Direct dsRNA preparation by promoter-free RCT and RNase H cleavage using one circular dsDNA template with a mismatched bubble.

Double-stranded RNA (dsRNA) has aroused widespread interest due to its effects on immunity and applications based on RNAi. However, the in vitro preparation of dsRNA is costly and laborious. In this study, we have developed a novel and interesting method designated as pfRCT (promoter-free rolling-circle transcription) for direct, facile, and efficient dsRNA preparation. This method generates equal amounts of sense and antisense strands simultaneously from a single circular dsDNA template. To initiate transcription by T7 RNA polymerase without directional preference, a 9-15-bp bubble (mismatched duplex with strong sequence symmetry) is introduced into the template. During RCT, all the necessary reagents, including the template, NTPs, RNA polymerase, RNase H, and Helpers, are present in one pot; and the just-transcribed RNA is immediately truncated by RNase H to monomers with the desired size. The ends of the dsRNA product can also be simply sealed by T4 RNA ligase 1 after pfRCT. This new approach is expected to promote the applications of dsRNA.

Selection and Characterization of a Novel DNA Aptamer, Apt-07S Specific to Hepatocellular Carcinoma Cells.

BACKGROUND: The efficacy of traditional therapeutic methods for liver cancer is unsatisfying because of the poor targeting, and inefficient drug delivery system. A recent study has proven that aptamers, developed through cell-SELEX, could specifically recognize cancer cells and show great potential in the development of a delivery system for anticancer drugs. PURPOSE: To develop a hepatocellular carcinoma specific aptamer using two kinds of hepatocellular carcinoma cell lines, HepG2 and SMMC-7721, as double targets and a normal hepatocyte, L02, as a negative control cell. METHODS: Hepatocellular carcinoma specific aptamer was developed via cell-SELEX. The enrichment of the library was monitored by flow cytometric analysis. The specificity, affinity, and distribution of the candidate aptamer were explored. Further study was carried to assess its potential in drug delivery. RESULTS: The library was enriched after 14 rounds of screening. Candidate aptamer Apt-07S can recognize four kinds of hepatocellular carcinoma cells and show little cell-binding ability to normal cells and four cell lines of different cancer types, revealing a high specificity of Apt-07S. Confocal imaging showed that Apt-07S distributed both on the surface and in the cytoplasm of the two target cells. Moreover, an anti-sense nucleotide to gene Plk1 (ASO-Plk1) was connected at the 3' end of Apt-07S to form an integrated molecule (Apt-07S-ASO-Plk1); the functional analysis indicated that the structure of Apt-07S may help ASO-Plk1 enter the cancer cells. CONCLUSION: The study indicates that Apt-07S can specifically target HCC and may have potential in the delivery of anticancer drugs.

Mutations in ASH1L confer susceptibility to Tourette syndrome.

Tourette syndrome (TS) is a childhood-onset neuropsychiatric disorder characterized by repetitive motor movements and vocal tics. The clinical manifestations of TS are complex and often overlap with other neuropsychiatric disorders. TS is highly heritable; however, the underlying genetic basis and molecular and neuronal mechanisms of TS remain largely unknown. We performed whole-exome sequencing of a hundred trios (probands and their parents) with detailed records of their clinical presentations and identified a risk gene, ASH1L, that was both de novo mutated and associated with TS based on a transmission disequilibrium test. As a replication, we performed follow-up targeted sequencing of ASH1L in additional 524 unrelated TS samples and replicated the association (P value = 0.001). The point mutations in ASH1L cause defects in its enzymatic activity. Therefore, we established a transgenic mouse line and performed an array of anatomical, behavioral, and functional assays to investigate ASH1L function. The Ash1l+/- mice manifested tic-like behaviors and compulsive behaviors that could be rescued by the tic-relieving drug haloperidol. We also found that Ash1l disruption leads to hyper-activation and elevated dopamine-releasing events in the dorsal striatum, all of which could explain the neural mechanisms for the behavioral abnormalities in mice. Taken together, our results provide compelling evidence that ASH1L is a TS risk gene.

Identification of Two Missense Mutations in DUOX1 (p.R1307Q) and DUOXA1 (p.R56W) That Can Cause Congenital Hypothyroidism Through Impairing H2O2 Generation.

Context: The DUOX/DUOXA systems play a key role in H2O2 generation in thyroid cells, which is required for iodine organification and thyroid hormone synthesis. DUOX2/DUOXA2 defects can cause congenital hypothyroidism (CH), but it is unknown whether DUOX1/DUOXA1 mutations can also cause CH. Objective: We aimed to identify DUOX1/DUOXA1 mutations and explore their role in the development of CH by investigating their functional impacts on H2O2 generation. Patients and Methods: Forty-three children with CH with goiter were enrolled, in whom all exons and flanking intronic regions of DUOX1/DUOXA1 were directly sequenced. We characterized the functional effects of identified mutations on the expression of DUOX1 and DUOXA1 and H2O2 generation. Results: We identified a heterozygous DUOX1 missense mutation (G > A base substitution at nucleotide 3920 in exon 31) that changed a highly conserved arginine to glutamine at residual 1307 (p.R1307Q) in patient 1. A heterozygous-missense mutation (c.166 C>T; p.R56W) was identified in DUOXA1 in patient 2. Functional studies demonstrated that both p.R1307Q mutant or p.R56W mutant decreased the DUOX1 expression at mRNA and protein levels, with a corresponding impairment in H2O2 generation (P < 0.01). The results also showed that intact DUOXA1 was required for full activity of DUOX1 and H2O2 generation. Conclusions: We have identified two heterozygous missense mutations in DUOX1 and DUOXA1 in two patients that can cause CH through disrupting the coordination of DUOX1 and DUOXA1 in the generation of H2O2. This study for the first time demonstrates that the DUOX1/DUOXA1 system, if genetically defective, can cause CH.

Fucoidan prevent murine autoimmune diabetes via suppression TLR4-signaling pathways, regulation DC/Treg induced immune tolerance and improving gut microecology.

BACKGROUND: This study was to investigate the effect and its possible mechanism of fucoidan on the development of spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice. METHODS: 7-week-old NOD mice were randomly divided into three groups: control group, low-dose (300 mg/kg) and high-dose (600 mg/kg) fucoidan-treatment groups. After 5 weeks of treatment, 10 mice per group were randomly selected to be sacrificed after feces collection. The remaining 12 mice per group were fed until 26 weeks of age to assess the incidence of diabetes. RESULTS: Treatment with fucoidan increased serum insulin level, delayed the onset and decreased the development of diabetes in NOD mice. Fucoidan reduced the levels of strong Th1 proinflammatory cytokines, but induced Th2-bias ed. cytokine response. And dentridic cells (DCs) in fucoidan treatment group were characterized as low expression of MHC class II and CD86 molecules. TLR4 expressions and the downstream molecules in pancreas were down-regulated in fucoidan-treated groups. There were significant differences in the composition of gut flora between NOD control group and fucoidan group. Lactobacillus and Akkermansia were significantly enriched in fucoidan group. CONCLUSIONS: Fucoidan could prevent the development of autoimmune diabetes in NOD mice via regulating DC/Treg induced immune tolerance, improving gut microecology, down-regulating TLR4 signaling pathway, and maintaining pancreatic internal environment.

The suppressive effect of co-inhibiting PD-1 and CTLA-4 expression on H22 hepatomas in mice.

OBJECTIVE: We investigated the suppressive effect of siRNA-mediated co-inhibition of PD-1 and CTLA-4 expression on H22 hepatomas in mice. METHODS: Murine H22 cells were cultured in vivo in ICR mice. An allograft tumor model was also established in another ICR mouse group. The tumor-bearing mice were randomly divided into four groups: control, single PD-1 siRNA, single CTLA-4 siRNA, and double PD-1 + CTLA-4 siRNAs. The survival time and physiological condition of the mice were observed after the injection of the siRNAs and placebo. The volume and weight of the solid tumor were measured to assess the inhibition of the tumor. To assess the effects of siRNAs on mouse immune function, the protein levels of IFN-γ and IL-10 in the blood and PD-L1 in the tumor and liver were determined using ELISA, and the mRNA levels of IFN-γ, PD-L1, PD-1, CTLA-4, IL-6 and Survivin in the tumor, liver and spleen were determined using quantitative RT-PCR. The ratios of Bax and Bcl-2 protein were determined via western blot to analyze the effect of siRNAs on tumor cell apoptosis. RESULTS: The anti-tumor effect appeared in all groups with siRNA-mediated inhibition. The tumor growth suppression was stronger in the group with double inhibition. The weight and volume of the tumors were significantly lower and the survival rate improved in the three siRNA groups. IFN-γ levels increased but IL-10 levels decreased in the blood of the siRNA group mice compared with the results for the control group. In the tumor and spleen tissue, the IFN-γ levels significantly increased, but in the liver tissue they significantly decreased in the three siRNA groups. The results of quantitative RT-PCR showed that the mRNAs for PD-1 and CTLA-4 were downregulated in spleen tissue in the three siRNA groups, while the PD-L1 mRNA and protein levels increased significantly in the tumor, but decreased in the liver. Survivin and IL-6 mRNA levels decreased in the tumor. Western blot results showed that ratio of Bax and Bcl-2 had significantly increased. These results indicated that downregulating PD-1 and CTLA-4 could increase the body's immune response and promote apoptosis of tumor cells. CONCLUSION: Co-inhibiting the expressions of PD-1 and CTLA-4 can effectively suppress the growth of H22 hepatoma and promote the apoptosis of tumor cells in mice. Blocking PD-1 and CTLA-4 can improve the vitality of T cells, and improve the immune environment and response.

RETRACTED: The function of long non-coding RNA MT1JP in the development and progression of gastric cancer.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Executive Editor (Chairman) as panels from Figures 3A,B and 4D are similar to panels from Figures 4A,B and 5E of the article published by Mingjun Bi, Hongmei Yu, Bin Huang and Cuiyan Tang in Gene 626 (2017) 337­343 http://dx.doi.org/10.1016/j.gene.2017.05.049. Also, Figures 3C and 4A are similar to Figures 4C and 5A of the Gene article. One of the conditions of submission of a paper for publication is that authors declare explicitly that their work is original and has not appeared in a publication elsewhere. As such this article represents an abuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process.

Effects of n-3PUFAs on autophagy and inflammation of hypothalamus and body weight in mice.

OBJECTIVE: Fat-1 transgenic mice were used as a model to study the effect of endogenous n-3 polyunsaturated fatty acids (n-3PUFAs) on the body weight, inflammatory factors and autophagy proteins in hypothalamus to explore the mechanism of n-3PUFAs inhibiting obesity. METHOD: The mice were divided into two groups after genotype identification: fat-1 transgenic mice and wild-type mice. The body weight and body length of mice were measured at 14th week, and calculated the Lee 's index. The autophagosome in arcuate nucleus neurons was observed through electron microscopy; the expression of autophagy protein P62, LC3 and ATG7 in hypothalamus were detected and analyzed quantitatively by immunofluorescence and Western blot techniques. The mRNAs of inflammatory factor TNF-α, IL-6, IL-1ß, NF-kB, chemokine MCP-1, CCL5, CXCL12, CX3CL1, microglia markers TMEM119, GFAP were detected by real-time fluorescence quantitative PCR. RESULT: The Lee's index of fat-1 transgenic mice was lower than that of wild-type mice(P < 0.05). The autophagosome of the arcuate nucleus in fat-1 transgenic mice were more than those in wild-type mice, and the expression of autophagy-related protein P62 was significantly decreased (P < 0.05) in hypothalamus of fat-1 transgenic mice, while the expression of autophagy related protein ATG7 was significantly up-regulated (P < 0.05), and the ratio of LC3 II/I was significantly increased (P < 0.05), The results of qPCR showed that the mRNAs of TNF-α, IL-6, IL-1ß, NF-kB, MCP-1, CCL5, CXCL12, and GFAP was significantly down regulated (P < 0.05), but CX3CL1 was significantly up-regulated (P < 0.05) in hypothalamus of fat-1 transgenic mice. CONCLUSION: Fat-1 gene or n-3 PUFAs possesses the function of reducing body weight, which involves the enhancement of autophagy and reduction of inflammatory factor in hypothalamus.

Caspase-dependent and caspase-independent induction of apoptosis in breast cancer by fucoidan via the PI3K/AKT/GSK3ß pathway in vivo and in vitro.

Purpose Fucoidan, a complex, sulfated polysaccharide obtained from brown seaweed, exerted anticancer activity through the down-regulation of ß-catenin signaling in mouse breast cancer cells in our previous study. This study examines the anti-cancer effects of fucoidan as well as its underlying molecular mechanisms in the human triple negative breast cancer (TNBC) cell line and in 7,12-dimethylbenz[a]anthracene (DMBA)-induced experimental mammary carcinogenesis in rats. Methods in vitro studies, fluorescent staining, flow cytometry and Western blotting were performed to analyze apoptosis and protein expression in human breast cancer MDA-MB-231 cells. In vivo intervention experiments were conducted with Sprague Dawley (SD) rats with DMBA-induced breast cancer. Tumor volumes and weights were measured. Results in vitro fucoidan treatment inhibited proliferation and induced apoptosis in MDA-MB-231 cells. Western blotting detected that Cyt C and Smac were released into the cell cytoplasm and that caspase-3 and caspase-9 were activated in MDA-MB-231 cells. The levels of AIF and EndoG were significantly increased in the cytoplasm and in the nuclei by fucoidan. These data show that fucoidan induced caspase-dependent and caspase-independent apoptosis. Moreover, fucoidan treatment down-regulated the expression of Bid, Bcl-2 and Bcl-xl and up-regulated the level of Bax. In vivo, fucoidan supplementation decreased the mean tumor weight. DISCUSSION: Results from the in vivo and in vitro experiments both showed that fucoidan decreased the levels of p-PI3K, p-AKT and p-GSK-3ß (Ser9) in breast cancer. The level of ß-catenin was also decreased. These results suggest that fucoidan can inhibit MDA-MB-231 human breast cancer cells and DMBA-induced tumors in rats by down-regulating the PI3 K/AKT/GSK3ß pathway. This study provides experimental evidence that elucidates the mechanism of antitumor effect of fucoidan and clarifies the mechanism of the effect of fucoidan on the regulation of ß-catenin.

Detection of Diverse N-Acyl-Homoserine Lactones in Vibrio alginolyticus and Regulation of Biofilm Formation by N-(3-Oxodecanoyl) Homoserine Lactone In vitro.

Quorum sensing (QS) is a cell-to-cell communication system based on the exchange of small intercellular signal molecules, such as N-Acyl homoserine lactones (AHLs), which act as cell-density mediators of QS gene expression, and are highly variable both in types and amounts in most Gram-negative Proteobacteria. Understanding the regulation of AHLs may contribute to the elucidation of cell density-dependent phenomena, such as biofilm formation. Vibrio alginolyticus is among the most frequently observed marine opportunistic Vibrio pathogens. However, AHL production of this species and its effects on biofilm formation remain to be understood. Here, our study reported the diverse AHL profiles of 47 marine-isolated V. alginolyticus strains and the effects of exogenous 3-oxo-C10-HSL on biofilm formation under different temperature conditions (16°C and 28°C). A total of 11 detected AHLs were produced by the isolates, of which 3-OH-C4-HSL, 3-oxo-C10-HSL and 3-oxo-C14-HSL comprised the largest proportions. We also observed that moderate levels of exogenous 3-oxo-C10-HSL (10 and 20 µM) could induce or enhance biofilm formation and alter its structure, while high levels (40 and 100 µM) did not significantly improve and even inhibited biofilm formation in V. alginolyticus. Further, regulation by exogenous 3-oxo-C10-HSL was both concentration- and temperature-dependent in V. alginolyticus.

Apoptosis is induced by docosahexaenoic acid in breast cancer cells via death receptor and mitochondria-mediated pathways.

In the present study, the antitumor effect of n­3 fatty acid was evaluated, and the effect of docosahexaenoic acid (DHA) on the induction of apoptosis and its underlying mechanism were examined. Flow cytometry and western blot analysis were performed to analyze apoptosis and the expression of protein factors in human breast cancer cells. The data revealed that DHA inhibited the viability of MCF­7 breast cancer cells in vitro, and promoted cell death by the induction of apoptosis. DHA decreased the expression of B­cell lymphoma 2 (Bcl­2), whereas the expression of Bcl­2­associated X protein was increased. DHA was also shown to promote the release of Smac/Diablo and cytochrome c from the mitochondria. DHA increased the levels of cleaved caspase­8, ­9 and ­3. Additionally, the protein expression of tumor necrosis factor­related apoptosis­inducing ligand, death receptor 4 and Fas were increased following DHA treatment. In conclusion, DHA caused apoptosis of the human breast cancer cells in vitro through the death receptor and mitochondria­mediated pathways. The results of this study encourage further investigation of the effect of fish oil on the prevention and treatment of human breast cancer.

Identification and characterization of novel PAX8 mutations in Congenital Hypothyroidism(CH) in a Chinese population.

OBJECTIVE: Based on mutations in PAX8 is associated with thyroid dysgenesis. We aim to identify and characterize PAX8 mutations in a large cohort of congenital hypothyroidism(CH) from thyroid dysgenesis in Chinese population. METHODS: We screened 453 unrelated Chinese patients with CH from thyroid dysgenesis for PAX8 mutations by sequencing the whole coding regions of PAX8 on genomic DNA isolated from blood. Cell transfection assays using various vector constructs and induced mutagenesis as well as electrophoretic mobility shift assays were used to investigate the effects of selected mutations on the transcribing and binding activities of PAX8 at the promoters of target genes for thyroglobulin (TG) and thyroperoxidase (TPO). RESULTS: Five PAX8 mutations were found, yielding a mutation prevalence of 5/453 (1.1%). We selected two mutations in the critical paired domain of PAX8 and generated mutants D94N and G41V. We demonstrated G41V was unable to bind the specific sequence in the promoters of TG and TPO and activate them. D94N could bind to TG and TPO promoters and normally activate the TG promoter transcription but not the TPO promoter transcription. We also demonstrated a dominant negative role of the PAX8 mutants in impairing the function of the wild-type PAX8. CONCLUSION: We for the first time documented the prevalence and characterized the function of PAX8 mutations in CH in Chinese population. The study specifically demonstrated the role of novel mutations D94N and G41V in impairing the function of PAX8, providing further evidence for genetic PAX8 defects as a disease mechanism in CH.

RNAi-mediated gene silencing of vascular endothelial growth factor C suppresses growth and induces apoptosis in mouse breast cancer in vitro and in vivo.

Vascular endothelial cell growth factor (VEGF)-C promotes tumorigenesis by allowing lymph node metastasis and lymphangiogenesis, among other actions. RNA interference (RNAi) is a novel technique for suppressing target gene expression and may increase the effectiveness of cancer treatments. The present study assessed the influence of VEGF-C RNAi on the apoptosis and proliferation of mouse breast cancer cells in vitro and in vivo. A total of three pairs of small interfering RNA (siRNA) targeting mouse VEGF-C were designed and synthesized prior to transfection into 4T1 cells via a liposomal approach. Reverse transcription polymerase chain reaction, western blot analysis, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst 33258 staining and flow cytometry were performed in vitro to analyze VEGF-C expression, cleaved caspase-3 protein expression and 4T1 cell proliferation and apoptosis. Experiments were also conducted in vivo on BALB/c mice with breast cancer. Tumor weight and volume were measured and the number of apoptotic cells in tumor tissues was assessed by a TUNEL assay. Immunohistochemical assays and an enzyme-linked immunosorbent assay were used to measure the expression of VEGF-C in tumor tissues. The results demonstrated that the three pairs of siRNA, particularly siV2, significantly reduced VEGF-C mRNA and protein levels in 4T1 cells. siV2 was deemed to be the most efficient siRNA and therefore was selected to be used in subsequent experiments. Furthermore, in vitro studies indicated that VEGF-C RNAi significantly decreased cell growth, induced apoptosis and upregulated the expression of cleaved caspase-3 protein. Tumor weight and volume in breast cancer in vivo models was reduced by the intratumoral injection of siV2. Antitumor efficacy was associated with decreased VEGF-C expression and increased induction of apoptosis. The present study therefore indicated that VEGF-C RNAi inhibited mouse breast cancer growth in vitro and in vivo and that it may be a novel targeted therapy for breast cancer.

Downregulation of survivin expression exerts antitumoral effects on mouse breast cancer cells in vitro and in vivo.

Metastasis constantly occurs in the majority of cases of primary breast cancer at late stage or following surgical treatment. Survivin, a member of the inhibitor of apoptosis protein family, has long been recognized as a promising anticancer target, but its antitumor effects remain largely unexplored. In order to elucidate the role of survivin in breast cancer metastasis, short interfering RNA (siRNA) was used in the present study to specifically downregulate survivin expression in the murine breast cancer cell line 4T1. The results demonstrated that blocking the expression of survivin by siRNA inhibited the proliferation, migration and invasion abilities of murine breast cancer cells in vitro. Vascular endothelial growth factor (VEGF)-C is a lymphatic endothelial cell-stimulating factor that may lead to the formation of lymphatic vessels in lymph nodes. In the present study, the inhibition of survivin by siRNA was able to reduce the overexpression of VEGF-C in 4T1 cells. Furthermore, intratumoral injections of the survivin-siRNA significantly inhibited the growth of orthotopically transplanted 4T1 tumors in vivo. In addition, the number of pulmonary metastases and the microlymphatic vessel density were significantly reduced in vivo, following transfection with survivin-siRNA. The results of the present study suggested that the Akt/hypoxia-inducible factor-1α signaling pathway participates in the survivin-mediated downregulation of VEGF-C expression observed in breast cancer cells treated with survivin-siRNA. Therefore, the use of siRNA specifically targeting survivin may be a potential anticancer method in the future.

Screening of HHEX Mutations in Chinese Children with Thyroid Dysgenesis.

OBJECTIVE: Congenital hypothyroidism (CH) is a frequent neonatal endocrine disease with an incidence of about 1:2500 worldwide. Although thyroid dysgenesis (TD) is the most frequent cause of CH cases, its pathogenesis remains unclear. The aim of this study was to screen the hematopoietically-expressedhomeobox gene (HHEX) mutations in Chinese children with TD. METHODS: Genomic deoxyribonucleic acid was extracted from peripheral blood leukocytes in 234 TD patients from Shandong Province. Mutations in all exons and nearby introns of HHEX were analyzed by direct sequencing after polymerase chain reaction amplification. RESULTS: Sequencing analysis of HHEX indicated that no causative mutations were present in the coding regionof the TD patients. However, a genetic variant (IVS2+ 127 G/T, 10.26%) was observed in the intron 2 in HHEX. CONCLUSION: Our results indicate that the frequency of HHEX mutation is very low and may not be the main causative factor in Chinese TD patients. However, these results need to be replicated using larger datasets collected from different populations.

Transgenic n-3 PUFAs enrichment leads to weight loss via modulating neuropeptides in hypothalamus.

Body weight is related to fat mass, which is associated with obesity. Our study explored the effect of fat-1 gene on body weight in fat-1 transgenic mice. In present study, we observed that the weight/length ratio of fat-1 transgenic mice was lower than that of wild-type mice. The serum levels of triglycerides (TG), cholesterol (CT), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c) and blood glucose (BG) in fat-1 transgenic mice were all decreased. The weights of peri-bowels fat, perirenal fat and peri-testicular fat in fat-1 transgenic mice were reduced. We hypothesized that increase of n-3 PUFAs might alter the expression of hypothalamic neuropeptide genes and lead to loss of body weight in fat-1 transgenic mice. Therefore, we measured mRNA levels of appetite neuropeptides, Neuropeptide Y (NPY), Agouti-related peptides (AgRP), Proopiomelanocortin (POMC), Cocaine and amphetamine regulated transcript (CART), ghrelin and nesfatin-1 in hypothalamus by real-time PCR. Compared with wild-type mice, the mRNA levels of CART, POMC and ghrelin were higher, while the mRNA levels of NPY, AgRP and nesfatin-1 were lower in fat-1 transgenic mice. The results indicate that fat-1 gene or n-3 PUFAs participates in regulation of body weight, and the mechanism of this phenomenon involves the expression of appetite neuropeptides and lipoproteins in fat-1 transgenic mice.

Support of positive association in family-based genetic analysis between COL27A1 and Tourette syndrome.

Recently, a genome-wide association study has indicated associations between single nucleotide polymorphisms in the Collagen Type XXVII Alpha 1 gene (COL27A1) and Tourette syndrome in several ethnic populations. To clarify the global relevance of the previously identified SNPs in the development of Tourette syndrome, the associations between polymorphisms in COL27A1 and Tourette syndrome were assessed in Chinese trios. PCR-directed sequencing was used to evaluate the genetic contributions of three SNPs in COL27A1(rs4979356, rs4979357 and rs7868992) using haplotype relative risk (HRR) and transmission disequilibrium tests (TDT) with a total of 260 Tourette syndrome trios. The family-based association was significant between Tourette syndrome and rs4979356 (TDT: χ2 = 4.804, P = 0.033; HRR = 1.75, P = 0.002; HHRR = 1.32, P = 0.027), and transmission disequilibrium was suspected for rs4979357 (TDT: χ2 = 3.969, P = 0.053; HRR = 1.84, P = 0.001; HHRR = 1.29, P = 0.044). No statistically significant allele transfer was found for rs7868992 (TDT: χ2 = 2.177, P = 0.158). Although the TDT results did not remain significant after applying the conservative Bonferroni correction (p = 0.005), the significant positive HRR analysis confirmed the possibility of showing transmission disequilibrium, which provides evidence for an involvement of COL27A1in the development of TS. However, these results need to be verified with larger datasets from different populations.

Digestion of Nucleic Acids Starts in the Stomach.

The ingestion of nucleic acids (NAs) as a nutritional supplement or in genetically modified food has attracted the attention of researchers in recent years. Discussions over the fate of NAs led us to study their digestion in the stomach. Interestingly, we found that NAs are digested efficiently by human gastric juice. By performing digests with commercial, recombinant and mutant pepsin, a protein-specific enzyme, we learned that the digestion of NAs could be attributed to pepsin rather than to the acidity of the stomach. Further study showed that pepsin cleaved NAs in a moderately site-specific manner to yield 3'-phosphorylated fragments and the active site to digest NAs is probably the same as that used to digest protein. Our results rectify the misunderstandings that the digestion of NAs in the gastric tract begins in the intestine and that pepsin can only digest protein, shedding new light on NA metabolism and pepsin enzymology.

PUFA diets alter the microRNA expression profiles in an inflammation rat model.

Omega­3 and ­6 polyunsaturated fatty acids (PUFAs) can directly or indirectly regulate immune homeostasis via inflammatory pathways, and components of these pathways are crucial targets of microRNAs (miRNAs). However, no study has examined the changes in the miRNA transcriptome during PUFA­regulated inflammatory processes. Here, we established PUFA diet­induced autoimmune­prone (AP) and autoimmune­averse (AA) rat models, and studied their physical characteristics and immune status. Additionally, miRNA expression patterns in the rat models were compared using microarray assays and bioinformatic methods. A total of 54 miRNAs were differentially expressed in common between the AP and the AA rats, and the changes in rno­miR­19b­3p, ­146b­5p and ­183­5p expression were validated using stem­loop reverse transcription­quantitative polymerase chain reaction. To better understand the mechanisms underlying PUFA­regulated miRNA changes during inflammation, computational algorithms and biological databases were used to identify the target genes of the three validated miRNAs. Furthermore, Gene Ontology (GO) term annotation and KEGG pathway analyses of the miRNA targets further allowed to explore the potential implication of the miRNAs in inflammatory pathways. The predicted PUFA­regulated inflammatory pathways included the Toll­like receptor (TLR), T cell receptor (TCR), NOD­like receptor (NLR), RIG­I­like receptor (RLR), mitogen­activated protein kinase (MAPK) and the transforming growth factor­ß (TGF­ß) pathway. This study is the first report, to the best of our knowledge, on in vivo comparative profiling of miRNA transcriptomes in PUFA diet­induced inflammatory rat models using a microarray approach. The results provide a useful resource for future investigation of the role of PUFA­regulated miRNAs in immune homeostasis.