PD-L1 siRNA hitched polyethyleneimine-elastase constituting nanovesicle induces tumor immunogenicity and PD-L1 silencing for synergistic antitumor immunotherapy.

BACKGROUND: PD-1/PD-L1 blockade has become a powerful method to treat malignant tumors. However, a large proportion of patients still do not benefit from this treatment, due to low tumor immunogenicity and low tumor penetration of the agents. Recently, neutrophil elastase has been shown to induce robust tumor immunogenicity, while the insufficient enzyme activity at the tumor site restricted its anti-tumor application. Here, we designed polyethyleneimine-modified neutrophil elastase (PEI-elastase) loaded with PD-L1small interfering RNA (PD-L1 siRNA) for improving enzymatic activity and delivering siRNA to tumor, which was expected to solve the above-mentioned problems. RESULTS: We first demonstrated that PEI-elastase possessed high enzymatic activity, which was also identified as an excellent gene-delivery material. Then, we synthesized anti-tumor lipopolymer (P-E/S Lip) by encapsulating PEI-elastase and PD-L1siRNA with pH-responsive anionic liposomes. The P-E/S Lip could be rapidly cleaved in tumor acidic environment, leading to exposure of the PEI-elastase/PD-L1 siRNA. Consequently, PEI-elastase induced powerful tumor immunogenicity upon direct tumor killing with minimal toxicity to normal cells. In parallel, PEI-elastase delivered PD-L1siRNA into the tumor and reduced PD-L1 expression. Orthotopic tumor administration of P-E/S Lip not only attenuated primary tumor growth, but also produced systemic anti-tumor immune response to inhibit growth of distant tumors and metastasis. Moreover, intravenous administration of P-E/S Lip into mice bearing subcutaneous tumors leaded to an effective inhibition of established B16-F10 tumor and 4T1 tumor, with histological analyses indicating an absence of detectable toxicity. CONCLUSIONS: In our study, a protease-based nanoplatform was used to cooperatively provoke robust tumor immunogenicity and down-regulate PD-L1 expression, which exhibited great potential as a combination therapy for precisely treating solid tumors.

Contribution of genetic polymorphism in ABCB1 to individual variations of imatinib plasma levels in patients with gastrointestinal stromal tumor.

Background: Imatinib mesylate (IM) is a first-line treatment option for the majority of patients diagnosed with gastrointestinal stromal tumors (GISTs). Although the clinical benefit is high, interindividual response is variable. This study thus aimed to assess how genetic polymorphisms can affect the blood levels of IM and treatment outcomes in patients with GIST. Methods: A total of 31 single-nucleotide polymorphisms (SNPs) in selected cytochrome P450 (P450), ATP-binding cassette transporter (ABC), solute carrier family (SLC), interleukin-4 receptor (IL4R), and vascular endothelial growth factor (VEGF) genes were genotyped using an SNP mass array platform. A total of 192 consecutive patients with GIST who received 400 mg of IM daily were enrolled into the study, with 1,485 blood samples being analyzed. According to genotypes, IM trough concentrations were tested and compared. Progression-free survival (PFS) and overall survival (OS) were also assessed. Results: With a mean follow-up of 75.99 months, trough concentrations of imatinib were examined at average time points of 7.73 for each patient. Polymorphism in ABCB1 rs1045642 was found to be associated with steady-state IM trough plasma levels (P=0.008). Patients with the C genotype (CT + CC) of rs1045642 exhibited higher IM trough concentrations (1,271.09±306.69 ng/mL) compared to those with the TT genotype (1,106.60±206.05 ng/mL). No statistically significant differences in IM plasma concentration were observed for the other SNPs tested. None of the tested SNPs displayed a significant association with patients' survival in this study. Conclusions: This is the largest cohort study evaluating the associations of SNP and imatinib blood trough levels. The ABCB1 rs1045642 genetic polymorphism may exert an effect on the pharmacokinetics of imatinib. The presence of the C allele in ABCB1 rs1045642 is predictive of a higher plasma concentration of IM.

The Prophylactic Protection of Salmonella Typhimurium Infection by Lentilactobacillus buchneri GX0328-6 in Mice.

Salmonellosis is a disease caused by non-typhoid Salmonella, and although some lactic acid bacteria strains have been shown previously to relieve Salmonellosis symptoms, little has been studied about the preventive mechanism of Lentilactobacillus buchneri (L. buchneri) against Salmonella infection in vivo. Therefore, the L. buchneri was fed to C57BL/6 mice for 10 days to build a protective system of mice to study its prevention and possible mechanisms. The results showed that L. buchneri GX0328-6 alleviated symptoms caused by Salmonella typhimurium infection among C57BL/6 mice, including low survival rate, weight loss, increase in immune organ index and hepatosplenomegaly, and modulated serum immunoglobulin levels and intrinsic immunity. Importantly, the L. buchneri GX0328-6 enhanced the mucosal barrier of the mouse jejunum by upregulating the expression of tight junction proteins such as ZO-1, occludins, and claudins-4 and improved absorptive capacity by increasing the length of mouse jejunal villus and the ratio of villus length to crypt depth and decreasing the crypt depth. L. buchneri GX0328-6 reduced the intestinal proliferation and invasion of Salmonella typhimurium by modulating the expression of antimicrobial peptides in the intestinal tract of mice, and reduced intestinal inflammation and systemic spread in mice by downregulating the expression of IL-6 and promoting the expression of IL-10. Furthermore, L. buchneri GX0328-6 increased the relative abundance of beneficial bacteria colonies and decreased the relative abundance of harmful bacteria in the cecum microflora by modulating the microflora in the cecum contents.

Mesomycoplasma ovipneumoniae from goats with respiratory infection: pathogenic characteristics, population structure, and genomic features.

BACKGROUND: Mycoplasma ovipneumoniae is a critical pathogen that causes respiratory diseases that threaten Caprini health and cause economic damage. A genome-wide study of M. ovipneumoniae will help understand the pathogenic characteristics of this microorganism. RESULTS: Toxicological pathology and whole-genome sequencing of nine M. ovipneumoniae strains isolated from goats were performed using an epidemiological survey. These strains exhibited anterior ventral lung consolidation, typical of bronchopneumonia in goats. Average nucleotide identity and phylogenetic analysis based on whole-genome sequences showed that all M. ovipneumoniae strains clustered into two clades, largely in accordance with their geographical origins. The pan-genome of the 23 M. ovipneumoniae strains contained 5,596 genes, including 385 core, 210 soft core, and 5,001 accessory genes. Among these genes, two protein-coding genes were annotated as cilium adhesion and eight as paralog surface adhesins when annotated to VFDB, and no antibiotic resistance-related genes were predicted. Additionally, 23 strains carried glucosidase-related genes (ycjT and group_1595) and glucosidase-related genes (atpD_2), indicating that M. ovipneumoniae possesses a wide range of glycoside hydrolase activities. CONCLUSIONS: The population structure and genomic features identified in this study will facilitate further investigations into the pathogenesis of M. ovipneumoniae and lay the foundation for the development of preventive and therapeutic methods.

Lactobacillus plantarum GX17 benefits growth performance and improves functions of intestinal barrier/intestinal flora among yellow-feathered broilers.

Lactobacillus plantarum has recently been found to be a natural source feed additive bacteria with great advantages in food safety and animal welfare. Discovering novel strains with commercial application potentiation could benefit the local poultry industry, and in particular support Chinese farmers. In this study, we tested a recently isolated novel strain of Lactobacillus plantarum GX17 as a feed additive on the growth performance and intestinal barrier functions of 1-day-old Chinese yellow-feather chicks. As good as other commercial probiotics, feeding with Lactobacillus plantarum GX17 showed significant improvements in humoral immune responses and enhanced the immune effect after vaccination for either the Newcastle disease vaccine or the avian influenza vaccine. This study also found that feeding with Lactobacillus plantarum GX17 improved the feed-to-weight ratio and caused a significant increase of the villus length to crypt depth ratio. Furthermore, Lactobacillus plantarum GX17 significantly up-regulated the mRNA expression of CLDN, MUC2, and TLR2, all of which are jejunum-associated barrier genes, indicating an improvement of the intestinal barrier functions by enhancing the tight junction between epithelia cells. These results are comparable to the effects of feeding the commercial complex probiotics that improve the expression levels of CLDN, ocludin, MUC2, TLR2, and TLR4. In terms of maintaining intestinal health, commercial complex probiotics increased the relative abundance of Parabacteroides and Romboutsia, while Lactobacillus plantarum GX17 increased the relative abundance of Pseudoflavonifractor. Our data suggest that Lactobacillus plantarum GX17 could enhance the intestinal absorption of nutrients and therefore improve the growth performance of Chinese yellow-feather chicks. In conclusion, compared with the commercial complex probiotics, Lactobacillus plantarum GX17 has more positive effects on the growth performance and intestinal barrier function of yellow-feather chickens, and can be used as a feed additive.

Isolation, Characterization and Whole Genome Analysis of an Avian Pathogenic Escherichia coli Phage vB_EcoS_GN06.

Escherichia coli (O78) is an avian pathogenic Escherichia coli (APEC). It can cause perihepatitis, pericarditis, septicemia and even systemic infections in the poultry industry. With the incidence of antibiotic resistance reaching a crisis point, it is important to find alternative treatments for multidrug-resistant infections. The use of phages to control pathogens is a promising therapeutic option for antibiotic replacement. In this study, we isolated a lytic phage called vB_EcoS_GN06 from sewage. It lysed APEC GXEC-N22. Transmission electron microscopy showed that the phage belongs to family Siphoviridae. Phage GN06 has a 107,237 bp linear double-stranded DNA genome with 39.2% GC content and 155 coding sequences. It belongs to the genus Tequintavirus, subfamily Markadamsvirinae. The multiplicity of infection of 0.01 and the one-step growth showed that the latent time is 60 min and the burst size is 434 PFU/cell. Temperature and pH stability tests showed that phage GN06 was stable in the range of 4 °C-60 °C and pH 5-9. GN06 showed significant inhibition of APEC both within the liquid medium and in biofilm formation. These results suggest that phage GN06 has the potential to control bacterial pathogens. Thus, GN06 has the potential to be a new potential candidate for phage therapy.

Immunological pathogenesis of Bovine E. coli infection in a model of C. elegans.

BACKGROUND: Cattle industry is critical for China's livestock industry, whereas E. coli infection and relevant diseases could lead huge economic loss. Traditional mammalian models would be costly, time consuming and complicated to study pathological changes of bovine E. coli. There is an urgent need for a simple but efficient animal model to quantitatively evaluate the pathological changes of bovine-derived E. coli in vivo. Caenorhabditis elegans (C. elegans) has a broad host range of diverse E. coli strains with advantages, including a short life cycle, a simple structure, a transparent body which is easily visualized, a well-studied genetic map, an intrinsic immune system which is conservable with more complicated mammalians. RESULTS: Here, we considered that O126 was the dominant serotype, and a total of 19 virulence factors were identified from 41 common E. coli virulence factors. Different E. coli strains with diverse pathogenicity strengths were tested in C. elegans in E. coli with higher pathogenicity (EC3/10), Nsy-1, Sek-1 and Pmk-1 of the p38 MAPK signaling pathway cascade and the expression of the antimicrobial peptides Abf-3 and Clec-60 were significantly up-regulated comparing with other groups. E. coli with lower pathogenicity (EC5/13) only activated the expression of Nsy-1 and Sek-1 genes in the p38 MAPK signaling pathway, Additionally, both groups of E. coli strains caused significant upregulation of the antimicrobial peptide Spp-1. CONCLUSION: Thirteen E. coli strains showed diverse pathogenicity in nematodes and the detection rate of virulence factors did not corresponding to the virulence in nematodes, indicating complex pathogenicity mechanisms. We approved that C. elegans is a fast and convenient detection model for pathogenic bacteria virulence examinations.

Gut Microbiota-Related Inflammation Factors as a Potential Biomarker for Diagnosing Major Depressive Disorder.

Objective: Although many works have been done, the objectively measured diagnostic biomarkers are not available. Thus, we conducted this study to identify potential biomarkers for objectively diagnosing depression and explore the role of gut microbiota in the onset of depression. Methods: Major depressive disorder (MDD) patients (n=56) and demographic data-matched healthy controls (HCs) (n=56) were included in this study. The gut microbiota in fecal samples and inflammation-related factors in serum were measured. Both univariate and multivariate statistical analyses were performed to identify the differential gut microbiota and inflammation-related factors. Results: Finally, 46 differential operational taxonomic units (OTUs) (60.9% OTUs belonging to Firmicutes) and ten differential inflammation-related factors were identified. Correlation analysis showed that there were significant correlations between 14 differential OTUs (9 OTUs belonging to Firmicutes and 5 OTUs belonging to family Lachnospiraceae under Firmicutes) and seven differential inflammation-related factors. Meanwhile, 14 differential OTUs (9 OTUs belonging to Firmicutes and 5 OTUs belonging to family Lachnospiraceae under Firmicutes) and five differential inflammation-related factors (adiponectin, apolipoprotein A1, alpha 1-antitrypsin, neutrophilicgranulocyte count/white blood cell count and basophil count) were significantly correlated to depression severity. A panel consisting of these five differential inflammation-related factors could effectively diagnose MDD patients from HCs. Conclusions: Our results suggested that Firmicutes, especially family Lachnospiraceae, might play a role in the onset of depression via affecting the inflammation levels of host, and these five differential inflammation-related factors could be potential biomarkers for objectively diagnosing MDD.

[Clinical Diagnosis and Prenatal Screening of Hb Lepore-BW Associated with IVS-II-654 Heterozygous Mutation].

OBJECTIVE: To identify one case of rare Hb Lepore-BW associated with IVS-II-654 heterozygous mutation in Sichuan area. METHODS: The blood routine examination and hemoglobin electrophoresis methods were used to analyze the blood routine parameters, HbA2 and HbF in the samples of peripheral blood in proband and his parents, as well as the cord blood of pregnant woman. The detection of thalassemia gene and Sanger sequencing methods were used to detect the hemoglobin mutations. RESULTS: The result showed that the Hb Lepore-BW heterozygous mutation was detected in the father of the proband, while a rare Hb Lepore-BW with IVS-II-654 heterozygous mutation was detected in the proband, as well as his mother and cord blood were both detected as IVS-II-654 heterozygous mutation. CONCLUSION: The study identified a rare Hb Lepore-BW with IVS-II-654 heterozygous mutation, which was characterized by intermediate ß-thalassemia. It is necessary to hemoglobin electrophoresis combined with routine blood testing in prenatal screening.

Comprehensive analysis of NGS and ARMS-PCR for detecting EGFR mutations based on 4467 cases of NSCLC patients.

BACKGROUND: By comparing the detection rate and type of targeted gene mutations in non-small cell lung cancer (NSCLC) between amplification refractory mutation system PCR (ARMS-PCR) and next-generation sequencing (NGS), the characteristics and application advantages of non-small cell lung cancer detection are explained, providing a basis for clinicians to effectively select the corresponding detection methods. METHODS AND MATERIALS: The cases of targeted genes for lung cancer were selected from the First Affiliated Hospital of Chongqing Medical University from January 2016 to October 2020. A sample of 4467 cases was selected, and they were diagnosed with NSCLC by Pathological biopsy. Sample sources include surgical resection, bronchoscope biopsy, metastatic biopsy, blood, sputum, cytology of pleural effusion. Among them, 3665 cases were detected by ARMS-PCR technique, and 802 cases were detected by NGS technology. The detection rate and type of ARMS-PCR and NGS techniques for EGFR gene mutations (including exon 18, exon 19, exon 20, exon 21 and so on) in different NSCLC samples were compared, respectively. RESULTS: The total mutation rate of EGFR gene detected by ARMS-PCR was 47.6% while 42.4% detected by NGS which indicated that there was a significant difference between the two methods in detecting total mutation of EGFR gene (P < 0.001). In different exons, the EGFR mutation rate detected by two methods is various. The mutation rate of exon 19 by ARMS-PCR detection was evidently higher than that of NGS detection, while the mutation rate of exons 20 and 21 by ARMS-PCR detection were statistically significantly lower than that of NGS detection. Moreover, the multiple mutation rate detected by NGS was 16.3% which was much higher than the 2.7% detected by ARMS-PCR with statistically different. CONCLUSION: It showed that NGS could direct the drug use for the resistant patients. However, some rare loci could be detected by NGS but the importance and directed meaning are still unknown and the number of rare mutations is rare too. Further research on new biomarkers and technique is still needed for early diagnosis, directing drug use and assessing the therapy prognosis.

Gut Microbiota-Derived Inflammation-Related Serum Metabolites as Potential Biomarkers for Major Depressive Disorder.

BACKGROUND: Although many works have been conducted to explore the biomarkers for diagnosing major depressive disorder (MDD), the widely accepted biomarkers are still not identified. Thus, the combined application of serum metabolomics and fecal microbial communities was used to identify gut microbiota-derived inflammation-related serum metabolites as potential biomarkers for MDD. METHODS: MDD patients and healthy controls (HCs) were included in this study. Both serum samples and fecal samples were collected. The liquid chromatography mass spectrometry (LC-MS) was used to detect the metabolites in serum samples, and the 16S rRNA gene sequencing was used to analyze the gut microbiota compositions in fecal samples. RESULTS: Totally, 60 MDD patients and 60 HCs were recruited. The 24 differential serum metabolites were identified, and 10 of these were inflammation-related metabolites. Three significantly affected inflammation-related pathways were identified using differential metabolites. The 17 differential genera were identified, and 14 of these genera belonged to phyla Firmicutes. Four significantly affected inflammation-related pathways were identified using differential genera. Five inflammation-related metabolites (LysoPC(16:0), deoxycholic acid, docosahexaenoic acid, taurocholic acid and LysoPC(20:0)) were identified as potential biomarkers. These potential biomarkers had significant correlations with genera belonged to phyla Firmicutes. The panel consisting of these biomarkers could effectively distinguish MDD patients from HCs with an area under the curve (AUC) of 0.95 in training set and 0.92 in testing set. CONCLUSION: These findings suggested that the disturbance of phyla Firmicutes might be involved in the onset of depression by regulating host's inflammatory response, and these potential biomarkers could be useful for future investigating the objective methods for diagnosing MDD.

Establishment and Validation of the Detection of TERT Promoter Mutations by Human Gliomas U251 Cell Lines.

Gliomas are the most common type of primary brain tumor, yet the prognosis for glioma patients remains poor. Mutations in the promoter region of the telomerase reverse transcriptase gene (TERTp) are associated with diagnosis and poor prognosis in gliomas. Here, we developed a precise and rapid Sanger sequencing assay to screen or TERTp mutations. We established the Sanger sequencing approach for the detection of TERTp mutations based on human glioma cell lines U251 and assessed the analytical validation by determining the accuracy, sensitivity, precision, and specificity. In our study, we verified the accuracy of Sanger sequencing by the real-time polymerase chain reaction method. Our data showed that TERTp mutations were detected at an analytical sensitivity of 10% per mutant. The precision and specificity validation also showed the desired results. In total, 147 glioma patients were investigated for TERTp mutations, and of each patient, clinical data and molecular characteristics were analyzed. We found that anaplastic oligodendroglioma had the highest frequency of TERTp mutations (66.7%). No differences in TERTp mutation frequency were observed between frozen tissue specimens and formalin-fixed and paraffin-embedded tissue. TERTp mutations were associated with older patients (≥45 years), whereas isocitrate dehydrogenase (IDH) mutations were inclined to a younger age (<45 years), frontal location, and pathologic stage II-III patients. IDH mutations were significantly associated with O6-methylguanine-DNA methyltransferase (MGMT) methylation (P = 0.003) and lower Ki-67 protein expression (P = 0.011). Moreover, MGMT methylation was enriched in IDH-mutant/TERTp-mutant gliomas, and Ki-67 protein expression was the highest in the IDH-wild type/TERTp-mutant group. Taken together, the findings of this study indicate the establishment of a rapid, precise, and practical Sanger sequencing technique for TERTp mutations in gliomas that may show promising results in clinical applications.

Potential Biomarkers for Diagnosing Major Depressive Disorder Patients with Suicidal Ideation.

BACKGROUND: Major depressive disorder (MDD) and suicide are two major health problems, but there are still no objective methods to diagnose MDD or suicidal ideation (SI). This study was conducted to identify potential biomarkers for diagnosing MDD patients with SI. METHODS: First-episode drug-naïve MDD patients with SI and demographics-matched healthy controls (HCs) were recruited. First-episode drug-naïve MDD patients without SI were also included. The serum lipids, C-reactive protein (CRP), transferring (TRSF), homocysteine (HCY) and alpha 1-antitrypsin (AAT) in serum were detected. The univariate and multivariate statistical analyses were used to identify and validate the potential biomarkers. RESULTS: The 86 HCs, 53 MDD patients with SI and 20 MDD patients without SI were included in this study. Four potential biomarkers were identified: AAT, TRSF, high-density lipoprotein cholesterol (HDLC), and apolipoprotein A1 (APOA1). After one month treatment, the levels of AAT and APOA1 were significantly improved. The panel consisting of these potential biomarkers had an excellent diagnostic performance, yielding an area under the ROC curve (AUC) of 0.994 and 0.990 in the training and testing set, respectively. Moreover, this panel could effectively distinguish MDD patients with SI from MDD patients without SI (AUC=0.928). CONCLUSION: These results showed that these potential biomarkers could facilitate the development of an objective method for diagnosing MDD patients with SI, and the decreased AAT levels in MDD patients might lead to the appearance of SI by resulting in the elevated inflammation.

Comprehensive evaluation of risk factors for lymph node metastasis in patients with papillary thyroid carcinoma.

With the increasing incidence of papillary thyroid cancer (PTC), it is important to risk-stratify patients who may have a more aggressive tumor biology. The present study aimed to evaluate the risk factors for lymph node metastasis (LNM) in patients with PTC, which may provide a significant reference for clinical diagnosis and treatment. In total, 1,045 patients with PTC [313 with PT microcarcinoma (PTMC) and 732 with non-PTMC] between August 2016 and August 2019 were investigated. The B-type Raf kinase (BRAF) V600E mutation was tested in all samples. The clinical data (sex, age, tumor location, sample type and pathological features) were retrospectively analyzed. Logistic regression analysis was performed to evaluate independent risk factors for LNM. A total of 181/313 (57.8%) PTMC cases and 145/732 (19.8%) non-PTMC cases had a BRAF V600E mutation. In the PTMC cases, significant differences in sex and sample type were identified (BRAF V600E mutation vs. wild-type). In the non-PTMC cases, significant differences in sex and age were identified (BRAF V600E mutation vs. wild-type). Female sex and tumor diameter ≤1 cm were significant independent predictors of LNM in PTC. In PTMC, female sex was a significant independent predictor of LNM. A bilateral tumor was an independent protective factor for LNM in PTC, PTMC and non-PTMC. The BRAF V600E mutation rate of ultrasound-guided fine-needle aspiration cytology was higher compared with FFPE in PTMC (P=0.018). In contrast to previous studies, the results of the present study suggested that being female and having a tumor of diameter ≤1 cm were risk factors for LNM, and that the BRAF wild-type of PTMC may be more aggressive than other types. Notably, the position of the tumor in the bilateral thyroid was also an independent protective factor for LNM. Therefore, ultrasound-guided fine-needle aspiration should be recommended for gene analysis (BRAF V600E) in PTMC. In addition, clinicians should consider an individualized treatment according to gene mutations, sex, age, tumor size and the location of the tumor, in order to achieve an improved therapeutic efficacy.

A double mutation of BRAF L597Q and V600E in situ and solitary brain metastasis of occult papillary thyroid carcinoma: A case report.

RATIONALE: The rare BRAF L597Q (c.T1790A) point mutation has been previously reported in childhood acute lymphoblastic leukemia. We present the first rare case of occult papillary thyroid carcinoma with BRAF L597Q mutation in a Tibetan patient. PATIENT CONCERNS: A 57-year-old male patient presented with a protruding mass on the left forehead for 2 years and numbness in the right limb for 3 weeks. DIAGNOSES: The patient had a double mutation of BRAF L597Q and V600E in 2 separate lesions at thyroid and brain, the immunohistochemical staining showed that the cytokeratin (CK), thyroglobulin (Tg) and thyroid transforming factor-1 (TTF-1) were immunoreactive. All the findings supported the diagnosis of solitary brain metastasis of occult papillary thyroid carcinoma. INTERVENTIONS: The patient underwent left frontal lobe metastasis (thyroid cancer) resection that involved craniectomy and artificial skull repair. OUTCOMES: During the 24-month follow-up, no postoperative complications or recurrence and metastasis were found. LESSONS: This is the first case of solitary brain metastasis of occult papillary thyroid carcinoma with double mutation of BRAF L597Q and V600E in 2 separate lesions reported in the literature. Our study extends the disease spectrum of occult papillary thyroid carcinoma and suggests that the BRAF L597Q mutation might play a specific role in inducing the solitary brain metastasis of occult papillary thyroid carcinoma in a Chinese Tibetan patient, but the detailed molecular mechanism remains to be confirmed by a large number of functional experiments and clinical research.

Association of methylenetetrahydrofolate reductase (MTHFR) variant C677T and risk of carotid atherosclerosis: a cross-sectional analysis of 730 Chinese Han adults in Chongqing.

BACKGROUND: Uncertainty still remains on the correlation of methylenetetrahydrofolate reductase (MTHFR) variant C677T with risk of carotid atherosclerosis (CAS), and there is a lack of reports on C677T/MTHFR in the Asian population. The association of C677T/MTHFR polymorphisms with CAS in the Chinese Han population in Chongqing was investigated in the present study. METHODS: Subjects (n = 730, 214 females and 516 males, Han ethnicity) who provided an informed consent were randomly selected from the general population of Chongqing, China. Polymerase chain reaction-restriction fragment length polymorphism and Sanger sequencing genotyping assays were used to determine the MTHFR genotypes. The atherosclerosis index of the intima-media thickness (IMT) was measured by high-resolution ultrasound to evaluate the CAS. Less than 1.0 mm was considered as normal for IMT, 1.0-1.5 mm was considered as thickening, and ≥ 1.5 mm and a local bulge thickened in the lumen was considered as CAS. According to the carotid ultrasonography results, these subjects were divided into two groups: CAS-group (IMT ≥ 1.0 mm) and control group (IMT < 1.0 mm). RESULTS: The frequency of C/T heterozygotes, T/T homozygotes genotype was significantly higher in the subjects with CAS (62% vs. 36.9%; 16.2% vs. 9.5%; 47.2% vs. 27.9%, P < 0.05), while the frequency of C/C homozygotes and C allele was significantly lower (21.8% vs. 53.7%; 52.8% vs. 72.1%, P < 0.05), when compared to the control group. The risk of CAS was higher for subjects with C/T heterozygotes and T/T homozygotes (OR = 4.06, 95% CI: 2.76-5.98, P < 0.001 and OR = 3.14, 95% CI: 1.73-5.69, P < 0.001, respectively), when compared to the subjects with the C/C genotype. In the model 1 (CT + TT versus CC), C677T/MTHFR was significantly associated with the prevalence of CAS, and the all adjusted OR values for CAS were 3.87 (95% CI, 2.67 to 5.62) in all, 17.18 (95% CI, 7.27 to 40.49) in women and 2.57 (95% CI, 1.65 to 3.99) in men after adjusting for potential confounding factors. CONCLUSIONS: The present study suggests that a mutation in the methylenetetrahydrofolate reductase gene is a risk factor of CAS in the Chinese Han population.

Evaluation of Concordance Between Deficient Mismatch Repair and Microsatellite Instability Testing and Their Association with Clinicopathological Features in Colorectal Cancer.

BACKGROUND: Microsatellite instability (MSI) is one of the most important molecular characteristics of colorectal cancer (CRC), which mainly results from defective DNA mismatch repair (MMR). This study was performed to investigate the concordance between deficient MMR and MSI testing, and to evaluate the association of these two results with clinicopathological characteristics in Chinese CRC patients. METHODS: A total of 738 CRC patients were included. Tumor tissues and paired peripheral blood specimens were obtained. Screening for MMR was investigated using immunohistochemical (IHC) technique, and multiple polymerase chain reaction-capillary electrophoresis (PCR-CE) method was performed to detect the MSI status. All clinicopathological data, immunohistochemistry and microsatellite instability analyses were then statistically analyzed. RESULTS: Of the 738 (17.75%) CRC patients, 131 expressed as deficient mismatch repair (dMMR) status, and postmeiotic segregation increased 2 (PMS2) deficiency was the most frequent deficiency among these four MMR proteins. MSI-high (MSI-H) status occurred in 74 of the 738 (10.03%) CRC patients, 55 of whom showed instability at all six mononucleotides repeat markers. dMMR was significantly associated with MSI-H and moderate concordance was observed between IHC and PCR-CE in evaluating deficient MMR/MSI through Kappa test. Statistically, dMMR was significantly associated with younger age, right-sided colon and poor differentiation. MSI-H was associated with younger age, right-sided colon, poor differentiation, mucinous type and tumor, node, metastasis (TNM) stage II. CONCLUSION: A moderate concordance between deficient MMR and MSI testing indicates that both IHC and PCR-CE methods should be routinely tested to provide reliable data for clinical treatment decisions.

Detection and genomic characterization of Bovine papillomavirus isolated from Chinese native cattle.

Bovine papillomaviruses (BPV) are small circular DNA viruses which can be widely spread in herd, inducing cattle tumours, therefore, leading economic losses in dairy and beef production industries. BPV-leads symptoms include cutaneous papillomas, fibropapillomas, urinary bladder and oesophageal carcinoma. As one of the most important producers of beef in the world, China has not provided systematic research to prevent the harm of BPV, particularly in papillomavirus molecular characterization which presents among Chinese native cattle which was known to have higher disease resistance. In this study, skin papilloma was observed and samples were collected following by histopathological analysis. We analysed all neoplasms samples and reviewed their degrees in acanthosis and/or hyperkeratosis. Full-length genomic sequencing was applied for all four isolated strains (JX180408, LA150909, HX160815, and BS160810) to exploring the molecular reason why BPV currently prevalent in Chinese native cattle. As a result, we identified that these four isolates were classified as BPV-1 and clustered into the Deltapapillomavirus genera. Our study also identified that BPV 1 isolates from Chinese indigenous cattle breeds belong to subtypes A which has a closer genetic background compare with their common ancestor and suggest it can be a more ancestral species. European isolates more recently diverged group (group B) contained almost exclusively European samples. In this study, we analysed the similarity of ORF between Chinese isolated BPV 1 and BPV 1 reference strains and listed results. This study provides the complete genomic characterization of BPVs circulating in Chinese native cattle breeds for the first time, which provide a detailed description of how diverse strains may cause skin tumour among Chinese local breed cattle therefore critical for further epidemiological study of relevant diseases.

High-sensitive immunosensing of protein biomarker based on interfacial recognition-induced homogeneous exponential transcription.

A novel and versatile immunosensing strategy was developed for ultrasensitive and specific detection of proteins by organically integrating interfacial specific target recognition and homogeneous transcription amplification. In principle, classic antigen-antibody sandwich structure on the microplate could realize the specific identification of target protein. Biotinylated DNA probe was subsequently introduced by streptavidin-biotin system as a bridge linking interfacial and homogeneous reaction. The biotinylated DNA initiated exponential transcription amplification in the solution, which converted per target recognition event on the interface to numerous single-stranded RNA products in solution for highly sensitive fluorescence immunosensing. The proposed immunoassay based on interfacial recognition-induced homogeneous exponential transcription (IR-HET) for vascular endothelial growth factor (VEGF) detection showed a good linear range from 0.01 to 1000 pg/mL and the limit of detection as low as 1 fg/mL, which was 3 orders lower than traditional ELISA method. The established strategy was also successfully applied to directly detect VEGF from culture supernatants of tumor cells and clinical body fluid samples, proving very high sensitivity, selectivity and low matrix effect. Therefore, IR-HET-based immunosensing strategy might become a potential powerful tool be applied in ultrasensitive detection of low abundance protein biomarker for clinical early diagnosis, treatment and prognosis.

Engineered zinc-finger transcription factors inhibit the replication and transcription of HBV in vitro and in vivo.

In the present study, an artificial zinc-finger transcription factor eukaryotic expression vector specifically recognizing and binding to the hepatitis B virus (HBV) enhancer (Enh) was constructed, which inhibited the replication and expression of HBV DNA. The HBV EnhI­specific pcDNA3.1­artificial transcription factor (ATF) vector was successfully constructed, and then transformed or injected into HepG2.2.15 cells and HBV transgenic mice, respectively. The results demonstrated that the HBV EnhI (1,070­1,234 bp)­specific ATF significantly inhibited the replication and transcription of HBV DNA in vivo and in vitro. The HBV EnhI­specific ATF may be a meritorious component of progressive combination therapies for eliminating HBV DNA in infected patients. A radical cure for chronic HBV infection may become feasible by using this bioengineering technology.