A mechanism for SARS-CoV-2 RNA capping and its inhibition by nucleotide analog inhibitors.

Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here, we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The NiRAN domain in polymerase catalyzes the covalent link of RNA 5' end to the first residue of nsp9 (termed as RNAylation), thus being an intermediate to form cap core (GpppA) with GTP catalyzed again by NiRAN. We also reveal that triphosphorylated nucleotide analog inhibitors can be bonded to nsp9 and fit into a previously unknown "Nuc-pocket" in NiRAN, thus inhibiting nsp9 RNAylation and formation of GpppA. S-loop (residues 50-KTN-52) in NiRAN presents a remarkable conformational shift observed in RTC bound with sofosbuvir monophosphate, reasoning an "induce-and-lock" mechanism to design inhibitors. These findings not only improve the understanding of SARS-CoV-2 RNA capping and the mode of action of NAIs but also provide a strategy to design antiviral drugs.

FoxO1 promotes ovarian cancer by increasing transcription and METTL14-mediated m6A modification of SMC4.

The transcription factor forkhead box protein O1 (FoxO1) is closely related to the occurrence and development of ovarian cancer (OC), however its role and molecular mechanisms remain unclear. Herein, we found that FoxO1 was highly expressed in clinical samples of OC patients and was significantly correlated with poor prognosis. FoxO1 knockdown inhibited the proliferation of OC cells in vitro and in vivo. ChIP-seq combined with GEPIA2 and Kaplan-Meier database analysis showed that structural maintenance of chromosome 4 (SMC4) is a downstream target of FoxO1, and FoxO1 promotes SMC4 transcription by binding to its -1400/-1390 bp promoter. The high expression of SMC4 significantly blocked the tumor inhibition effect of FoxO1 knockdown. Furtherly, FoxO1 increased SMC4 mRNA abundance by transcriptionally activating methyltransferase-like 14 (METTL14) and increasing SMC4 m6A methylation on its coding sequence region. The Cancer Genome Atlas dataset analysis confirmed a significant positive correlation between FoxO1, SMC4, and METTL14 expression in OC. In summary, this study revealed the molecular mechanisms of FoxO1 regulating SMC4 and established a clinical link between the expression of FoxO1/METTL14/SMC4 in the occurrence of OC, thus providing a potential diagnostic target and therapeutic strategy.

Metabolomic analysis of hydroxycinnamic acid inhibition on Saccharomyces cerevisiae.

Ferulic acid (FA) and p-coumaric acid (p-CA) are hydroxycinnamic acid inhibitors that are mainly produced during the pretreatment of lignocellulose. To date, the inhibitory mechanism of hydroxycinnamic acid compounds on Saccharomyces cerevisiae has not been fully elucidated. In this study, liquid chromatography-mass spectrometry (LC-MS) and scanning electron microscopy (SEM) were used to investigate the changes in S. cerevisiae cells treated with FA and p-CA. In this experiment, the control group was denoted as group CK, the FA-treated group was denoted as group F, and the p-CA-treated group was denoted as group P. One hundred different metabolites in group F and group CK and 92 different metabolites in group P and group CK were selected and introduced to metaboanalyst, respectively. A total of 38 metabolic pathways were enriched in S. cerevisiae under FA stress, and 27 metabolic pathways were enriched in S. cerevisiae under p-CA stress as identified through Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis. The differential metabolites involved included S-adenosine methionine, L-arginine, and cysteine, which were significantly downregulated, and acetyl-CoA, L-glutamic acid, and L-threonine, which were significantly upregulated. Analysis of differential metabolic pathways showed that the differentially expressed metabolites were mainly related to amino acid metabolism, nucleotide metabolism, fatty acid degradation, and the tricarboxylic acid cycle (TCA). Under the stress of FA and p-CA, the metabolism of some amino acids was blocked, which disturbed the redox balance in the cells and destroyed the synthesis of most proteins, which was the main reason for the inhibition of yeast cell growth. This study provided a strong scientific reference to improve the durability of S. cerevisiae against hydroxycinnamic acid inhibitors. KEY POINTS: • Morphological changes of S. cerevisiae cells under inhibitors stress were observed. • Changes of the metabolites in S. cerevisiae cells were explored by metabolomics. • One of the inhibitory effects on yeast is due to changes in the metabolic network.

Subacute exposure to DEHP leads to impaired decidual reaction and exacerbates the risk of early miscarriage in mice. / 孕早期增塑剂DEHPäºšæ€¥æ€§æš´éœ²å½±å“èœ•è†œååº”å¹¶å¢žåŠ æµäº§é£Žé™©.

OBJECTIVES: To investigate the effect of subacute exposure of Di (2-ethylhexyl) phthalate (DEHP) on endometrial decidualization and early pregnancy miscarriage in mice. METHODS: CD1 mice were orally administrated with 300 (low-dose group), 1000 (medium-dose group), or 3000 mg·kg－1·d－1 DEHP (1/10 LD50, high-dose group) for 28 days, respectively. An early natural pregnancy model and an artificially induced decidualization model were established. The uterine tissues were collected on D7 of natural pregnancy and D8 of artificially induced decidualization, respectively. The effects of a subacute exposure to DEHP on the decidualization of mice were detected by HE staining, Masson staining, TUNEL assay, and Western blotting. A model of spontaneous abortion was constructed in mice after subacute exposure to 300 mg·kg－1·d－1 DEHP, and the effect of impaired decidualization on pregnancy was investigated by observing the pregnancy outcome on the 10th day of gestation. RESULTS: Compared with the control group, the conception rate was significantly decreased in the high-dose DEHP subacute exposure group (P<0.05). HE staining showed that, compared with the control group, the decidual stromal cells in the low- and medium-dose exposure groups were disorganized, the nuclei of the cells were irregular, the cytoplasmic staining was uneven, and the number of polymorphonuclear cells was significantly reduced. Masson staining showed that compared with the control group, the collagen fibers in the decidua region of the DEHP low-dose group and the medium-dose group were more distributed, more abundant and more disorderly. TUNEL assay showed increased apoptosis in the decidua area compared to the control group. Western blotting showed that the expression of BMP2, a marker molecule for endometrial decidualization, was significantly reduced (P<0.05 or P<0.01). The abortion rate and embryo resorption rate were increased, and the number of embryos, uterine wet weight, uterine area and placenta wet weight were decreased in DEHP low-dose group compared to the control group stimulated by mifepristone, an abortifacient drug (P<0.05 or P<0.01). CONCLUSIONS: Subacute exposure to DEHP leads to impaired endometrial decidualization during early pregnancy and exacerbates the risk of adverse pregnancy outcomes in mice.

Effects of Five-Step Nutritional Interventions Conducted by a Multidisciplinary Care Team on Gastroenteric Cancer Patients Undergoing Chemotherapy: A Randomized Clinical Trial.

The present study aims to report a five-step nutritional intervention conducted by a multidisciplinary care team as well as to investigate its effects on the nutritional status and quality of life of gastroenteric cancer patients undergoing chemotherapy. A total of 176 patients with newly diagnosed gastroenteric cancer were enrolled in the observational study. The nutritional status of the patients was assessed using Patient-Generated Subjective Global Assessment (PG-SGA) and Nutritional Risk Screening-2002 (NRS-2002), and anthropometry and biological tests were performed. Patients were randomly divided into intervention group (n = 40) and control group (n = 38). Patients in the intervention group received five-step nutrition intervention, while the control group received routine nutrition management. In the newly diagnosed patients with gastroenteric cancer, 50% presented mild to moderate malnutrition, 29.5% presented severe malnutrition, while only 20.5% of patients were in good nutritional status. Nutritional interventions reduced the progression of malnutrition after 10 weeks. Anthropometric parameters increased as well as function and symptoms improved; therefore, controlled the decline in quality of life. To sum up, five-step nutritional interventions conducted by a multidisciplinary care team improved the nutritional status of patients with gastroenteric cancer undergoing chemotherapy, and showed positive impacts on quality of life.

Plasticizer DEHP exposure in early pregnancy affects the endometrial decidualization in mice through reducing lncRNA RP24-315D19.10 expression. / 孕早期增塑剂DEHPæš´éœ²é™ä½Žé•¿é“¾éžç¼–ç RNA RP24-315D19.10è¡¨è¾¾å½±å“å­å®«å† è†œèœ•è†œååº”.

OBJECTIVES: To explore the effect of exposure to di (2-ethyl) hexyl phthalate (DEHP) in early pregnancy on endometrial decidualization in mice and its relation with lncRNA RP24-315D19.10. METHODS: Early pregnancy mice were exposed to DEHP (1000 mg·kg－1·d－1) to construct the model. The uterus was collected on day 6 of pregnancy to detect its effect on decidualization by HE staining and immunofluorescence. A decidualization induction model of mouse endometrial stromal cells exposed to DEHP (0.1, 0.5, 2.5, 12.5, 62.5 µmol/L) was constructed. The changes of cell morphology were observed by light microscopy and phalloidin staining, and the expression of decidual reaction related molecular markers were detected by immunofluorescence, realtime RT-PCR and Western blotting. The expression of RP24-315D19.10 in decidua tissue and cells was detected by realtime RT-PCR. Cellular localization of RP24-315D19.10 was determined by lncLocator database and RNA FISH. AnnoLnc2 database was used to predict miRNAs bound to RP24-315D19.10. RESULTS: The number of embryo implantation sites, uterine weight and uterine area were significantly lower in the DEHP exposed group than those in the control group, and the expression of the decidual reaction related molecular markers matrix metalloprotein 9 and homeobox A10 in the DEHP exposure group were also significantly lower than those in the control group (all P<0.05). With the increase of DEHP concentration, the expression of dtprp in decidua cells was gradually decreased. 2.5 µmol/L DEHP exposed stromal cells failed to be fully decidualized in vitro, andphalloidin staining showed abnormal cytoskeleton morphology. The expression levels of homeobox A10, bone morphogenetic protein 2 and proliferating cell nuclear antigen in the DEHP exposure group were significantly lower than those in the control group (all P<0.05). The expression of RP24-315D19.10 in DEHP exposed decidua tissue and cells was significantly reduced (both P<0.05). RP24-315D19.10 is mainly localized in the cytoplasm and RP24-315D19.10 might bind to 45 miRNAs, among them, miR-138-5p, miR-155-5p, miR-183-5p and miR-223-3p were associated with endometrial decidualization. CONCLUSIONS: DEHP exposure in early pregnancy may impair endometrial decidualization, and the damage may be associated with the down-regulation of RP24-315D19.10.

Catalytic Asymmetric Construction of Axially and Centrally Chiral Heterobiaryls by Minisci Reaction.

Axially chiral biaryls and heterobiaryls constitute the most represented subclass of atropisomers with prevalence in natural products, bioactive compounds, privileged chiral ligand/catalysts, and optically pure materials. Despite many ionic protocols for their construction, radical-based variants represent another highly desirable and intriguing strategy but are far less developed. Moreover, efficient synthesis of axially chiral heterobiaryl molecules, especially ones having multiple heteroatoms and other types of chiral elements, through radical routes remains extremely limited. We herein disclose the first catalytic asymmetric, metal-free construction of axially and centrally chiral heterobiaryls by Minisci reaction of 5-arylpyrimidines and α-amino acid-derived redox-active esters. This is enabled by the use of 4CzIPN as an organic photoredox catalyst in conjunction with a chiral phosphoric acid catalyst. The reaction achieved a variety of interesting 5-arylpyrimidines featuring the union of an axially chiral heterobiaryl and a centrally chiral α-branched amine with generally excellent regio-, diastereo-, and enantioselectivity (up to 82% yield; >19:1 dr; >99% ee). This finding also builds up a new platform for the development of desymmetrization methods via radical-involved atroposelective functionalization at heteroarene of prochiral heterobiaryls.

The correlation between proteoglycan 2 and neuropsychiatric systemic lupus erythematosus.

The proposed pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) mainly includes ischemia and neuroinflammation mechanisms. Protein encoded by Proteoglycan 2 (PRG2) mRNA is involved in the immune process related to eosinophils, also being found in the placenta and peripheral blood of pregnant women. We evaluated the correlation between PRG2 and NPSLE for the first time and found that PRG2 protein was overexpressed in the serum of patients with NPSLE and correlated with the SLE disease activity index (SLEDAI) subset scores of psychosis. Moreover, we investigated the correlation between hippocampal PRG2 level and hippocampally dependent learning and memory ability in MRL/lpr mice, and discovered that the number of PRG2+GFAP+ astrocytes in the cortex and hypothalamus and the number of PRG2+IBA-1+ microglia in the hippocampus and cortex significantly increased in the MRL/lpr mice. These data provided a reference for the follow-up exploration of the role of PRG2 in SLE or other diseases.

The expression of lincRNA AC027700.1 in mouse decidualization.

Long non-coding RNAs (lncRNAs), which belong to the non-protein-coding RNAs, are greater than 200 nt in length. Although they have been found to play crucial roles in the regulation of cell growth and development, cell metabolism and the development of diseases, they are rarely reported in decidualization. The objective of our study is to explore the expression of lincRNA AC027700.1 in the endometrium of early pregnant mice and its role in decidualization. The expression of AC027700.1 in uterine tissues at implantation sites and inter implantation sites on the 6th day of pregnancy were detected by qRT-PCR. The relative expression of AC027700.1 in an in vivo model of induced decidualization in pseudopregnant mice and in in vitro model of induced decidualization in primary stromal cells and nucleus/cytoplasmic fractions were detected by qRT-PCR. GO and KEGG analysis of downstream target genes were performed by GOseq and KOBAS, respectively. The results show that AC027700.1 expression is significantly increased in tissues at implantation sites on the 6th day of pregnancy and in decidualized endometrial tissues and stromal cells. Furthermore, AC027700.1 localizes in the nuclear fraction and the downstream targeted genes are mainly involved in autophagy, cell cycle and RNA transport pathways. This study revealed that lincRNA AC027700.1 may be involved in decidualization of endometrium in early pregnancy, but the specific role and regulatory mechanism remain to be further studied.

Pyruvate kinase isoform M2 impairs cognition in systemic lupus erythematosus by promoting microglial synaptic pruning via the ß-catenin signaling pathway.

BACKGROUND: Neuropsychiatric systemic lupus erythematosus (NPSLE) is a severe complication, which involves pathological damage to the brain and cognitive function. However, its exact mechanism of action still remains unclear. In this study, we explored the role of microglia in the cognitive dysfunction of NPSLE mice. We also analyzed and compared the metabolites in the hippocampal tissues of the lupus model and control mice. METHODS: MRL/MpJ-Faslpr (MRL/lpr) female mice were used as the NPSLE mouse model. Metabolomics was used to assess hippocampal glycolysis levels. Glucose, lactic acid, IL-6, and IL-1ß of the hippocampus were detected by ELISA. Based on the glycolysis pathway, we found that pyruvate kinase isoform M2 (PKM2) in the hippocampus was significantly increased. Thus, the expression of PKM2 was detected by qRT-PCR and Western blotting, and the localization of PKM2 in microglia (IBA-1+) or neurons (NeuN+) was assessed by immunofluorescence staining. Flow cytometry was used to detect the number and phenotype of microglia; the changes in microglial phagocytosis and the ß-catenin signaling pathway were detected in BV2 cells overexpressing PKM2. For in vivo experiments, MRL/lpr mice were treated with AAV9-shPKM2. After 2 months, Morris water maze and conditional fear tests were applied to investigate the cognitive ability of mice; H&E and immunofluorescence staining were used to evaluate brain damage; flow cytometry was used to detect the phenotype and function of microglia; neuronal synapse damage was monitored by qRT-PCR, Western blotting, and immunofluorescence staining. RESULTS: Glycolysis was elevated in the hippocampus of MRL/lpr lupus mice, accompanied by increased glucose consumption and lactate production. Furthermore, the activation of PKM2 in hippocampal microglia was observed in lupus mice. Cell experiments showed that PKM2 facilitated microglial activation and over-activated microglial phagocytosis via the ß-catenin signaling pathway. In vivo, AAV9-shPKM2-treated mice showed decreased microglial activation and reduced neuronal synapses loss by blocking the ß-catenin signaling pathway. Furthermore, the cognitive impairment and brain damage of MRL/lpr mice were significantly relieved after microglial PKM2 inhibition. CONCLUSION: These data indicate that microglial PKM2 have potential to become a novel therapeutic target for treating lupus encephalopathy.

Genetic variability, phylogeny and functional implication of the long control region in human papillomavirus type 16, 18 and 58 in Chengdu, China.

BACKGROUND: Long control region (LCR) of human papillomavirus (HPV) has shown multiple functions on regulating viral transcription. The variations of LCR related to different lineages/sub-lineages have been found to affect viral persistence and cervical cancer progression differently. In this study, we focused on gene polymorphism of HPV16/18/58 LCR to assess the effect variations caused on transcription factor binding sites (TFBS) and provided more data for further study of LCR in Southwest China. METHODS: LCR of HPV16/18/58 were amplified and sequenced to do polymorphic and phylogenetic anlysis. Sequences of each type were aligned with the reference sequence by MEGA 6.0 to identify SNPs. Neighbor-joining phylogenetic trees were constructed using MEGA 6.0. Transcription factor binding sites were predicted by JASPAR database. RESULTS: The prevalence of these three HPVs ranked as HPV16 (12.8%) > HPV58 (12.6%) > HPV18 (3.5%) in Chengdu, Southwest China. 59 SNPs were identified in HPV16-LCR, 18 of them were novel mutations. 30 SNP were found in HPV18-LCR, 8 of them were novel. 55 SNPs were detected in HPV58-LCR, 18 of them were novel. Also, an insertion (CTTGTCAGTTTC) was detected in HPV58-LCR between position 7279 and 7280. As shown in the neighbor-joining phylogenetic trees, most isolates of HPV16/18/58 were clustered into lineage A. In addition, one isolate of HPV16 was classified into lineage C and 3 isolates of HPV58 were classified as lineage B. JASPAR results suggested that TFBS were potentially influenced by 7/6 mutations on LCR of HPV16/18. The insertion and 5 mutations were shown effects in LCR of HPV58. CONCLUSION: This study provides more data for understanding the relation among LCR mutations, lineages and carcinogenesis. It also helps performing further study to demonstrate biological function of LCR and find potential marker for diagnosis and therapy.

PIWI-interacting RNA-36712 restrains breast cancer progression and chemoresistance by interaction with SEPW1 pseudogene SEPW1P RNA.

BACKGROUND: Breast cancer is one of the most common malignancies and the major cause of cancer-related death in women. Although the importance of PIWI-interacting RNAs (piRNAs) in cancer has been increasingly recognized, few studies have been explored the functional mechanism of piRNAs in breast cancer development and progression. METHODS: We examined the top 20 highly expressed piRNAs based on the analysis of TCGA breast cancer data in two patient cohorts to test the roles of piRNAs in breast cancer. The effects of piRNA-36,712 on the malignant phenotypes and chemosensitivity of breast cancer cells were detected in vitro and in vivo. MS2-RIP and reporter gene assays were conducted to identify the interaction and regulation among piRNA-36,712, miRNAs and SEPW1P. Kaplan-Meier estimate with log-rank test was used to compare patient survival by different piRNA-36,712 expression levels. RESULTS: We found piRNA-36,712 level was significantly lower in breast cancer than in normal breast tissues and low level was correlated with poor clinical outcome in patients. Functional studies demonstrated that piRNA-36,712 interacts with RNAs produced by SEPW1P, a retroprocessed pseudogene of SEPW1, and subsequently inhibits SEPW1 expression through competition of SEPW1 mRNA with SEPW1P RNA for microRNA-7 and microRNA-324. We also found that higher SEPW1 expression due to downregulation of piRNA-36,712 in breast cancer may suppress P53, leading to the upregulated Slug but decreased P21 and E-cadherin levels, thus promoting cancer cell proliferation, invasion and migration. Furthermore, we found that piRNA-36,712 had synergistic anticancer effects with the paclitaxel and doxorubicin, two chemotherapeutic agents for breast cancer. CONCLUSIONS: These findings suggest that piRNA-36,712 is a novel tumor suppressor and may serve as a potential predictor for the prognosis of breast cancer patients.

Synthesis of 6-Fluoroalkyl 6 H-Benzo[ c]chromenes via Visible-Light-Promoted Radical Addition/Cyclization of Biaryl Vinyl Ethers.

A novel visible-light-promoted cascade cyclization reaction for the synthesis of 6-fluoroalkyl 6 H-benzo[ c]chromenes has been successfully realized, which was initiated by intermolecular radical addition to biaryl vinyl ethers using easily available fluoroalkylated reagents BrCF2CO2Et or 2-bromo-2,2-difluoroamides as the sources of fluorinated radicals, followed by the cyclization onto an aromatic ring process. This protocol tolerated a wide range of functional groups and provided the desired products in moderate to good yields.

Chromatogram-Bioactivity Correlation-Based Discovery and Identification of Three Bioactive Compounds Affecting Endothelial Function in Ginkgo Biloba Extract.

Discovery and identification of three bioactive compounds affecting endothelial function in Ginkgo biloba Extract (GBE) based on chromatogram-bioactivity correlation analysis. Three portions were separated from GBE via D101 macroporous resin and then re-combined to prepare nine GBE samples. 21 compounds in GBE samples were identified through UFLC-DAD-Q-TOF-MS/MS. Correlation analysis between compounds differences and endothelin-1 (ET-1) in vivo in nine GBE samples was conducted. The analysis results indicated that three bioactive compounds had close relevance to ET-1: Kaempferol-3-O-α-l-glucoside, 3-O-{2-O-{6-O-[P-OH-trans-cinnamoyl]-ß-d-glucosyl}-α-rhamnosyl} Quercetin isomers, and 3-O-{2-O-{6-O-[P-OH-trans-cinnamoyl]-ß-d-glucosyl}-α-rhamnosyl} Kaempferide. The discovery of bioactive compounds could provide references for the quality control and novel pharmaceuticals development of GRE. The present work proposes a feasible chromatogram-bioactivity correlation based approach to discover the compounds and define their bioactivities for the complex multi-component systems.

B-type brain natriuretic peptide as a measure of the severity of hand-foot-mouth disease: a case-control study.

BACKGROUND: Hand-foot-mouth disease (HFMD) is an acute infectious disease caused by enteroviruses, and HFMD complicated by cardiopulmonary failure has a high mortality. B type natriuretic peptide (BNP) is widely applied in monitoring cardiovascular disorders, and thus, we investigated whether this index was associated with the severity of HFMD and the outcome in severe HFMD. METHODS: Serum BNP, lactate, and glucose levels as well as white blood cell (WBC) count, PaO2/FiO2, and cardiac output (CO) were analyzed in the 83 enrolled HFMD patients according to different conditions (common, severe, and critical; with and without complication; and survivors and non-survivors). The control group consisted of 29 patients with respiratory tract infections. RESULTS: No significant differences in CO were observed between the groups. Serum lactate, glucose, BNP, and WBC levels in the critical group were significantly higher than those in the severe, common, and control groups (p < 0.01 or 0.05). The PaO2/FiO2 ratio was significantly lower in the critical group (214.286 ± 154.346) than in the other groups. According to logistic regression analysis, the areas under the curve for serum BNP, glucose, and PaO2/FiO2 of the patients with complications were 0.774, 0.738, and 0.75, respectively. Moreover, the BNP level was significantly higher in HFMD patients with complications and non-survivors. CONCLUSION: Our findings indicate that BNP could be a biochemical indicator for severe (critical) HFMD and used for prognosis in terms of complications and death. Combined with Glu and PaO2/FiO2 and clinical symptoms of HFMD, the value of BNP as an indicator became more precise and specific. Our results may provide another valuable, objective biochemical indicator for severe HFMD. TRIAL REGISTRATION NUMBER: ChiCTR-DDT-14004576 . Name of registry: Chinese Clinical Trial Registry. Date of registration: 2014-09-21.

PCR-RFLP method to detect zoonotic and host-specific Giardia duodenalis assemblages in dog fecal samples.

Giardia duodenalis is a zoonotic protozoan that parasitizes the upper small intestine of human and many mammals including dogs. To develop a restriction fragment length polymorphism (RFLP) method for typing zoonotic (A, B) and host-specific (C, D) assemblages of G. duodenalis from dog, ß-giardin gene was amplified with design primer pairs B3 and B4. The PCR products were digested with restriction enzyme Afa I and Msp I; then, PCR-RFLP method was compared with HRM genotyping and sequencing method for G. duodenalis from dog. The results showed that each of assemblages A-D had unique restriction pattern, which was consistent with the predictive results. Among 21 samples tested by PCR-RFLP, 1 human-derived and 8 dog-derived G. duodenalis were identified as assemblage A; 5 dog-derived G. duodenalis as assemblage C; 7 dog-derived G. duodenalis as assemblage D, which were coincided with the HRM genotyping and sequencing results. It is concluded that the PCR-RFLP is quick, easy, and accurate method for the sequence typing of G. duodenalis zoonotic and specific assemblages from dogs.

Development of a rapid HRM genotyping method for detection of dog-derived Giardia lamblia.

Giardia lamblia is a zoonotic flagellate protozoan in the intestine of human and many mammals including dogs. To assess a threat of dog-derived G. lamblia to humans, the common dog-derived G. lamblia assemblages A, C, and D were genotyped by high-resolution melting (HRM) technology. According to ß-giardin gene sequence, the qPCR-HRM primers BG5 and BG7 were designed. A series of experiments on the stability, sensitivity, and accuracy of the HRM method were also tested. Results showed that the primers BG5 and BG7 could distinguish among three assemblages A, C, and D, which Tm value differences were about 1 °C to each other. The melting curves of intra-assay reproducibility were almost coincided, and those of inter-assay reproducibility were much the same shape. The lowest detection concentration was about 5 × 10(-6)-ng/µL sample. The genotyping results from 21 G. lamblia samples by the HRM method were in complete accordance with sequencing results. It is concluded that the HRM genotyping method is rapid, stable, specific, highly sensitive, and suitable for clinical detection and molecular epidemiological survey of dog-derived G. lamblia.

Genetic association analyses of fast plasma glucose level in pre-menopausal Chinese women: potential interaction between osteocalcin and oestrogen receptor α.

BACKGROUND: Fasting plasma glucose (FPG) levels are usually tightly regulated within a narrow physiologic range. Variation of FPG levels is clinically important and is strongly heritable. Several lines of evidence suggest the importance of the oestrogen receptor α (ER-α) and osteocalcin (also known as BGP, for bone Gla protein) in determining FPG; however, whether their polymorphisms are associated with FPG variation is not well understood. AIM: To investigate whether ER-a PvuII and BGP HindIII genetic polymorphisms and their potential interaction are associated with FPG variation. SUBJECTS AND METHODS: The study subjects were 328 unrelated pre-menopausal Chinese women aged 21 years and over (mean age ± SD, 33.2 ± 5.9 years), with an average FPG of 4.92 (SD = 0.81). All subjects were genotyped at the ER-α PvuII and BGP HindIII loci using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). RESULTS: The ER-α PvuII genotypes were significantly associated with FPG (p = 0.007). In addition, a significant interaction was observed of the ER-α PvuII polymorphism with BGP HindIII polymorphism on FPG variation (p = 0.013), although the BGP HindIII polymorphism was not shown to be individually associated with FPG. CONCLUSION: The PvuII polymorphism of the ER-α gene and its potential interaction with the HindIII polymorphism of the BGP gene were associated with FPG in pre-menopausal Chinese women.

Occurrence and molecular identification of Giardia duodenalis from stray cats in Guangzhou, southern China.

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), ß-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.

Genotyping of Giardia duodenalis isolates from dogs in Guangdong, China based on multi-locus sequence.

This study aimed to identify the assemblages (or subassemblages) of Giardia duodenalis by using normal or nested PCR based on 4 genetic loci: glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), ß-giardin (bg), and small subunit ribosomal DNA (18S rRNA) genes. For this work, a total of 216 dogs' fecal samples were collected in Guangdong, China. The phylogenetic trees were constructed with MEGA5.2 by using the neighbor-joining method. Results showed that 9.7% (21/216) samples were found to be positive; moreover, 10 samples were single infection (7 isolates assemblage A, 2 isolates assemblage C, and 1 isolate assemblage D) and 11 samples were mixed infections where assemblage A was predominant, which was potentially zoonotic. These findings showed that most of the dogs in Guangdong were infected or mixed-infected with assemblage A, and multi-locus sequence typing could be the best selection for the genotype analysis of dog-derived Giardia isolates.