BRIX1 promotes ribosome synthesis and enhances glycolysis by selected translation of GLUT1 in colorectal cancer.

BACKGROUND: Ribosome biogenesis protein BRX1 homolog (BRIX1) is critically required for the synthesis of the 60S ribosome subunit. However, the role and mechanism of BRIX1 in colorectal cancer (CRC) remain unclear. METHODS: Kyoto Encyclopedia of Gene and Genome pathway and Gene Ontology analyses were used for bioinformatics analysis. The rRNA levels were detected in CRC tissues and cells. Nascent RNA synthesis was detected via cellular immunofluorescence. The correlation was analyzed between patient Positron Emission Tomography-Computed Tomography (PET-CT) values and their BRIX1 expression. The extracellular acidification rate (ECAR) and oxygen consumption rate were determined via live metabolic analyses. Polysome fractions were collected for BRIX1 mRNA used in translation. The orthotopic model and Cell Counting Kit-8 (CCK8) assay were used to assess BRIX1 function in CRC. RESULTS: BRIX1 is a core protein involved in ribosome-related pathway changes in CRC. Gene Ontology analysis showed that BRIX1 was primarily enriched in ribosome assembly and ribosome biogenesis pathways. In fresh CRC tissue, rRNA levels (5S, 5.8S, 18S and 28S) were higher in the BRIX1 high-expression group than in the BRIX1 low-expression group. Similarly, BRIX1 knockdown significantly decreased rRNA levels for 5S, 5.8S, 18S and 28S in CRC cells, whereas overexpression of BRIX1 significantly increased these levels. In addition, BRIX1 knockdown inhibited nascent RNA synthesis in CRC cells. In clinical data analysis, BRIX1 expression was related to the glucose uptake in PET-CT. BRIX1 knockdown significantly decreased the ECAR value, glucose uptake and lactic acid production in CRC cells, whereas BRIX1 overexpression significantly increased these. Furthermore, BRIX1 knockdown significantly decreased the protein expression of GLUT1, whereas BRIX1 overexpression significantly increased this; however, expression of BRIX1 mRNA was unaffected in either case. Blocking glycolysis by si-GLUT1 or galactose reversed BRIX1 promotion of glycolysis and cell proliferation in CRC cells.

Efficacy and safety of non-ergot dopamine-receptor agonists as an adjunct to levodopa in advanced Parkinson's disease: A network meta-analysis.

BACKGROUND AND PURPOSE: Non-ergot dopamine agonists (NEDAs) have been used as an adjunct therapy to levodopa in advanced Parkinson's disease (PD) for many years. However, there is no strong evidence that a given NEDA is more potent than another. To compare and rank the efficacy, tolerability, and safety of six commonly used NEDAs as an adjunct to levodopa in advanced PD, which includes long-acting and standard formulations, a network meta-analysis was performed. METHODS: The MEDLINE, Embase, Cochrane Central Register of Controlled Trials, China National Knowledge Infrastructure, and Wanfang databases were searched from January 1996 to June 2022 for eligible randomized controlled trials (RCTs). Six NEDAs, including rotigotine transdermal patch, ropinirole immediate-release (IR)/prolonged-release (PR), pramipexole IR/extended-release (ER), and piribedil, were investigated. RESULTS: A total of 34 RCTs (7868 patients) were included in the current study. The surface under the cumulative ranking curve indicated that ropinirole PR was associated with the best improvement in Unified Parkinson's Disease Rating Scale (UPDRS)-II, UPDRS-III, and UPDRS-II + III (0.811, 0.742, and 0.827). For OFF time reduction, pramipexole IR ranked first (0.979), and ropinirole PR ranked first in OFF time responder rate (0.927). Pramipexole ER ranked first in overall withdrawals, and rotigotine transdermal patch ranked first in the incidence of adverse events (≥1 AEs). CONCLUSIONS: This network meta-analysis suggests six commonly used NEDAs are effective as an adjunct to levodopa in advanced PD. In comprehensive consideration of better symptomatic management, ropinirole PR may be a better choice than other NEDAs in advanced PD. Six NEDAs showed different profiles of AEs.

Repeated intravenous thrombolysis in recurrent ischemic stroke within 3 months: a systematic review.

BACKGROUND: Repeated intravenous thrombolysis (RIVT) within 3 months is an off-guideline therapy, however, may be an effective and safe way to treat early recurrent ischemic stroke. This study was conducted to assess the potential influencing factors on the efficacy and safety of RIVT in recurrent ischemic stroke within 3 months and to explore the strategy of RIVT within 3 months. METHODS: PubMed, Cochrane Library, Embase, China National Knowledge Infrastructure, and Wanfang Database were searched for cases of RIVT in recurrent ischemic stroke within 3 months up to February 1, 2023. Clinical characteristics were compared and analyzed between the good-outcome and poor-outcome groups and between the symptomatic intracranial hemorrhage (sICH) and non-sICH groups respectively. RESULTS: A total of 16 studies including 24 cases of RIVT within 3 months were retrospectively analyzed in the present study. The patients' ages ranged from 42 to 87 years (median 73.5 years) and the intervals between thrombolysis were from 0.25 to 90 days (median 9.5 days). Comparing the clinical characteristics between the good-outcome group and the poor-outcome group, no statistically significant differences were found (P > 0.05), but the differences in baseline National Institutes of Health stroke scale (NIHSS) score of the recurrent stroke (P = 0.056) and good outcome after the previous IVT (P = 0.054) nearly reached statistical significance. Comparing the data between the non-sICH group and the sICH group, statistically significant differences were found in terms of the proportion of cardiogenic embolism (P = 0.036), baseline NIHSS score in the recurrent stroke (P = 0.007) and the interval between thrombolysis (P = 0.041), but no significant difference was found by regression analysis. CONCLUSION: In patients with recurrent ischemic stroke within 3 months, those with a good outcome after the previous IVT and a low baseline NIHSS score in the recurrent stroke may be considered for RIVT, whereas those with a high baseline NIHSS score, a short interval between thrombolysis, and cardiogenic embolism may suffer a higher risk of sICH. Due to sample size and publication bias, more studies with larger sample sizes and more rigorous designs are needed to confirm this conclusion.

In silico development and clinical validation of novel 8 gene signature based on lipid metabolism related genes in colon adenocarcinoma.

BACKGROUND: Changes in lipid metabolism pathways play a major role in colon carcinogenesis and development. Hence, we conducted a systematic analysis of lipid metabolism-related genes to explore new markers that predict the prognosis of colon adenocarcinoma (COAD). METHODS: The non-negative Matrix Factorization (NMF) algorithm was applied to identify the molecular subtypes based on lipid metabolism-related genes. A weighted correlation network analysis (WCGNA) was used to identify co-expressed genes, and Lasso multivariate Cox analysis was performed to build a risk prognosis model. A timer database was used to analyze the immune infiltration of the gene signature and the GSCALite database was used for genome-wide analysis of the gene signature. RESULTS: TCGA-COAD samples were divided into 3 subtypes based on lipid metabolism-related genes. 2739 genes were identified by WGCNA analysis. Finally, an 8-gene signature (RTN2, FYN, HEYL, FAM69A, FBXL5, HMGN2, LGALS4, STOX1) was constructed that demonstrated good robustness in different datasets, as well as an independent risk factor for colon cancer patients' prognosis. In addition, our model's predictive efficacy overall was higher than that of the other published models, and the 8 genes' expression analysis indicated that RTN2, HEYL, and STOX1 were all expressed highly significantly in COAD, while FAM69A, FBXL5, LGALS4, FYN and HMGN2 were expressed significantly poorly in cancer tissues, which was confirmed in immunohistochemistry. The 8 genes were expressed significantly differently in COAD immune subtypes and correlated with clinical variables. Genome-wide analysis revealed that the STOX1 mutation frequency was the highest, and genome methylation influenced HEYL, FAM69A, and STOX1 gene expression significantly; further, the expression of HEYL and FBXL5 was correlated positively with Copy number variation (CNV) and was regulated significantly by CNV in most cancers. FBXL5 was correlated significantly with austocystin d and bafilomycin and played an important role in anti-tumor and immunotherapy. The HEYL, FYN, FAM69A, and RTN2 genes' expression was associated with the EMT pathway's activation, while LGALS4 and STOX1 were associated significantly with the EMT pathway's inhibition. CONCLUSION: This study constructed an 8-gene signature as a novel marker to predict colon cancer patients' survival.

A Multidisciplinary Approach for Tophi Wounds Caused by Glycogen Storage Disease Type 1a: A Rare Case.

ABSTRACT: Glycogen storage diseases (GSDs) are a group of rare inherited metabolic disorders caused by enzyme deficiencies in glycogen catabolism. The more common type, GSD type Ia, is caused by glucose-6-phosphatase deficiency and often complicated by gout from hyperuricemia. Here, the authors report a rare case of a tophi wound caused by GSD type Ia in a Chinese patient. Difficulties in this case included the control of abnormal blood markers, especially uric acid; removal of tophi deposited in the tissues; restoration of hand function after wound healing; and patient adherence to treatment and follow-up. A multidisciplinary team was set up consisting of experts from the authors' wound care center and the departments of endocrinology, orthopedics, and rehabilitation. The wound healed in 53 days and was followed up for about 7 months. During follow-up, the patient's hand function returned to normal, and no new tophi formed. Because GSDs are a congenital lifelong condition, regular follow-ups are especially important.

Comparison of the direct and indirect reduction techniques during the surgical management of posterior malleolar fractures.

BACKGROUND: The optimal method for the reduction and fixation of posterior malleolar fracture (PMF) remains inconclusive. Currently, both of the indirect and direct reduction techniques are widely used. We aimed to compare the reduction quality and clinical outcome of posterior malleolar fracture managed with the direct reduction technique through posterolateral approach or the indirect reduction technique using ligamentotaxis. METHODS: Patients with a PMF involving over 25% of the articular surface were recruited and assigned to the direct reduction (DR) group or the indirect reduction (IR) group. Following reduction and fixation of the fracture, the quality of fracture reduction was evaluated in post-operative CT images. Clinical and radiological follow-ups were performed at 6 weeks, 3 months, 6 months, 12 months, and then at 6 month-intervals postoperatively. Functional outcome (AOFAS score), ankle range of motion, and Visual Analog Scale (VAS) were evaluated at the last follow-up. Statistical differences were compared between the DR and IR groups considering the patient demographics, quality of fracture reduction, AOFAS score, and VAS. RESULTS: Totally 116 patients were included, wherein 64 cases were assigned to the DR group and 52 cases were assigned to the IR group. The quality of fracture reduction was significant higher in the DR group (P = 0.038). In the patients who completed a minimum of 12 months' follow-up, a median AOFAS score of 87 was recorded in the DR group, which was significantly higher than that recorded in the IR group (a median score of 80). The ankle range of motion was slightly better in the DR group, with the mean dorsiflexion restriction recorded to be 5.2° and 6.1° in the DR and IR group respectively (P = 0.331). Similar VAS score was observed in the two groups (P = 0.419). CONCLUSIONS: The direct reduction technique through a posterolateral approach provide better quality of fracture reduction and functional outcome in the management of PMF over 25% of articular surface, as compared with the indirect reduction technique using ligamentotaxis. TRIAL REGISTRATION: NCT02801474 (retrospectively registered, June 2016, ClinicalTrails.gov).

Dopamine agonists versus levodopa monotherapy in early Parkinson's disease for the potential risks of motor complications: A network meta-analysis.

BACKGROUND: Compared with levodopa, dopamine agonists (DAs) as initial treatment are associated with lower incidences of motor complications in early Parkinson's disease (PD). There is no strong evidence that a given DA is more potent in lower incidences of motor complications than another. OBJECTIVE: We performed a network meta-analysis of levodopa versus DAs as monotherapy in early PD to access the risk of motor complications. METHODS: Databases were searched up to June 2022 for eligible RCTs. Levodopa and four DAs (pramipexole, ropinirole, bromocriptine and pergolide) were investigated. The incidences of motor complications and efficacy, tolerability and safety outcomes were analyzed. RESULTS: Nine RCTs (2112 patients) were included in the current study. The surface under the cumulative ranking curve (SUCRA) indicated that levodopa ranked first in the incidence of dyskinesia (0.988), followed by pergolide, pramipexole, ropinirole, and bromocriptine (0.704, 0.408, 0.240, 0.160). Pramipexole was least prone to wearing-off (0.109) and on-off fluctuation (0.041). Levodopa performed best in improvements of UPDRS-II, UPDRS-III, and UPDRS-II + III (0.925, 0.952, 0.934). Bromocriptine ranked first in total withdrawals and withdrawals due to adverse events (0.736, 0.751). Four DAs showed different adverse events profiles. CONCLUSION: In the two non-ergot DAs, ropinirole is associated with a lower risk of dyskinesia while pramipexole is associated with lower risks of wearing-off and on-off fluctuations. Our research may facilitate head-to-head research, larger sample sizes, long following-up time RCTs to confirm the findings of this network meta-analysis.

Pathogenic free-living amoebic encephalitis from 48 cases in China: A systematic review.

Background: Free-living amoebae (FLA) including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris can become pathogenic and cause severe cerebral infections, named primary amoebic meningoencephalitis (PAM), granulomatous amoebic encephalitis (GAE), and balamuthia amoebic encephalitis (BAE), respectively. FLA encephalitis has been reported across China, but the clinical data descriptions and analytical results of these different reports vary widely. Currently, no consensus treatment has been established. We conduct a systematic review to evaluate the exposure location, clinical symptoms, diagnosis, treatment, and prognosis of three FLA encephalitis and aim to reveal the differences between three FLA encephalitis in China. Methods: We used MEDLINE (PubMed interface), EMBASE, China National Knowledge Infrastructure (CNKI), Wanfang database, and China Biology Medicine disc (CBMdisc) databases for literatures published and manually retrieve the hospital records of our hospital. The search time was up to August 30, 2022, with no language restrictions. Results: After excluding possible duplicate cases, a total of 48 patients of three FLA encephalitis were collected. One from the medical records of our hospital and 47 patients from 31 different studies. There were 11 patients of PAM, 10 patients of GAE, and 27 patients of BAE. The onset of PAM is mostly acute or subacute, and the clinical symptoms are acute and fulminant hemorrhagic meningoencephalitis. Most patients with GAE and BAE have an insidious onset and a chronic course. A total of 21 BAE patients (77.8%) had skin lesions before onset of symptoms. Additionally, 37 cases (77.1%) were diagnosed with FLA encephalitis before death. And there were 4 of PAM, 2 of GAE, and 10 of BAE diagnosed using next generation sequencing. No single agent can be proposed as the ideal therapy by itself. Only 6 cases were successfully treated. Conclusions: This review provides an overview of the available data and studies of FLA encephalitis in China and identify some potential differences. FLA encephalitis is a rare but pathogenic infection, and physicians should early identify this encephalitis to improve survival.

Comparative efficacy and safety of six non-ergot dopamine-receptor agonists in early Parkinson's disease: a systematic review and network meta-analysis.

Background: Non-ergot dopamine agonists (NEDAs) have been used as monotherapy or as an adjunctive therapy to levodopa for many years. Novel long-acting formulations of NEDAs including pramipexole extended-release (ER), ropinirole prolonged-release (PR), and rotigotine transdermal patch have been developed. However, there is no strong evidence that a given NEDA is more potent than another. We performed a systematic review and network meta-analysis to evaluate the efficacy, tolerability and safety of six commonly used NEDAs in early Parkinson's disease (PD). Methods: Six NEDAs including piribedil, rotigotine transdermal patch, pramipexole immediate-release (IR)/ER, and ropinirole IR/PR were investigated. The efficacy outcomes including Unified Parkinson's Disease Rating Scale activities in daily life (UPDRS-II), motor function (UPDRS-III), and their subtotal (UPDRS-II + III), tolerability and safety outcomes were analyzed. Results: A total of 20 RCTs (5,355 patients) were included in the current study. The result indicated that compared with placebo, all six investigated drugs had statistically significant differences in the improvement of UPDRS-II, UPDRS-III, and UPDRS-II + III (except ropinirole PR in UPDRS-II). There were no statistically significant differences between six NEDAs for the UPDRS-II and UPDRS-III. For UPDRS-II + III, the improvement of ropinirole IR/PR and piribedil were higher than that of rotigotine transdermal patch, and piribedil was higher than that of pramipexole IR. The surface under the cumulative ranking curve (SUCRA) indicated that piribedil resulted in best improvement in UPDRS-II and UPDRS-III (0.717 and 0.861, respectively). For UPDRS-II + III, piribedil and ropinirole PR exhibited similar improvement and both had high rates (0.858 and 0.878, respectively). Furthermore, piribedil performed better as monotherapy, ranking first in the improvement of UPDRS-II, III, and II + III (0.922, 0.960, and 0.941, separately). With regard to tolerability, there was a significant increase in overall withdrawals with pramipexole ER (0.937). In addition, the incidence of adverse reaction of ropinirole IR was relatively high (nausea: 0.678; somnolence: 0.752; dizziness: 0.758; fatigue: 0.890). Conclusions: In this systematic review and network meta-analysis of six NEDAs, piribedil exhibited better efficacy, especially as monotherapy, and ropinirole IR was associated with a higher incidence of adverse events in patients with early PD.

Computed Tomography Angiography of Gastrocolic Vein Trunk by Morphological Filtering Technique in Right Colon Cancer.

BACKGROUND: This study was to evaluate CT vein image segmentation and three-dimensional imaging in the anatomical structure of gastrocolic venous trunk before operation for colon cancer. METHODS: CT scanning images by Philips Brilliance CT 256CT scanner. Vein position was segmented and calculated by grey value through algorithm flow. Intensity measurement of selected image area by a calculate through noise cancellation and missing pixel filling, based on numerical morphology. RESULTS: The direction of the right colonic vein could be clearly displayed in all 96 patients by morphological filtering technique. Among these patients, there were 78 patients with gastrocolic vein trunk, with an occurrence rate of 81.25%. According to the classification of GVT, there were 36 cases of type A (46.2%), 22 cases of type B (28.2%), 12 cases of type C (15.4%) and 6 cases of type D (7.7%). CONCLUSION: CT vein image segmentation and three-dimensional imaging can effectively evaluate the anatomical variation of gastrocolic vein trunk before operation, which is helpful for operators to correctly understand its anatomical structure and choose a reasonable anatomical approach.

Decreased Expression of MPC2 Contributes to Aerobic Glycolysis and Colorectal Cancer Proliferation by Activating mTOR Pathway.

Mitochondrial Pyruvate Carrier 1 (MPC1), one of the rate-limiting proteins involved in glycolysis metabolism, has been demonstrated as a tumor inhibitor in several cancers. This study was conducted with the aim of exploring the role and underlying mechanisms of MPC2 in colorectal cancer (CRC). Here, we found that MPC2 expression was decreased in CRC samples. According to the analysis on our TMA data, lower expression of MPC2 is correlated with a higher incidence of distant metastasis and lymph node invasion, bigger tumor size, low survival rate of patients, and advanced T stages. Functionally, in vivo/vitro experiments showed that MPC2 knockdown induced CRC cell proliferation and growth, while MPC2 overexpression inhibited the proliferation and growth of CRC. Further study demonstrated that MPC2 knockdown resulted in aerobic glycolysis in CRC cells. Similarly, MPC2 overexpression in CRC cells also caused inhibited aerobic glycolysis. Further study found that MPC2 knockdown in CRC cell lines activated the mTOR signaling pathway, and the addition of rapamycin reversed the promoting effect of MPC2 knockdown on CRC proliferation and glycolysis. Likewise, the addition of MHY1485 also reversed the MPC2 overexpression's role in hindering aerobic glycolysis in CRC cells. Collectively, our study established that low expression of MPC2 led to CRC growth as well as aerobic glycolysis through the regulation of the mTOR pathway in CRC cells, indicating a potential biomarker and therapy target for CRC.

Thrombospondin 2/Toll-Like Receptor 4 Axis Contributes to HIF-1α-Derived Glycolysis in Colorectal Cancer.

BACKGROUND: Aerobic glycolysis is a typical metabolic reprogramming in tumor cells, which contributes to the survival and proliferation of tumor cells. The underlying mechanisms controlling this metabolic switch in colorectal cancer (CRC), however, remain only partially understood. METHODS: The Cancer Genome Atlas (TCGA) dataset and Gene Expression Omnibus (GEO) (GDS4382, GSE6988, GSE35834) were used to analyzed the mRNA expression of THBS2. 392 paired samples of CRC and adjacent non-cancerous tissues were collected to detect the expression of THBS2 by IHC. The correlation of THBS2 expression with categorical clinical variables in patients with CRC was evaluated using chi-square analysis or Student's t-test. CCK-8, colony formation, and animal CT scan were used to functional analysis of THBS2 in CRC. RESULTS: Thrombospondin 2 (THBS2) is aberrantly upregulated and linked to a poor prognosis in CRC. Subsequent experiments also showed that THBS2 promotes the proliferation of CRC cells. In terms of mechanism, THBS2 interacted with Toll-like receptor 4 (TLR4), but not with the other toll-like receptors (TLRs), which upregulated the mRNA expression of GLUT1, HK2, ALDOA, PKM2, and LDHA and enhanced glycolytic capacity in CRC cells. Moreover, THBS2/TLR4 axis significantly increased the protein level of HIF-1α and blocking HIF-1α by siRNA reversed the enhanced glycolytic capacity and the upregulated expression of glycolytic enzymes in CRC cells. CONCLUSION: Our findings revealed that the THBS2/TLR4 axis contributes to HIF-1α derived glycolysis and eventually promotes CRC progress.

Usefulness of serum D­dimer for preoperative diagnosis of infected nonunion after open reduction and internal fixation.

PURPOSE: Infected nonunion after open reduction internal fixation (ORIF) is a serious complication. The aim of this study was to evaluate the usefulness of serum D-dimer for preoperative diagnosis of infected nonunion. PATIENTS AND METHODS: Patients undergoing debridement and external fixation for infected nonunion (n=32) and replacement of internal fixation due to aseptic failure (n=34) were enrolled and compared in this retrospective study. The optimum cutoff value of D-dimer for identification of infected nonunion was determined by calculating the Youden J statistic. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of four preoperative laboratory parameters-serum D-dimer level, white blood cell (WBC) count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP)-for diagnosis of infected nonunion were compared. RESULTS: Serum D-dimer level was significantly higher in patients with infected nonunion than in patients with aseptic nonunion: 2.62 mg/mL (range, 0.13-11.90 mg/mL) vs 0.35 mg/mL (range, 0.07-6.46 mg/mL; p<0.001). WBC count, CRP, and ESR demonstrated sensitivity of 12.5% (95% CI: 4.08-29.93), 40.6% (95% CI: 24.22-59.21), and 56.3% (95% CI: 37.88-73.16), respectively, and specificity of 94.1% (95% CI: 78.94-98.97), 88.2% (95% CI: 71.61-96.16), and 85.3% (95% CI: 68.17-94.46), respectively. Using the Youden index, 1.70 mg/mL was determined as the optimal threshold value for serum D-dimer for the diagnosis of infected nonunion. The sensitivity and specificity of serum D-dimer (>1.70 mg/mL) were 75.0% (95% CI: 56.25-87.87) and 91.2% (95% CI: 75.19-97.69). CONCLUSIONS: Serum D-dimer level may be useful for preoperative prediction of infected nonunion in patients after ORIF.

[DNA microarrays and their application in detecting and identifying intestinal pathogens].

DNA microarrays offer many advantages of high throughout, automation, rapid detection, and so on. Therefore, this technology had been used in many fields such as molecular epidemiology of bacteria, microbial gene identification, disease mechanism, gene mutation, gene expression identification, DNA sequencing and medicine screening etc. The assays for identifying pathogens using DNA microarrays reported aboard recently are introduced. The application of DNA microarrays in detecting and identifying intestinal pathogens mainly includes three aspects: the identification of toxin and characteristic genes of pathogens, the identification of bacterial DNA or RNA directly, the simultaneous detection of a large number of intestinal pathogens with the target - gene of ribosomal RNA. Because of its high efficiency, DNA microarrays is superior to other biological method. Obviously DNA microarrays technology may be useful in identifying intestinal pathogens and have a wide prospect.

Fabrication and optimization of the multiplex PCR-based oligonucleotide microarray for detection of Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum.

To detect and identify the pathogens responsible for sexually transmitted diseases (STDs) at the early stage of infection and with a high throughput, a new microarray with a bifunctional probe modification was prepared using Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum as a model system. During the fabrication of the microarray, an asymmetric fluorescently labeled multiplex PCR was introduced. The fabrication optimization proved that the best hybridization results would be obtained by spotting N. gonorrhoeae probe at a position near the side of the fluorescently labeled reverse primer within its target gene and spotting each probe at a concentration of 50 microM onto the aldehyde-derived glass slides using spotting solution S1 and using hybridization solution H2 for hybridization. The probes designed by our laboratory could specifically discriminate the pathogens of N. gonorrhoeae, C. trachomatis and U. urealyticum in the presence of the internal control on the microarray simultaneously and separately. By incorporating the key features of DNA microarray with those of multiplex PCR, the microarray provides a fast high throughput platform for multiple infections and multiple samples to be detected and identified simultaneously for STD clinics. It also provides a new platform for other diseases and gene mutations to be detected and identified at a high throughput.

Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

[Influence of cytochrom P450 CYP2C9 polymorphism on the pharmacokinetics of tolbutamide metabolism using oligonucleotide genotyping microarray].

AIM: To investigate the influence of cytochrom P450 CYP2C9 polymorphism on the pharmacokinetics of tolbutamide. METHODS: An oligonucleotide microarray was designed and fabricated to genotype the CYP2C9 accurately and quickly. 137 healthy volunteers were genotyped with the array to investigate the frequency of CYP2C9 functional SNPs. Moreover, 1 homozygous mutant, 9 heterozygous and 10 wild-genotypes subjects in the assay were selected randomly and sequenced directly. After orally taking tolbutamide, blood samples and urine samples were collected, and their pharmacokinetics was studied with HPLC. RESULTS: CYP2C9 *1/*3 were found in 9 of 137 volunteers, CYP2C9 *3/*3 in only one, others were all CYP2C9 *1/*1 wild types. CYP2C9 *2, CYP2C9 *4 and CYP2C9 *5 alleles were not detected. Direct sequencing of the purified PCR products of the heterozygotes, mutant homozygotes and ten wild type individuals gave a corresponding result to that genotyped by microarray. Pharmacokinetic outcome showed that the individuals with CYP2C9 *1/*3 or CYP2C9 *3/*3 had slower metabolic elimination of tolbutamide than those with CYP2C9 *1/*1. CONCLUSION: CYP2C9 genetic polymorphism has a significant influence on the pharmacokinetics of tolbutamide. Pharmacogenomic study will be helpful in guiding rational and individualized medication. Key words: tolbutamide; cytochrom P450 CYP2C9; allele; single nucleotide polymorphism; genotyping

[Study on PBPs genes of penicillin-resistant Streptococcus pneumonia in Zhejiang province].

To elucidate alternations in gene/amino acid sequence of penicillin-binding proteins (PBPs) 1A, 2B, 2X from clinical isolates of penicillin-resistant Streptococcus pneumonia (PRSP) in Zhejiang Province, 26 strains of Streptococcus pneumonia were collected from November 2001 to January 2004. The antibiotics susceptibility of these strains was detected. PCR amplification and direct sequencing of PBP1A, 2B, 2X genes were performed. The sequence variations of PBP genes of the PRSPs in this region were studied by sequence BLAST analysis. It was shown that the main alternations of PBP1A were the four consecutive amino acid substitutions (Thr574Ala, Ser575Thr, Gln576Gly, Phe577Tyr) following the conservative motif KTG and the amino acid substitution Thr371Ser in the conservative motif STMK. The main alternation of PBP2B was Thr451Ala following the conservative motif SSN, and the main alternation of PBP2X was Thr338Ala in conservative motif STMK. The above mutation sites and drug resistant level were consistent to the data reported previously. Neither new gene mutation specific to these strains nor certain amino acid substitutions related to penicillin resistance reported was identified in the genes.

Rapid detection of the known SNPs of CYP2C9 using oligonucleotide microarray.

AIM: Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. Individuals with mutant enzymes may risk serious side effects under routine therapy with certain drugs metabolized by CYP2C9. In order to facilitate the detection of the known SNPs of CYP2C9, an allele-specific oligonucleotide (ASO) based microarray was made. METHODS: An oligonucleotide microarray was made to facilitate the SNP (single nucleotide polymorphism) screening and was applied for the detection of CYP2C9 polymorphism in 62 high blood pressure (HBP) patients who received Irbesartan for treatment. Part of the genotyping results was confirmed by direct sequencing. And the relation between CYP2C9 polymorphism and therapeutic outcome of Irbesartan was statistically analyzed. RESULTS: Heterozygous alleles of CYP2C9*1/*3 were found in 7 out of 62 subjects. No mutant alleles of CYP2C9*2, *4 and *5 and no homozygous mutant alleles were detected. The 7 heterozygous CYP2C9*1/*3 and 13 random wild type DNA samples were subjected to direct sequencing with purified PCR products and same genotyping results were obtained with the 20 DNA samples. There was no significant difference in the odds of effectiveness of Irbesartan between the wild type (normal) group and CYP2C9*1/*3 (mutant) group (P>0.05). CONCLUSION: The oligonucleotide microarray made in this study is a reliable assay for detecting the CYP2C9 known alleles and the heterozygous CYP2C9*1/*3 has no significant effects on the therapeutic outcome of Irbesartan.