METTL3 Is Involved in the Development of Graves' Disease by Inducing SOCS mRNA m6A Modification.

Objective: Epigenetic modifications in RNA are known to play critical roles in cell differentiation through regulating expressions of some key genes including members of the suppressor of cytokine signaling (SOCS) family. The present study aimed to unveil the relationship of SOCS mRNA methylation induced by methyltransferase like 3 (METTL3) with Graves' disease (GD). Methods: Differently expressed genes (DEG) in GD tissues were identified using microarray analysis and further validated using CD4+ T cell microarray of GD tissues and isolated peripheral blood mononuclear cells (PBMCs). Furthermore, expressions of METTL3 targeted genes were detected using METTL3 knock-down experiment in RAW264.7 cells. Results: High throughput microarrays revealed that METTL3 and SOCS molecules were aberrantly expressed in thyroid tissues and CD4+T cells of GD compared to the controls. Bioinformatic analysis was undertaken by searching databases of found genes of the SOCS family that possessed many mRNA m6A modification loci. METTL3 knock-down experiment revealed that expressions of SOCS family members SOCS1, SOCS2, SOCS4, SOCS5, and SOCS6 were increased after METTL3 knock-down. Conclusions: For the first time, the present study revealed the relationship between m6A modification and GD and indicated that METTL3 may be involved in the development of GD by inducing mRNA m6A methylation modification of SOCS family members.

METTL3 gene polymorphisms contribute to susceptibility to autoimmune thyroid disease.

PURPOSE: Autoimmune thyroid disease (AITD) is a classic autoimmune disorder that mainly includes Graves' disease (GD) and Hashimoto's thyroiditis (HT). In this study, we explored the potential relationship between single-nucleotide polymorphisms (SNPs) of methyltransferase like 3 (METTL3) gene and the development of AITD. METHODS: The distribution of METTL3 genotypes at seven loci (rs1139130, rs1263790, rs1263791, rs17197156, rs2242526, rs3752411, and rs4417466) in 960 AITD (599 GD and 361 HT) patients and 732 unrelated healthy volunteers was examined using high-throughput sequencing technology in a case-controlled manner and their correlations with AITD development were statistically analyzed. RESULTS: METTL3 genotypes at these seven SNPs were not correlated with both GD and HT except a borderline association between rs3752411and GD after adjusted for age, sex, and thyroid function under the recessive model. Subgroup analysis demonstrated that the minor allele frequencies of rs2242526 and rs4417466 were higher in male AITD patients than in healthy volunteers before adjusted for confounding factors and the genotype distribution of rs4417466 was significantly different between the two groups. Additionally, the genotype frequencies of rs1139130, rs1263791, rs2242526, and rs4417466 were positively related with GD in male patients. Likewise, the allele distribution of rs1263791, rs2242526, and rs4417466 in male GD patients differed significantly from that in male controls. Multivariate logistic regression analyses revealed a significant association between allele frequencies of these three loci and GD in male patients after adjusted for the confounding factors. Moreover, the genotype of rs3752411 was strongly associated with GD in females as well. Furthermore, distribution of rs3752411 genotype was significantly associated with hypothyroidism in HT patients. CONCLUSION: Our study for the first time revealed a strong correlation between METTL3 mutations and AITD predisposition, implying that METTL3 may be a new candidate gene for AITD treatment.

Randomized multicenter study on long-term complications of peripherally inserted central catheters positioned by electrocardiographic technique.

BACKGROUND: The intracavitary electrocardiogram (IC-ECG) method has been used for the tip location of central venous access devices for the advantage of being safe, accurate and highly cost effective. However, long-term follow-up is rare. This randomized clinical trial aimed to evaluate the long-term complications of peripherally inserted central catheters (PICCs) positioned by the IC-ECG method. METHODS: We randomized 2250 patients who needed PICC placement to either a landmark length estimation supplemented by IC-ECG positioned group (ECG group) or the traditional landmark length estimation alone group (control group) in a 2:1 allocation. Post-procedural chest X-rays were applied to confirm tip position. Follow-up was performed monthly to six months. Standard statistics analyses were performed with the SAS 9.13 software, and p < 0.05 was considered significant. RESULTS: As evaluated by post-procedural chest X-ray, tip location in the ECG group had a first-attempt success (catheter tip located at optimal position) of 91.7% (95% confidence interval (CI): 90.3%-93.1%), significantly higher than 78.9% (95% CI: 76.0%-81.9%) observed in the control group (p < 0.001). At six-month follow-up, in the control group, frequency of total complications was 9.5%, including the exit site infection (4.0%), phlebitis (1.3%), deep venous thrombosis (1.5%), liquid extravasation (2.9%) and mechanical failure (1.9%). The IC-ECG group had significantly lower rates of complications (6.4%, p < 0.001), including the exit site infection (2.7%, p > 0.05), phlebitis (1.1%, p > 0.05), deep venous thrombosis (1.2%, p > 0.05), liquid extravasation (2.4%, p > 0.05) and mechanical failure (1.2%, p > 0.05). In the univariable logistic regression analysis, ECG method, other diseases and upper arms were the independent protective factors, and the number of adjustment procedures (n ≥ 2) were the independent risk factors of the complications. CONCLUSIONS: The intra-procedural tip location by IC-ECG is more safe and accurate than the traditional method of verifying tip location only post-procedurally, by chest X-ray.

A scale-down model of 4000-L cell culture process for inactivated foot-and-mouth disease vaccine production.

The anticipated increasing demand for inactivated foot-and-mouth (FMD) disease vaccine calls for its larger production capacity, while development of a large-scale process typically requires high running cost and has very limited experimental throughput at manufacturing scale. Thus, an economic scale-down model of representing a large-scale process becomes necessary and essential. In this study, we used a systematic approach to establish a scale-down model representing a 4000-L culture process for FMD vaccine production by suspension BHK-21 cells. In detail, we firstly compared hydrodynamic properties of three bioreactors (14-L, 800-L and 4000-L) under three different conditions (equivalent mixing time, equivalent shear stress and equivalent volumetric power). We figured out equivalent volumetric power (P/V) potentially as an appropriate scale-down strategy, since it resulted in comparable calculated hydrodynamic parameters among three bioreactors. Next, we used computational fluid dynamics (CFD) simulation to provide more details about hydrodynamic environments inside the bioreactors, which supports the reliability of this scale-down strategy. Finally, we compared cell growth, metabolites, vaccine productivity and product quality attributes during FMD vaccine production by BHK-21 cells and observed very close performances among three bioreactors, which once again demonstrates the robustness of this scale-down model. This scale-down strategy can be applied to study variations and critical quality attributes (CQAs) in the resultant production process based on quality by design (QbD) principles, aiming at further more efficient optimization of vaccine production.

Insertion of peripherally inserted central catheters with intracavitary electrocardiogram guidance: A randomized multicenter study in China.

INTRODUCTION: Ultrasound-guided venipuncture and tip location by intracavitary electrocardiogram have many advantages during the insertion of peripherally inserted central catheters, both in terms of safety and cost-effectiveness. Recently, a new tip-conductive peripherally inserted central catheters and new Doppler ultrasound device integrated with intracavitary electrocardiogram have been introduced into clinical practice in China. A randomized multicenter study (clinical trial no. NCT03230357) was performed to verify the feasibility and accuracy of intracavitary electrocardiogram, as performed with this new peripherally inserted central catheters and device. METHODS: Our study enrolled a total of 2250 adult patients in 10 different Chinese hospitals. The patients were randomly assigned to either the study group (intracavitary electrocardiogram) or the control group (anatomical landmark guidance) in a 2:1 allocation. Ultrasound was used in both groups for venipuncture and tip navigation. All patients underwent chest X-ray after the procedure to verify the position of the catheter tip. RESULTS: No insertion-related complications were reported in either group. In the study group, first-attempt successful tip location was 91.7% (95% confidence interval: 90.3%-93.1%), significantly higher than 78.9% (95% confidence interval: 76.0%-81.9%) observed in the control group (p < 0.001). As evaluated by post-procedural chest X-ray, tip location in the study group had a sensitivity of 99.3% (95% confidence interval: 98.8%-99.7%), significantly higher than 86.8% (95% confidence interval: 84.4%-89.2%) observed in the anatomical landmark group (p < 0.001). CONCLUSION: These results indicated that during peripherally inserted central catheters insertion in adult patients, tip location with intracavitary electrocardiogram guidance, as carried out by a new tip-conductive peripherally inserted central catheters and intracavitary electrocardiogram integrated ultrasound device, was more effective and more accurate than tip location using anatomical landmarks.

Downregulated Tim-3 expression is responsible for the incidence and development of colorectal cancer.

The present study aimed to investigate the role of T cell immunoglobulin domain and mucin-3 (Tim-3) in its gene and protein forms in colorectal cancer (CRC), and to verify the significance of Tim-3 expression in patients with CRC. A prospective analysis of 258 patients with CRC and 246 normal controls was conducted between December 2012 and June 2015. Intestinal samples were collected, including of CRC tissues, paracancerous tissues and normal colon mucosa tissues. Peripheral venous blood samples were also collected. Polymerase chain reaction (PCR) amplification, reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis was performed for the detection and evaluation of Tim-3 gene and protein in various tissues. PCR analysis indicated that the T and G alleles of -882C/T and 4259T/G are associated with a significantly increased risk of CRC. Following the confirmation of Tim-3 expression in CRC tissues, RT-qPCR detection and western blot analysis revealed clear downregulation of Tim-3 mRNA and protein expression in the blood and tissue samples obtained from patients with CRC, as compared with in the corresponding control samples. Similar trends of decreased Tim-3 mRNA levels and protein expression were observed in CRC tissues compared with in the paracancerous and the normal colon mucosa tissues. In addition, the mRNA and protein expression levels in the paracancerous tissues were lower than those in the normal colon mucosa tissues. Furthermore, significantly lower Tim-3 mRNA levels were observed in patients with a tumor size >5 cm, a poor differentiation degree, higher tumor-node-metastasis stage (stage III-IV), and lymph node and distant metastasis. Collectively, genetic changes to Tim-3, expressed as polymorphisms in Tim-3, and decreased mRNA/protein expression may be partially responsible for the incidence and development of CRC.

[Deletion of IV a2 gene from adenoviral genome by lambda-Red recombinase system and packaging of the recombinant adenovirus].

This investigation is to delete the most of the coding sequence (1104 bp) of the IV a2 gene in an adenovirus genome by a lambda-Red recombinase system-mediated PCR-targeting approach and rescue a recombinant adenovirus with IV a2 deletion. First, the template pAK of PCR targeting, containing kanamycin cassette, was constructed. Then, a linear fragment for PCR targeting, which had 39 bp homologous arms at both of its terminus, was amplified by PCR from the pAK. The pFG140 and the linear fragment were electroporated into E. coli BW25113/pIJ790 sequentially and the recombinant pFG140-deltaIV a2 (1104) was established by homologous recombination between the linear fragment and the pFG140 with aid of X-Red recombinase. The precise deletion of 1 104 bp fragment from IV a2 was confirmed by restriction endonucleases digestion and DNA sequencing. ORF of IV a2 was amplified by PCR from pFG140 and then cloned into the pAAV2neo vector. The recombinant adenovirus Ad5delta IV a2 (1104) was rescued by co-transfection of pFG140-deltaIV a2 (1104) and pAAV2neo-IV a2 into HEK293 cells. It was shown by Western Blot that IV a2 could not be detected in the Ad5deltaIV a2 (1104)- infected HEK293 cells. This study established a PCR-targeting strategy for manipulating adenovirus genome directly by a lambda-Red recombinase system, and a recombinant adenovirus with IV a2 deletion was obtained.

[Effects of Lin28a and Lin28b on let-7 family activity].

In this report, we study the effects of over-expression of Lin28a and Lin28b on let-7 family activity in HeLaS3. Firstly, we constructed pAAV2neo-Lin28a and pAAV2neo-Lin28b to express Lin28a and Lin28b, respectively. Then, pAAV2neo-Lin28a and pAAV2neo-Lin28b were transfected into HeLaS3, selected with G418 and obtained cell lines, HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b, to express Lin28a and Lin28b stably. Thereafter, we constructed eight plasmid vectors for detection of let-7 family activity based on pAAV2neo-Gluc-(Fluc). These vectors were further packaged into recombinant adeno-associated viral vectors (rAAV) which were used as sensors, nominated as Asensors, to detect inhibition activity of miRNA at post-transcriptional level. Subsequently, with HeLaS3 as a control, we assayed expression levels of Lin28a and Lin28b by Western blot, detected expression levels of let-7 family by QRT-PCR, and tested let-7 family activity by Asensors in HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b. Results demonstrated that both HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b could express Lin28a and Lin28b effectively. Compared with HeLaS3, the expression level of let-7 family except let-7e declined in HeLaS3/pAAV2neo-Lin28a. But declining extent among members of let-7 family was different. The let-7 family activity also decreased while the decreasing extent varied among members. Furthermore, the activity level was not consistent with its expression level for the same member in let-7 family. Compared with HeLaS3, both expression level and activity level of let-7 family in HeLaS3/ pAAV2neo-Lin28b were decreased. However, the decreasing extent of let-7 family expression changes was larger than that of HeLaS3/pAAV2neo-Lin28a while the decreasing extent of activity changes was similar. In this study, we established a method to detect and compare post-transcriptional inhibition level mediated by miRNA complementary targets. We firstly clarified the effect of Lin28a and Lin28b on let-7 family activity profile and found that this effect was not the same as that at expression level of let-7 family, suggesting that it was more comprehensive to understand miRNA regulation roles to detect both miRNA expression and activity. This paves a way for further research on mechanism of regulation of let-7 family.

[Study on severity, status of awareness and therapy regarding urinary incontinence in elderly people from rural areas in Jixian county, Tianjin].

OBJECTIVE: To understand the prevalence of urinary incontinence (UI) and its severity in rural elderly people, as well as to investigate the awareness on UI in the elderly and health-care service seeking behavior. METHODS: A cross-sectional study was carried out in two townships of Jixian county, Tianjin. A total of 743 people aged 60 years and over were selected under cluster sampling method. All the information was collected with a standardized structured questionnaire by face-to-face interview. Prevalence, severity, the awareness on UI in the elderly and their utilization of health-care service for its diagnosis and treatment were analyzed. RESULTS: Prevalence of UI was 33.38% among people aged 60 years and over in two townships of Jixian, higher in females than in males (43.15% vs. 22.75%, χ(2) = 34.70, P < 0.0001). The prevalence rates of UI in 60- age group, 65- age group, 70- age group, 75- age group, 80- age group, 85 - 95 age group were 28.64%, 32.12%, 34.08%, 35.45%, 47.76%, 30.00%, respectively, and increased with age (for trend χ(2) = 2.19, P = 0.029). Stress urinary incontinence (SUI) and urge urinary incontinence (UUI) were mild, while mixed urinary incontinence (MUI) were mainly moderate. The differences of severity of SUI, UUI, MUI between men and women did not show statistical significance (all P > 0.05). In 743 elderly people, more than half of the respondents had never heard of UI (50.20%, 373/743) and only 170(22.88%) elderly people considered UI as a disease. 630 (84.79%) and 665 (89.50%) elderly people in our research group did not know that such condition was curable and preventable. In 248 elderly people with UI, only 12 (4.84%) of them ever seeking community health-care services in the health-care centers or hospitals. For the ones who did visit the centers, the purpose was only to seek for drug treatment. CONCLUSION: Prevalence of UI appeared to be high among the elderly people in rural areas of Jixian county. Most of the elderly people were lack of knowledge about UI that hindered them from seeking for diagnosis and treatment in the clinics, plus the treatment program for UI was not standardized. Knowledge on UI and health care seeking behavior should be popularized and strengthened among elderly people living in the rural areas.

[Study on the prevalence of urinary incontinence and its related factors among elderly in rural areas, Jixian county, Tianjin].

OBJECTIVE: To understand the prevalence of urinary incontinence (UI) and its related factors so as to develop a three-tier program for prevention of the disease. METHODS: A cross-sectional study was carried out in two townships of Jixian county, Tianjin, during July to November 2007. A total of 743 people aged 60 years and over were selected under cluster sampling, and all information were collected with a standardized structured questionnaire by face-to-face interview. All the data were analyzed with multivariate logistic regression method to explore the related factors for UI in the elderly. RESULTS: The overall prevalence of UI was 33.38 percent among people aged 60 years and over in two townships. Risk factors for UI in men would include older age (OR = 1.39), occupation (OR = 5.00), awareness of UI (OR = 1.91), having in chronic respiratory diseases (OR = 2.23), prostate (OR = 11.47), neurological (OR = 11.76), or motor systems (OR = 2.48), while protective factors would include high educational level (taking primary school or below as control group), OR for the junior middle school group appeared to be 0.35, for senior middle school group it was 0.77, and of undergraduate group it was 0.53. Risk factors for UI in women would include older age (OR = 1.31), constipation (OR = 1.46), awareness of UI (OR = 1.94), increased body mass index (when normal weight group served as control group, OR in the overweight group was 1.03 and in the obesity group OR was 1.54), suffering from chronic respiratory diseases (OR = 4.84), diabetes mellitus (OR = 2.36), or motor system diseases (OR = 1.37), more gravidity (OR = 1.03), more parity (OR = 1.02), suffering from perinea laceration (OR = 1.72) and wound infection during delivery (OR = 1.65), while protective factors would include physical exercises (OR = 0.64). CONCLUSION: Prevalence of UI was higher among the elderly people in rural areas of Jixian county, Tianjin. UI in the elderly might have been influenced by various factors which suggesting the intervention strategy should be targeted at those related factors as well as focusing on primary prevention.

[To assess the demands of community-based health care service among 717 elderly].

UNLABELLED: OBJECTIVE To understand the demands and influencing factors on the community-based health care service (CHS) among the elderly and to provide evidence of implementing health service for them METHODS: Cluster sampling method was used on 717 persons aged 60 and above in Yanjiao Developing Area. A survey was carried out, using questionnaire and focus group discussion approaches. RESULTS: Data showed that a two-week prevalence of diseases was 56.3% while the overall prevalence of chronic diseases was 92.1% with 70.0% of them suffering from more than two kinds of chronic diseases. The rates of ADL and IADL loss were 1.8% and 7.0% respectively with 37.0% of them sought medical consultation in the past two weeks. Major reason affecting the use of health service was low income. 57.7% of the elderly expressed their willingness of accepting CHS with the strongest demands as regular physical check-up, health education and consultation, specialized care services, home care service while major reasons hindering the acceptance of CHS could be referred to self-perception on good stamina and lack of awareness to CHS. CONCLUSION: The demands of CHS in the elderly were greater than those in general population that called for CHS programs to be acceptable and relevant to meet the different needs.

[Evaluation on the effectiveness for self-management of hypertensive patients in a community].

OBJECTIVE: To evaluate the acceptability of self-management project for patients with hypertension and the short term result, and provide the evidence of preventing and controlling hypertension using chronic disease self-management. METHODS: Voluntary patients with hypertension were grouped into the control and experimental groups after matching age, sex, education levels and the number of chronic diseases between the two groups. Data collected through questionnaire after intervention were compared with the baseline data through analysis of covariance and chi(2) test. RESULTS: Rate of awareness for the diagnostic criteria of hypertension, risk factors and associated diseases increased by 56.4%, 50.4% and 37.6% respectively, and the rate of high salt diet intake, body mass index and the times of visiting doctors decreased by 27.1%, 0.8 kg/m(2) and 2.7 times on average in experimental group, as compared with control group. The health status (energy, fatigue, shortness of breathe, pain) improved (P < 0.05). In the similar comparison, the rate in which the hypertensive patients took antihypertensive medicine increased by 20%, and the scores of cognitive symptoms management and of patient communication with physician increased by 3.9 and 1.1 points respectively. The rate of compliance to medication increased by 21.5% compared with themselves. CONCLUSION: The awareness rate of hypertensive patients on knowledge and on unhealthy life style improved after implementing the self-management program. Their emotion, healthy behavior, health status and symptoms related to hypertension improved. Data showed that it was effective for hypertensive patients to use chronic diseases self-management.