Circ_0082878 contributes to prostate cancer progression via the miR-455-3p/WTAP axis.

Circ_0082878 has been found to be strongly expressed in prostate cancer (PCa). However, its roles and potential mechanism in PCa have not been investigated. This study aims to clarify it. RNase R digestion method was adopted for verifying the circular structure of circ_0082878. RT-qPCR assay is aimed to detect the expressions of circ_0082878, miR-455-3p and WTAP in PCa tissues and cells. For identifying cell proliferation, migration and invasion abilities, CCK-8 and transwell assay were used. To show the correlation between miR-455-3p and WTAP or circ_0082878, the luciferase reporter gene, RNA RIP and RNA pull-down experiments were employed. We employed western blot to detect protein level of WTAP. In addition, the impact of circ_0082878 on PCa cells in vivo was also studied. It was found that circ_0082878 and WTAP were highly expressed in PCa tissues and cells, whereas miR-455-3p was lowly expressed. Inhibition of circ_0082878 restrained the growth of PCa in vitro and in vivo. Regarding mechanism, miR-455-3p was the target of circ_0082878, and WTAP was the target of miR-455-3p. Circ_0082878 could downregulate the level of miR-455-3p, and inhibiting of miR-455-3p expression could partially eliminate the inhibitory impact of low expression of circ_0082878 on the proliferation and migration of PCa cells. Additionally, over-expression of miR-455-3p resulted in the reduced level of WTAP, and WTAP over-expression counteracted the tumor suppressive impact of miR-455-3p in PCa cells. Moreover, the obtained findings indicated that circ_0082878 may exert tumor-promoting activity in PCa via sponging miR-455-3p and then upregulating WTAP. This indicates that the circ_0082878/miR-455-3p/WTAP axis can probably become the possible therapeutic target for PCa.

Stereoselective behaviors and enantiomeric effects of paclobutrazol on microorganisms during Chinese cabbage pickling process.

Studies on the differences between chiral pesticide enantiomers have caused widespread concern in the last decade. In the current work, the selective behaviors and different biological activities of paclobutrazol enantiomers during Chinese cabbage pickling process were evaluated. Results of degradation kinetics indicated that when paclobutrazol reside in raw material (Chinese cabbage) and was introduced into the pickling process, the degradation rates of the two paclobutrazol enantiomers were significantly different, the half-lives of (2R, 3R)-paclobutrazol (R-paclobutrazol) and (2S, 3S)-paclobutrazol (S-paclobutrazol) were 18.24 and 6.19 d, respectively. Besides, the conversion between the two enantiomers could also be observed, and the conversion rate of R-paclobutrazol to S-paclobutrazol was slower than that of reverse process. In addition, from the analysis of 16S rRNA and ITS sequencing, we inferred that the degradation of paclobutrazol was probably due to the presence of Pseudomonas and Serratia. Moreover, there has a significant difference in biological activity between R-paclobutrazol and S-paclobutrazol and shown an obviously enantiomeric effects on microbial community composition of pickling system. Besides, the analysis of microbial community displayed R-paclobutrazol might inhibit the growth of Erwinia (a sort of plant pathogens). Results from this study served to enhance our understanding of chiral pesticide residues on food safety and the potential risks to human health.

Cancer-Associated Fibroblasts Boost Tumorigenesis of Clear Cell Renal Cell Carcinoma via Exosome-Mediated Paracrine SNHG1.

Despite the dominant roles of cancer-associated fibroblasts (CAFs) have attached much attention in tumorigenesis, the CAFs-derived molecular determinants that regulate renal cell carcinoma (RCC) development remains elusive. Our previous study uncovered an oncogenic SNHG1 in the immune escape of RCC, whereas CAFs-derived exosomes could be a source accounting for increasing SNHG1 in RCC cells, this is still a mystery. The obtained CAFs and normal fibroblast (NFs) from fresh RCC and adjacent tissues were firstly identified using western blot and immunofluorescent staining. The enrichment of SNHG1 was validated by RT-qPCR. CAFs-derived exosomes were isolated from conditioned medium using ultracentrifugation method and ExoQuick-TC system. The internalization of exosomes, transfer of SNHG1, was measured by immunofluorescence. Regulation of conditioned medium or exosomal SNHG1 from CAFs on RCC biological functions was evaluated by CCK-8, EdU incorporation, colony formation, and transwell assays to assess the RCC cell proliferation, migration, and invasion. SNHG1 was significantly upregulated in CAFs isolated from RCC stroma. Exosomes derived from CAFs transferred SNHG1 to RCC cells and resulted in an increased SNHG1 expression in RCC cells. The exosomes excreted by CAFs promoted RCC cell proliferation, migration, and invasion, whereas the promotion effect of CAFs-exosomes on RCC progression was attenuated by SNHG1 knockdown. The present study revealed a new mechanism of exosomal SNHG1 extracted from CAFs enhanced RCC progression and may provide a potential target for the treatment of RCC.

Weber Ankle Fracture Classification System Yields Greatest Interobserver and Intraobserver Reliability Over AO/OTA and Lauge-Hansen Classification Systems Under Time Constraints in an Asian Population.

No previous studies have evaluated the intra- and interobserver reliability between the Weber, Lauge-Hansen and AO Foundation/Orthopaedic Trauma Association (AO/OTA) classification systems under time constraints. This study compares the interobserver and intraobserver reliability of the aforementioned classification systems under simulated time constraints. Anteroposterior and lateral radiographs of ankle malleolar fractures from 80 consecutive patients from 2015 to 2016 were classified by 2 independent observers according to Weber, Lauge-Hansen and AO/OTA. Classifications were conducted over 4 successive weeks under timed (25-seconds) and untimed conditions, with 1-week gaps between each classification. Cohen's kappa and percentage agreement were calculated. Cohen's kappa for interobserver agreement ranged 0.67 to 0.67 and 0.59 to 0.73 for untimed and timed classifications for Weber; 0.38 to 0.47 and 0.44 to 0.50 for Lauge-Hansen; 0.28 to 0.49 and 0.13 to 0.37 for AO/OTA. Intraobserver agreement ranged from 0.83 to 0.85 and 0.78 to 0.79 for untimed and timed classifications for Weber; 0.46 to 0.65 and 0.59 to 0.73 for Lauge-Hansen; 0.42 to 0.63 and 0.40 to 0.51 for AO/OTA. Based on the Landis and Koch's benchmark scale, there was substantial agreement in the inter- and intraobserver variables for Weber; moderate agreement in inter- and intraobserver variables for Lauge-Hansen; fair and moderate agreement in inter- and intraobserver variables respectively for AO/OTA. Interobserver and intraobserver reliability was the most substantial for Weber, followed by Lauge-Hansen and AO/OTA. Time constraint did not have a statistically significant effect on the reliability of classifications. We recommend concurrent usage of the Weber and Lauge-Hansen system, since they demonstrate the greatest reliability and reproducibility, and confer better understanding of the fracture type, respectively.

Total Synthesis of Yuzurine-type Alkaloid Daphgraciline.

The first total synthesis of daphgraciline has been achieved, which also represents the first example of the synthesis of Daphniphyllum yuzurine-type alkaloids (∼50 members). The unique bridged azabicyclo[4.3.1] ring system in the yuzurine-type subfamily was efficiently and diastereoselectively assembled via a mild type II [5+2] cycloaddition for the first time. The compact tetracyclic [6-7-5-5] skeleton was installed efficiently via an intramolecular Diels-Alder reaction, followed by a benzilic acid-type rearrangement. The synthetically challenging spiro tetrahydropyran moiety in the final product was installed diastereoselectively via a TiIII-mediated reductive epoxide coupling reaction. Potential access to enantioenriched daphgraciline is presented.

[Effect of Social Support on Multimorbidity and Related Outcomes of Middle-Aged and Older Adults in China].

Objective: To analyze the current status of social support for middle-aged and older adults with multimorbidity and to explore the correlation between different dimensions of social support and multimorbidity and the related outcomes on the basis of China Health and Retirement Longitudinal Study (CHARLS) 2015 survey data so as to reveal the complex social background of multimorbidity and the impact of social support on multimorbidity. Methods: A total of 9168 valid samples, with an average age of 59.60 years, were included in the study. Using the social support-related variables of the respondents, we conducted factor analysis and constructed regression models of common factors of social support and multimorbidity-related outcomes, intending to analyze the impact of common factors of social support on multimorbidity in the middle-aged and older adults. Results: The multimorbidity of middle-aged and older adults in China was related to multiple factors of social support, and the differences were statistically significant. Logistic regression showed that social support in the form of activity/recreational facilities and medical resources was a protective factor of multimorbidity, that family emotional support and economic support had a positive effect on life satisfaction of comorbid patients, and that social support in the form of education, social life and housing conditions was negatively correlated with catastrophic medical expenditure of the comorbid population ( P<0.05). Conclusion: Social support for middle-aged and older adults in China is unevenly distributed. Social support in the form of activity/recreational facilities and medical resources may reduce the risks of multimorbidity among middle-aged and older adults. Good family economic and emotional support can improve the life satisfaction of middle-aged and older adults with multimorbidity. Social support in the form of education, social life and housing conditions may reduce the risk of catastrophic medical expenditure in middle-aged and older adults with multimorbidity.

Upregulated hepatokine fetuin B aggravates myocardial ischemia/reperfusion injury through inhibiting insulin signaling in diabetic mice.

Patients with type 2 diabetes mellitus (T2DM) are more susceptible to acute myocardial ischemia/reperfusion (MI/R) injury. However, the mechanism remains largely elusive. Clinical observation showed that high levels of hepatokine fetuin-B (FetB) in plasma are significantly associated with both diabetes and coronary artery diseases. This study was aimed to determine whether FetB mostly derived from liver exacerbates MI/R-induced injury and the underlying mechanisms in T2DM. Mice were given high-fat diet and streptozotocin to induce T2DM model and subjected to 30 min MI followed by reperfusion. Diabetes caused increased hepatic FetB expression and greater myocardial injury as evidenced by increased apoptosis and myocardial enzymes release following MI/R. In T2DM hearts, insulin-induced phosphorylations of insulin receptor substrate 1 at Tyr608 site and Akt at Ser473 site and glucose transporter 4 membrane translocation were markedly reduced. Interaction between FetB and insulin receptor-ß subunit (IRß) was enhanced assessed by immunoprecipitation analysis. More importantly, FetB knockdown via AAV9 alleviated MI/R injury and improved cardiac insulin-induced signaling in T2DM mice. Conversely, upregulation of FetB in normal mice caused exacerbated MI/R injury and impairment of insulin-mediated signaling. In cultured neonatal mouse cardiomyocytes, incubation of FetB significantly reduced tyrosine kinase activity of IR and insulin-induced glucose uptake, and increased hypoxia/reoxygenation-induced apoptosis. Furthermore, FoxO1 knockdown by siRNA suppressed FetB expressions in hepatocytes treated with palmitic acid. In conclusion, upregulated FetB in diabetic liver contributes to increased MI/R injury and cardiac dysfunction via directly interacting with IRß and consequently impairing cardiac insulin signaling.

LncRNA SNHG1 regulates immune escape of renal cell carcinoma by targeting miR-129-3p to activate STAT3 and PD-L1.

Immune escape of renal cell carcinoma (RCC) impacts patient survival. However, the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) in RCC immune escape remains unclear. Quantitative real-time PCR and western blotting results revealed that the expression of lncRNA SNHG1 and STAT3 were upregulated in RCC tissues and cells and that the expression of miR-129-3p was downregulated. Enzyme-linked immunosorbent assay results revealed the increased levels of immune-related factors (interferon-γ, tumour necrosis factor α, and interleukin-2) in RCC tissues. SNHG1 knockdown or miR-129-3p overexpression inhibited the proliferation and invasion of A498 and 786-O cells, while the proliferation and cytotoxicity of CD8+ T cells increased, which promoted the secretion of immune-related factors. STAT3 overexpression decreased the protective effect of miR-129-3p overexpression on RCC cell immune escape. In addition, miR-129-3p knockdown and STAT3 overexpression decreased the protective effect of lncRNA SNHG1 knockdown on RCC cell immune escape. In addition, PD-L1 expression was downregulated after lncRNA SNHG1 knockdown but upregulated after miR-129-3p knockdown and STAT3 overexpression. Dual-luciferase assays showed that lncRNA SNHG1 targets miR-129-3p, and miR-129-3p targets STAT3. RNA pull-down and RNA immunoprecipitation assays verified the regulatory relationship between SNHG1 and STAT3. In vivo, shSNHG1 prolonged the overall survival of RCC tumour model mice and inhibited RCC tumour growth and immune escape but increased CD8+ T cell infiltration in mice. Our findings provide an experimental basis for elucidating the molecular mechanisms of immune escape by RCC and reveal a novel target to treat this disease.

Continuous reference intervals for leukocyte telomere length in children: the method matters.

OBJECTIVES: Children with very short telomeres commonly develop bone marrow failure and other severe diseases. Identifying the individuals with short telomeres can improve outcome of bone marrow transplantation, with accurate diagnosis requiring the use of age-matched reference intervals (RIs). This study aimed to establish RIs for telomere length (TL) in children using three commonly used methods for TL measurement. METHODS: Healthy children aged 30 days to 18 years were recruited for assessment using age as a continuous variable. Venous blood samples were collected and leukocyte TL was measured using terminal restriction fragment (TRF) analysis, quantitative PCR (QPCR) and flow cytometry with fluorescence in situ hybridization (Flow-FISH). Fractional polynomial model and quantile regression were performed to generate continuous RIs. Factors that might contribute to variation in TL, such as gender, were also examined. RESULTS: A total of 212 samples were analyzed. Continuous RIs are presented as functions of age. TRF analysis and QPCR showed significant negative correlation between TL and age (r=-0.28 and r=-0.38, p<0.001). In contrast, Flow-FISH showed no change in TL with age (r=-0.08, p=0.23). Gender did not have significant influence on TL in children. CONCLUSIONS: This study provides three options to assess TL in children by establishing method-specific continuous RIs. Choosing which method to use will depend on several factors such as amount and type of sample available and required sensitivity to age-related change.

Calcium Carbonate@silica Composite with Superhydrophobic Properties.

In this paper, spherical calcium carbonate particles were prepared by using CaCl2 aqueous solution + NH3·H2O + polyoxyethylene octyl phenol ether-10 (OP-10) + n-butyl alcohol + cyclohexane inverse micro emulsion system. Then, nanoscale spherical silica was deposited on the surface of micron calcium carbonate by Stöber method to form the composite material. Scanning electron microscope (SEM), X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS) were used to characterize the morphology and structure of the composite material. It is found that the surface of the composite material has a micro-nano complex structure similar to the surface of a "lotus leaf", making the composite material show hydrophobicity. The contact angle of the cubic calcium carbonate, spherical calcium carbonate and CaCO3@SiO2 composite material were measured. They were 51.6°, 73.5°, and 76.8°, respectively. After modification with stearic acid, the contact angle of cubic and spherical CaCO3 were 127.1° and 136.1°, respectively, while the contact angle of CaCO3@SiO2 composite was 151.3°. These results showed that CaCO3@SiO2 composite had good superhydrophobicity, and the influence of material roughness on its hydrophobicity was investigated using the Cassie model theory.

[Analysis of the Status of Depression and the Influencing Factors in Middle-Aged and Older Adults in China].

OBJECTIVE: To understand the status of depression and its influencing factors in the middle-aged and older adult populations aged 45 and above in China on the basis of data from the 2018 China Family Panel Studies (CFPS), and to provide empirical evidence for the improvement of the mental health of the middle-aged and older adults and the alleviation of their depressive symptoms. METHODS: The source of the research data was the 2018 CFPS. The Center for Epidemiologic Studies Depression Scale (CES-D) was used to assess the prevalence of depression. A two-level two-category unconditional logistics regression method was used to analyze the influencing factors of the prevalence of depressive symptoms. RESULTS: The 80th percentile interval score of depression score was used as the critical value, and the detection rate of depressive symptoms was 23.61%. It was more likely for women to suffer from depressive symptoms than it was for men. Widowed individuals were at an even higher risk for having depression. The more education one had, the lower the possibility of developing depression. Middle-aged and older adults in rural areas were more likely to suffer from depression. Middle-aged and older adults with chronic diseases and self-rated poor health were at higher risk of depression. Sleep time is a protective factor that suppressed symptoms. After controlling the above-mentioned individual-level factors, middle-aged and older adults in coastal and economically developed areas were less likely to suffer from depression than those from inland and economically underdeveloped areas did. CONCLUSION: The health departments concerned should focus on the depressive symptoms of women, widowed individuals, and middle-aged and older adults with chronic diseases. In rural areas and underdeveloped inland regions, the state should invest more health resources in the prevention and improvement of depression among middle-aged and older adults.

[Preparation of nanoemulsion spray from Moslae Herba volatile oil and its antibacterial activity].

Moslae Herba is a commonly used aromatic Chinese medicinal with volatile oil as the main effective component and exhibits broad-spectrum antibacterial and antiviral effects. However, the irritation and instability of Moslae Herba volatile oil necessitate the preparation into a specific dosage form. In this study, the steam distillation method was employed to extract the Moslae Herba volatile oil. The content of thymol and carvacrol in Moslae Herba volatile oil was determined by HPLC as(0.111 9±0.001 0) and(0.235 4±0.004 7) mg·mL~(-1), respectively. Pseudo-ternary phase diagrams and surfactants compounding were applied in the selection of the optimal excipients(surfactant and cosurfactant). On this basis, a nanoemulsion was prepared from the Moslae Herba volatile oil and then loaded into pressure vessels to get sprays, whose stability and antibacterial activity were evaluated afterward. With clarity, viscosity, smell and body feeling as comprehensive indexes, the optimal formulation of the Moslae Herba volatile oil nanoemulsion was determined as follows: Moslae Herba volatile oil∶peppermint oil∶cremophor EL∶absolute ethanol∶distilled water 7.78∶1.58∶19.26∶6.15∶65.23. The as-prepared nanoemulsion was a light yellow transparent liquid, with Tyndall effect shown under the irradiation of parallel light. It has the pH of 5.50, conductivity of 125.9 µS·cm~(-1), average particle size of 15.45 nm, polydispersity index(PDI) of 0.156, and Zeta potential of-17.9 mV. Under a transmission electron microscope, the Moslae Herba volatile oil nanoemulsion was presented as regular spheres without adhesion and agglomeration. Stability test revealed that the Moslae Herba volatile oil nanoemulsion was stable at 4-55 ℃, which was free from demulsification and stratification within 30 days. After the centrifugation at 12 000 r·min~(-1) for 30 min, there was no stratification either. The nanoemulsion had good inhibitory effects on Escherichia coli, Staphylococcus aureus and resistant S. aureus strains, with the minimum inhibitory concentrations of 0.39, 3.12 and 1.56 mg·mL~(-1), respectively. The above results demonstrated that the nanoemulsion was prepared feasibly and showed stable physical and chemical properties and good antibacterial effects. This study provides a practicable technical solution for the development of anti-epidemic and anti-infection products from Moslae Herba volatile oil.

Targeting USP1-dependent KDM4A protein stability as a potential prostate cancer therapy.

The histone demethylase lysine-specific demethylase 4A (KDM4A) is reported to be overexpressed and plays a vital in multiple cancers through controlling gene expression by epigenetic regulation of H3K9 or H3K36 methylation marks. However, the biological role and mechanism of KDM4A in prostate cancer (PC) remain unclear. Herein, we reported KDM4A expression was upregulation in phosphatase and tensin homolog knockout mouse prostate tissue. Depletion of KDM4A in PC cells inhibited their proliferation and survival in vivo and vitro. Further studies reveal that USP1 is a deubiquitinase that regulates KDM4A K48-linked deubiquitin and stability. Interestingly, we found c-Myc was a key downstream effector of the USP1-KDM4A/androgen receptor axis in driving PC cell proliferation. Notably, upregulation of KDM4A expression with high USP1 expression was observed in most prostate tumors and inhibition of USP1 promotes PC cells response to therapeutic agent enzalutamide. Our studies propose USP1 could be an anticancer therapeutic target in PC.

[Evaluation of Reliability and Validity of EQ-5D-5L Based on Residents in Southwest China].

OBJECTIVE: To explore the reliability and validity of the EQ-5D-5L scale in the population of southwest China. METHODS: The internal consistency reliability is measured by Cronbach's α coefficient and the structural validity is measured by factor analysis. The difference in health utility value of different characteristic populations is compared by t test and analysis of variance. RESULTS: Cronbach's α coefficient was 0.857. Exploratory factor analysis extracts two common factors whose cumulative contribution rate is 77.311%. The first common factor represents mobility, self-care and uaual activities. The second common factor represents pain/discomfort and anxiety/depression. The results of confirmatory factor analysis showed that the correlation of the two common factors was 0.659, the average variance of the first common factor was 0.862 and the combination reliability was 0.949, and the average variance extracted of the second common factor was 0.587 and the composite reliability was 0.739. The factor loadings for mobility, self-care and uaual activities on the first common factor were 0.871, 0.945 and 0.967, respectively. The loadings for pain/discomfort and anxiety/depression on the second common factor were 0.708 and 0.820, respectively. CONCLUSION: EQ-5D-5L has good reliability and validity when it is applied to the measurement of healthy life quality of residents in Southwest China.

[Application of whole transcriptomics in animal husbandry].

RNA is a polymeric molecule which is involved in various biological processes including the coding, decoding, regulation, and expression of genes. Whole transcriptome sequencing is the dominant method for studying RNA functions which assists researchers to deepen the exploration and analysis of gene structure and function and to reveal intrinsic links between gene expression and life phenomena. To date, extensive research has been done in animal husbandry models including swine and poultry by using high-throughput RNA sequencing technology. These studies included transcript structure, gene expression level and non-coding region function. In this review, we briefly introduce the general processes and strategies of RNA-seq and small RNA-seq technologies, and summarize the various achievements of the application of whole transcriptomics in animal husbandry.

[The Effects of Social Capital on Depressive Symptoms of Elderly Patients with Chronic Diseases in Urbanized Communities].

OBJECTIVE: To understand the effects of social capital on depressive symptoms of elderly patients with chronic diseases in urbanized communities, and to explore preventive measures to promote their mental health. METHODS: A multi-stage stratified cluster sampling method was used to extract 740 elderly patients with chronic diseases in the urbanized communities in Chengdu and Kunming. The questionnaire survey was conducted by using the center of depression rating scale (CES-D) and the self-made social capital scale. Multivariate unconditional logistic regression was used to analyze the impact of urbanized residents' social capital on depressive symptoms. RESULTS: The self-made social capital scale has good reliability and validity. The incidence of depressive symptoms in this study was 24.9%. The incidence of depressive symptoms in elderly females with chronic diseases was higher (P < 0.05); the residents with high "sense of social trust and security" had lower risk of incidence of depressive symptoms 〔odds ratio (OR)=0.489〕; the residents with higher "community belonging" had a lower risk of incidence of depressive symptoms (OR=0.570), and the residents with higher "social support" scores had a lower risk of incidence of depressive symptoms (OR=0.233). CONCLUSION: Targeted measures should be taken to intervene in the social capital factors affecting the depressive symptoms of elderly patients with chronic diseases in urbanized communities to improve their mental health.

[Multilevel Modelling on the Prevalence and Determinants of Depressive Symptoms in Middle and Old-aged Rural-to-urban Immigrants in Chengdu].

OBJECTIVE: To determine the prevalence and determinants of depressive symptoms in middle and old-aged rural-to-urban immigrants in Chengdu. METHODS: A total of 1 645 middle and old-aged rural-to-urban immigrants aged over 45 yr. were selected to participate in a questionnaire survey through a multi-stage random sampling method in Chengdu. Multilevel (households and individuals) models were established to identify predictors of depressive symptoms. RESULTS: About 14.5% of respondents reported depressive symptoms. The multilevel model indicated that family clustering of depressive symptoms existed. Household income and length of urban life at the household level, and age, chronic diseases, smoking, and social support at the individual level were significant predictors of depressive symptoms. CONCLUSION: The prevalence of depressive symptoms in middle and old-aged rural-to-urban immigrants deserves increasing policy attention for the purpose of promoting mental health in the population.

[Application of next-generation sequencing in the molecular diagnosis of Duchenne muscular dystrophy].

The purpose of this study is to analyze the family's clinical data of 22 children who were given an intended clinical diagnosis of Duchenne muscular dystrophy (DMD), and to explore the clinical value of next-generation sequencing (NGS) in the molecular diagnosis of DMD. The probands were simultaneously tested by NGS for a gene panel associated with hereditary neuromuscular disease and multiplex ligation-dependent probe amplification (MLPA) for the Dystrophin gene. The exon deletion/repetition mutations of the Dystrophin gene determined by both methods were compared and the point mutations of the Dystrophin gene were verified by Sanger sequencing. Dystrophin gene mutations were found in all the 22 probands, including 14 exon deletion/repetition mutations and 8 point mutations/minor variations. The results of MLPA detection were consistent with those of NGS. The results of Sanger sequencing showed that the point mutations and minor variations determined by NGS were correct. One missense mutation (c.6290G>T), 1 nonsense mutation (c.3487C>T) and 4 minor deletion-induced frameshift mutations (c.1208delG, c.7497_7506delGGTGGGTGAC, c.9421_9422delAA and c.8910_8913delTCTC) had not been reported in the Human Gene Mutation Database, and thus were considered as novel mutations of the Dystrophin gene. The results of this study showed that NGS can detect variations in the Dystrophin gene, including exon deletion/repetition, point mutation, minor deletion and intron mutation. Therefore, NGS is of certain clinical value in the molecular diagnosis of DMD and is worthy of recommendation.

Rho Kinase Type 1 (ROCK1) Promotes Lipopolysaccharide-induced Inflammation in Corneal Epithelial Cells by Activating Toll-Like Receptor 4 (TLR4)-Mediated Signaling.

BACKGROUND Rho kinases (ROCKs) are the typical downstream effectors of RhoA, which can regulate corneal epithelial wound healing. In this study, the role of ROCK1 in lipopolysaccharide (LPS)-induced cornea inflammation was investigated. MATERIAL AND METHODS The expression of ROCK1 in human corneal epithelial cells (HCECs) was bilaterally modulated with ROCK1 expression vector and ROCK1 inhibitor Y-27632. The effects of ROCK1 modulation on the inflammatory response, cell viability, cell apoptosis, and cell cycle distribution were detected by ELISA assay, MTT assay, and flow cytometry, respectively. The pathways involved in the effect of ROCK1 in HCECs was preliminarily explained by detecting changes of TLR4-mediated NF-kB and ERK signaling using western blotting and electrophoretic mobility shift assays. RESULTS Overexpression of ROCK1 promoted LPS-induced production of IL-6, IL-8, IL-1ß, and TNF-α, and the apoptotic process in HCECs. Augmented inflammation and apoptosis were associated with stronger activation of TLR4-mediated signal transduction; the phosphorylation of IkBa, JNK, ERK1/2, and p38, and nuclear translocation of NF-κB p65 induced by LPS were further increased by overexpression of ROCK1. Inhibition of ROCK1 function by Y-27632 blocked the effect of LPS on HCECs; both LPS-induced inflammation and apoptosis was alleviated by Y-27632, which was associated with suppression of TLR4-mediated NF-κB and ERK signaling. CONCLUSIONS LPS-induced inflammation and apoptosis in HCECs depended on the function of ROCK1, inhibition of which would attenuate impairments on cornea cells due to LPS.

Comparative Transcriptome Profiling Reveals Defense-Related Genes against Meloidogyne incognita Invasion in Tobacco.

Root-knot nematodes Meloidogyne incognita are one of the most destructive pathogens, causing severe losses to tobacco productivity and quality. However, the underlying resistance mechanism of tobacco to M. incognita is not clear. In this study, two tobacco genotypes, K326 and Changbohuang, which are resistant and susceptible to M. incognita, respectively, were used for RNA-sequencing analysis. An average of 35 million clean reads were obtained. Compared with their expression levels in non-infected plants of the same genotype, 4354 and 545 differentially expressed genes (DEGs) were detected in the resistant and susceptible genotype, respectively, after M. incognita invasion. Overall, 291 DEGs, involved in diverse biological processes, were common between the two genotypes. Genes encoding toxic compound synthesis, cell wall modification, reactive oxygen species and the oxidative burst, salicylic acid signal transduction, and production of some other metabolites were putatively associated with tobacco resistance to M. incognita. In particular, the complex resistance response needed to overcome M. incognita invasion may be regulated by several transcription factors, such as the ethylene response factor, MYB, basic helix⁻loop⁻helix transcription factor, and indole acetic acid⁻leucine-resistant transcription factor. These results may aid in the identification of potential genes of resistance to M. incognita for tobacco cultivar improvement.