tumor efficacy and favorable safety profile. Either depletion or pharmacological inhibition of ADAR1 dramatically suppress PCa growth, inhibit metastasis, and potentiate immune response. We further reveal that the translation of MTDH is repressed by ADAR1 in an editing-dependent manner, which drives cell proliferation and invasion in PCa. Collectively, these results shed light on ADAR1 as a novel druggable target in PCa therapy and highlight the widespread applicability of ADAR1 inhibitors for a broad spectrum of malignancies.

Anti-nuclear antibodies to speckled 100 kDa (sp100) and glycoprotein 210 (gp210) are specific serologic markers of primary biliary cholangitis (PBC) of uncertain/controversial clinical or prognostic significance. To study the genetic determinants associated with sp100 and gp210 autoantibody subphenotypes, we performed a genome-wide association analysis of 930 PBC cases based on their autoantibody status, followed by a replication study in 1,252 PBC cases. We confirmed single-nucleotide polymorphisms rs492899 (P = 3.27 × 10-22 ; odds ratio [OR], 2.90; 95% confidence interval [CI], 2.34-3.66) and rs1794280 (P = 5.78 × 10-28 ; OR, 3.89; 95% CI, 3.05-4.96) in the human major histocompatibility complex (MHC) region associated with the sp100 autoantibody. However, no genetic variant was identified as being associated with the gp210 autoantibody. To further define specific classical human leukocyte antigen (HLA) alleles or amino acids associated with the sp100 autoantibody, we imputed 922 PBC cases (211 anti-sp100-positive versus 711 negative cases) using a Han Chinese MHC reference database. Conditional analysis identified that HLA-DRβ1-Asn77/Arg74, DRβ1-Ser37, and DPβ1-Lys65 were major determinants for sp100 production. For the classical HLA alleles, the strongest association was with DRB1*03:01 (P = 1.51 × 10-9 ; OR, 2.97; 95% CI, 2.06-4.29). Regression analysis with classical HLA alleles identified DRB1*03:01, DRB1*15:01, DRB1*01, and DPB1*03:01 alleles can explain most of the HLA association with sp100 autoantibody. Conclusion: This study indicated significant genetic predisposition to the sp100 autoantibody, but not the gp210 autoantibody, subphenotype in PBC patients. Additional studies will be necessary to determine if these findings have clinical significance to PBC pathogenesis and/or therapeutics.

Prostate cancer (PCa) is one of the most prevalent cancers in men worldwide, and hormonal therapy plays a key role in the treatment of PCa. However, the inevitable drug resistance of hormonal therapy makes it urgent and necessary to identify novel targets for PCa treatment. Herein, dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) was found and confirmed to be highly expressed in the PCa tissues and cells, and knock-down of DYRK2 remarkably reduces PCa burden in vitro and in vivo. On the base of DYRK2 acting as a promising target, we further discovered a highly selective DYRK2 inhibitor YK-2-69 and solved its co-crystal structure. Especially, YK-2-69 displayed great selectivity over 370 kinases and exhibited more potent anti-PCa efficacy than the first-line drug enzalutamide in vivo. Meanwhile, YK-2-69 displayed excellent safety properties with maximal tolerable dose of more than 10,000 mg/kg and great pharmacokinetic profiles with 55.78% bioavailability. In summary, we identified DYRK2 as a novel drug target and verified its critical roles in PCa. Meanwhile, we discovered a highly selective DYRK2 inhibitor with favorable druggability for the treatment of PCa.

In the current blockchain system, the transaction costs and network congestion are increasing, thus the transaction rate is lower, which limits the wide application of blockchain system. In order to address this problem, this paper proposes a secure large-scale multi-party payment (SLMP) system. When our SLMP is applied to the current blockchain system, its capacity is expanding, thus the forementioned problem is solved. The SLMP employs an off-chain payment channel and aggregating multi-signatures with public-key aggreation (AMSP). The security of the proposed system is similar to Bitcoin network. And the proposed system can support large-scale off-chain transactions, and the vendor can conduct aggregate signature on all final payment transactions of payers before the specified time. This modification allow nodes (miner) to validate all transactions at once, thus SLMP is efficient, we also give security proof of our SLMP. Moreover, SLMP system allows offline and off-chain transactions, which means our system is flexible in remote or geographically disastrous areas where communications infrastructure is inadequate or damaged.

With the rapid development of the vehicular ad hoc networks (VANETs), vehicle services are increasingly diversified. it will inevitably lead to authentication across data centers, privacy and security are crucial issues due to the unique characteristics of VANETs. The traditional cross-datacenter authentication mechanisms are mostly not anonymous or rely on a completely trusted third party (TTP) to guarantee the conditional anonymity of the user while TTP can easily obtain users' privacy. In this article, we introduce an lightweight anonymous authentication mechanism based on the combination of ring signature and blockchain technologies for VANETs. Our mechanism can achieve the following advantages: 1) achieving condition anonymity without TTP; 2) realizing a flexible anonymous authentication, in which a vehicle can decides whether to reauthentication or increases the intensity of anonymous according to the situation; 3)our mechanism is lightweight by recording signed pseudonym on blockchain to reduce the time consumption of anonymous authentication in different data centers, user only prove that pseudonym is recorded on the blockchain rather execute ring signature again; Our mechanism achieves these advantages by leveraging conditionally anonymous ring signature and extended blockchain technologies. These security features are demonstrated by carrying out security analysis. Meanwhile, extensive simulations are conducted to validate the efficiency and practicality of our mechanism.

There is an urgent need for new antibiotics and alternative strategies to combat bacterial pathogens. Molecular docking, antibacterial evaluation in vitro and in vivo, cytotoxicity assessment and enzyme inhibition analyses were performed. Compound 12 exhibited antimicrobial activity against Staphylococcus aureus (MIC: 4 μg/ml), various clinically isolated strains of MRSA (MIC: 4–16 μg/ml) and Acinetobacter baumannii (MIC: 4 μg/ml) when combined with subinhibitory concentrations of colistin B. Compound 12 (20 mg/kg) yielded mild improvement in survival of methicillin-resistant Staphylococcus aureus (MRSA)-infected mice. Additionally, enzyme inhibition tests showed that compound 12 exhibited inhibitory effects against S. aureus dihydrofolate reductase (105.1 μg/ml) and DNA gyrase (122.8 μg/ml). Compound 12 is a promising antibacterial candidate for further development.

The detection of autoantibody to glycoprotein 210 (gp210 Ab) against a 15 amino-acid peptide epitope by enzyme-linked immunosorbent assay (ELISA) has been widely used in the diagnosis of primary biliary cholangitis (PBC). However, this small peptide antigen presents spatial limitations for antibody access, which reduces the sensitivity of autoantibody detection. A recombinant gp210 antigen was constructed for increased sensitivity in antibody detection is described here.The gp210 C terminal 18 amino acid coding sequence was ligated to the modified C-terminal 108 amino acid coding sequence of human serum albumin (mHSA108) and produced as a recombinant gp210 antigen mHSA108-gp210-C18. Measurements of gp210 Ab using the gp210 C-terminal 25 amino acid peptide (gp210-C25) and mHSA108-gp210-C18 by in-house ELISA were compared. ELISAs with mHSA108-gp210-C18 and commercial INOVA kit for gp210 Ab detection were also compared in PBC patients and healthy controls. The correlation between the two assays was analyzed and their efficiency in diagnosing was compared.Of 86 PBC samples, 35 (40.70%) and 44 (52.33%) positive samples were detected for anti-gp210 Ab using gp210-C25 and mHSA108-gp210-C18, respectively. Of 252 samples from PBC, 114 (45.24%) were positive for mHSA108-gp210-C18 ELISA whereas 94 (37.3%) for commercial ELISA (INOVA). All positive samples detected with commercial ELISA kit were also tested positive in mHSA108-gp210-C18 ELISA. Among 374 patients with other autoimmune diseases, anti-gp210 Ab were detected by mHSA108-gp210-C18 ELISA in 0.95% systemic lupus erythematosus (SLE) patients (2/210), 13.04% rheumatoid arthritis (RA) patients (13/97), and 1.47% of Sjögren's Syndrome (SS) patients (1/67).Compared to the gp210 peptide antigen, the sensitivity of the ELISA system using mHSA108-gp210-C18 antigen was improved. The novel gp210 antigen could be useful for screening patients known to be at increased risk of developing PBC.

The genetic association of primary biliary cholangitis with major histocompatibility complex (MHC) has been widely confirmed among different ethnicities. To map specific MHC region variants associated with PBC in a Han Chinese cohort, we imputed HLA antigens and amino acids (AA) in 1126 PBC cases and 1770 healthy control subjects using a Han-MHC reference database. We demonstrate that HLA-DRB1 and/or HLA-DQB1 contributed the strongest signals, and that HLA-DPB1 was a separate independent locus. Regression analyses with classical HLA alleles indicate that HLA-DQB1*03:01 or HLA-DQβ1-Pro55, HLA-DPB1*17:01 or HLA-DPβ1-Asp84 and HLA-DRB1*08:03 could largely explain MHC association with PBC. Forward stepwise regression analyses with HLA amino acid variants localize the major signals to HLA-DRβ1-Ala74, HLA-DQβ1-Pro55 and HLA-DPβ1-Asp84. Electrostatic potential calculations implicated AA variations at HLA-DQβ1 position 55 and HLA-DPβ1 position 84 as critical to peptide binding properties. Furthermore, although several critical Han Chinese AA variants differed from those shown in European populations, the predicted effects on antigen binding are likely to be very similar or identical and underlie the major component of MHC association with PBC.

Table S4. Sequences of siRNA against specific target in this study (DOC 32 kb)

Figure S1. CeRNET_CC promotes the stemness of MCF-7 cells in vitro. (A) The expression of CYP4Z2P and CYP4Z1 in MCF-7 and MCF-7-tumorsphere cells was detected by qRT-PCR. (B and C) The infection efficiency of MCF-7 cells with CYP4Z1- or CYP4Z2P-3′UTR stable overexpression (B) or knockdown (C) was detected by qRT-PCR. (D) Phase contrast images of mammospheres formed by stable expression cells depicted in B and C and quantification of spheres. (E) Representative FACS profile of cells described in B with CD24− and CD44+ markers. (F and G) The mRNA and protein expression of stemness markers (ALDH1, SOX2, OCT4 and Nanog) in cells described in B and C were examined by qRT-PCR and western blot analysis, respectively. The data are presented as the means ± SDs, n = 3, *P

Table S6. Sequences of primers used for Luciferase reporter assay. (DOC 46 kb)

Table S2. Primary antibodies used in this study. (DOC 35 kb)

Figure S4. The correlation between six2 expression and the survival of breast cancer patients. (A) KM-plotter survival curves showed the disease-free survival probability of patients separated into low and high six2 levels. (B) KM-plotter survival curves showed the OS survival probability of patients separated into low and high six2 level. (C and F) KM-plotter survival curves showed the RFS probability of patients separated into low and high six2 levels ( http://hgserver1.amc.nl/cgi-bin/r2/main.cgi ). (PDF 576 kb)

Table S5. Sequences of primers used for plasmid constructions. (DOC 40 kb)

Table S3. Sequences of primers used for ChIP qRT-PCR in this study. (DOCX 16 kb)

Figure S3. Transcriptional factor six2 promotes the expression of CYP4Z1 and the pseudogene CYP4Z2P. (A) Computational analysis of the CYP4Z2P and CYP4Z1 promoters showed potential binding sites for six2. (B and C) Fragments of the CYP4Z2P and CYP4Z1 promoters were cloned into the luciferase reporter vector pGL3. MCF-7 cells were co-transfected with six2 and the luciferase constructs or the control construct. 72 h later, luciferase activity was measured. (D) Relative luciferase activity was detected in MCF-7 cells co-transfected with the six2 overexpression vector and CYP4Z1 and CYP4Z2P promoter vectors with six2 binding sites or mutated six2 binding sites. (E and F) The expression of CYP4Z2P, CYP4Z1, and six2 in MCF-7-six2 (E) and 231-six2 (F) cells was examined by qRT-PCR. (G and H) CYP4Z1 protein expression in MCF-7-six2 (G) and MCF7-Plko-six2 (H) cells was detected by western blot. The data were presented as the means ± SDs, n = 3, **P

Background The expression of CYP4Z1 and the pseudogene CYP4Z2P has been shown to be specifically increased in breast cancer by our group and others. Additionally, we previously revealed the roles of the competitive endogenous RNA (ceRNA) network mediated by these genes (ceRNET_CC) in breast cancer angiogenesis, apoptosis, and tamoxifen resistance. However, the roles of ceRNET_CC in regulating the stemness of breast cancer cells and the mechanisms through which ceRNET_CC is regulated remain unclear. Methods Transcriptional factor six2, CYP4Z1-3′UTR, and CYP4Z2P-3′UTR were stably overexpressed or knocked down in breast cancer cells via lentivirus infection. ChIP-sequencing and RNA-sequencing analysis were performed to reveal the mechanism through which ceRNET_CC is regulated and the transcriptome change mediated by ceRNET_CC. Clinical samples were used to validate the correlation between six2 and ceRNET_CC. Finally, the effects of the six2/ceRNET_CC axis on the stemness of breast cancer cells and chemotherapy sensitivity were evaluated by in vitro and in vivo experiments. Results We revealed that ceRNET_CC promoted the stemness of breast cancer cells. Mechanistically, six2 activated ceRNET_CC by directly binding to their promoters, thus activating the downstream PI3K/Akt and ERK1/2 pathways. Finally, we demonstrated that the six2/ceRNET_CC axis was involved in chemoresistance. Conclusions Our results uncover the mechanism through which ceRNET_CC is regulated, identify novel roles for the six2/ceRNET_CC axis in regulating the stemness of breast cancer cells, and propose the possibility of targeting the six2/ceRNET_CC axis to inhibit breast cancer stem cell (CSC) traits.

The original article [1] contained an error in Fig. 7c whereby the same flow image was accidentally misused for the second and fourth group.

A variety of molecular pathways, such as generation of advanced glycation end products, inflammation, and oxidative stress, are involved in the development of diabetic nephropathy (DN). Recently, a protective effect of omega-3 polyunsaturated fatty acids on the kidney has been reported. This study aimed to determine serum docosahexaenoic acid (DHA) level and its association with inflammation factors in patients with DN.One hundred patients with type 2 diabetic mellitus were divided into 3 groups of non-DN, early DN, and clinical DN, based on 24-hour urinary albumin levels. Hemoglobin A1c, biochemical indicators, β2-microglobulin, and 24-hour urine albumin levels were assessed. Enzyme linked immunosorbent assay was applied to determine the serum concentrations of DHA, advanced glycation end products, fractalkine, superoxide dismutase, and tumor necrosis factor-α.Lower serum DHA and superoxide dismutase and higher serum β2-microglobulin and 24-hour urine albumin levels were associated with clinical DN, compared to no DN and early DN. The reductions in serum DHA levels were different among the patients with early and clinical DN, stratified by sex, body mass index, and serum lipid levels. Serum DHA significantly correlated positively with superoxide dismutase and negatively with fractalkine and tumor necrosis factor-α in the patients with DN.Docosahexaenoic acid may suppress the expression and secretion of fractalkine through inhibiting the tumor necrosis factor-α signaling pathway in DN patients, which improves inflammation and oxidative stress of the kidney, and in turn, delaying the development of DN.

High-sensitivity C-reactive protein (hsCRP) is an inflammatory marker for the prediction and prognosis of ischemic stroke but there is an absence of evidence for cerebral hemorrhagic events. The aim of this study is to investigate the effects of elevated plasma hsCRP and CRP genetic variants on hemorrhagic stroke (HS). Two hundred thirty-six inpatients with HS and 993 age-matched controls from a community-based population were included in a case-control study and four tagging single nucleotide polymorphsims (tagSNPs) at CRP were genotyped. The association of hsCRP elevation and CRP variants with HS was evaluated by multiple logistic regression. HS cases had a higher median (interquartile) of hsCRP with 5.40 (1.30-10.7) mg/L and a proportion of hsCRP elevation (≥3 mg/L, 63.4%) than controls [1.20 (0.80-2.20) mg/L, 16.6%], respectively (P 0.05 for all). No significant difference of genotype or allele frequency of the four SNPs was observed between HS patients and controls (P 0.05). Further stratified analysis by gender showed that the variants of rs3093059 (T/C) and rs3091244 (C/T/A) were significantly associated with the decreased risk of HS in men and odds ratios (ORs) and 95% confidence intervals (95% CIs) for additive models were 0.515 (0.294-0.903) and 0.578 (0.349-0.96), respectively, after adjusting for covariates. In HS patients, rs3091244 was positively associated with the hsCRP elevation and rs2794521 was negatively associated with hsCRP elevation (P 0.05). Our findings suggest that hsCRP elevation is associated with the risk of HS and CRP contributes genetic susceptibility to HS in men as well as hsCRP elevation in HS.

Primary biliary cholangitis (PBC) is an autoimmune liver disease with a strong hereditary component. Here, we report a genome-wide association study that included 1,122 PBC cases and 4,036 controls of Han Chinese descent, with subsequent replication in a separate cohort of 907 PBC cases and 2,127 controls. Our results show genome-wide association of 14 PBC risk loci including previously identified 6p21 (HLA-DRA and DPB1), 17q12 (ORMDL3), 3q13.33 (CD80), 2q32.3 (STAT1/STAT4), 3q25.33 (IL12A), 4q24 (NF-κB) and 22q13.1 (RPL3/SYNGR1). We also identified variants in IL21, IL21R, CD28/CTLA4/ICOS, CD58, ARID3A and IL16 as novel PBC risk loci. These new findings and histochemical studies showing enhanced expression of IL21 and IL21R in PBC livers (particularly in the hepatic portal tracks) support a disease mechanism in which the deregulation of the IL21 signalling pathway, in addition to CD4 T-cell activation and T-cell co-stimulation are critical components in the development of PBC., Primary biliary cholangitis is an autoimmune liver disease. Here, the authors show that variants in interleukin genes which potentially deregulate their expression are associated with this condition, and suggest that the IL21 signalling pathway may have a role in disease aetiology.

Although the RAG2 core domain is the minimal region required for V(D)J recombination, the noncore region also plays important roles in the regulation of recombination, and mutations in this region are often related to severe combined immunodeficiency. A complete understanding of the functions of the RAG2 noncore region and the potential contributions of its individual residues has not yet been achieved. Here, we show that the zinc finger motif within the noncore region of RAG2 is indispensable for maintaining the stability of the RAG2 protein. The zinc finger motif in the noncore region of RAG2 is highly conserved from zebrafish to humans. Knock-in mice carrying a zinc finger mutation (C478Y) exhibit decreased V(D)J recombination efficiency and serious impairment in T/B-cell development due to RAG2 instability. Further studies also reveal the importance of the zinc finger motif for RAG2 stability. Moreover, mice harboring a RAG2 noncore region mutation (N474S), which is located near C478 but is not zinc-binding, exhibit no impairment in either RAG2 stability or T/B-cell development. Taken together, our findings contribute to defining critical functions of the RAG2 zinc finger motif and provide insights into the relationships between the mutations within this motif and immunodeficiency diseases.

High-sensitivity C-reactive protein (hsCRP) was reported as a strong, independent predictor of future myocardial infarction and stroke. It is of importance to illustrate the conformance of CRP genetic variation, increment of plasma hsCRP and cerebral events. A case-control study including 548 patients with acute ischemic stroke and 993 age-matched controls from community-based population was conducted and four tagging SNPs (tagSNPs) were genotyped. Multiple logistic regression was applied to evaluate the association of CRP gene and stroke hsCRP elevation with adjustment for covariates. The results indicated that rs3093059 and rs3091244 presented statistical associations with ischemic stroke. Odds ratios (ORs) (95 % confidence interval [CI]) of additive model, dominant model and minor allele at rs3093059 were 0.697 (0.528-0.921), 0.671 (0.487-0.923) and 0.811 (0.666-0.988), and ORs (95 % CI) of dominant model at rs3091244 was 0.728 (0.536-0.988), after adjusting for covariates. But there were no significant differences of genotype or allele frequencies of the four SNPs observed between hypertension (HT) and normal blood pressure (NBP) groups. Further analyses indicated the genetic variations of rs876537 and rs3093059 were positively associated with increased square root transformed hsCRP and hsCRP elevation (≥3 mg/l) in ischemic stroke patients, and rs876537 and rs3091244 were associated with hsCRP elevation in controls as well. Our finding suggests that the CRP genetic polymorphisms were associated with decreased risk of ischemic stroke and elevated plasma hsCRP and further replication study and functional research would be warranted.