Reaction Restriction Enzyme Research Articles

Identification of Nontuberculous Mycobacterium Species by Polymerase Chain Reaction - Restriction Enzyme Analysis (PCR-REA) of rpoB gene in Clinical Isolates.

Nontuberculous mycobacteria (NTM) infections are an emerging global health concern with increasing incidence. Conventional identification methods for NTM species in clinical settings are prone to errors. This study evaluates a newer method, polymerase chain reaction-restriction enzyme analysis (PCR-REA) of the rpoB gene, for NTM species identification. The study identified NTM species in clinical samples using conventional biochemical techniques and compared the results with PCR-REA of the rpoB gene. This cross-sectional study was conducted at a tertiary health-care center in North India over 18 months, analyzing both pulmonary and extrapulmonary samples. Two hundred and forty-seven NTM isolates were identified using phenotypic and biochemical methods. The same isolates were subjected to rpoB gene amplification by PCR followed by REA using Msp I and Hae III enzymes. Conventional methods identified 12 different NTM species (153 slow-growing and 94 rapid-growing), whereas PCR-REA identified 16 species (140 slow-growing, 107 rapid-growing). The Mycobacterium avium intracellulare complex was the most common species isolated. PCR-REA demonstrated higher resolution in species identification, particularly in differentiating within species complexes. PCR-REA of the rpoB gene proves to be a simple, rapid, and more discriminative tool for NTM species identification compared to conventional methods. This technique could significantly improve the diagnosis and management of emerging NTM infections in clinical settings.

Sphingomyelin and Phosphatidylserine Derived Compounds Found into RBCs of Two Unrelated Individuals with Idiopathic Erythrocytosis with Altered Hemoglobin (Hb) Function Properties and No Hemoglobinopathies - Preliminary Results

Sphingomyelin and phosphatidylserine derived compounds were found into Red Blood Cells (RBCs) of two unrelated individuals with idiopathic erythrocytosis, whom were referred to the Laboratory of Hemoglobinopathies - State University of Campinas (State of São Paulo, Brazil) to investigate the presence of hemoglobin (Hb) variants with high oxygen affinity. Both unrelated individuals were 5-year-old age Caucasian Brazilian boys with the following counts: RBC (x 10/6mm3) 6.15 and 7.69, Hb (g/dL) 20.1 and 20, Htc (%) 54 and 58.7, respectively. Most common causes of erythrocytosis, including polycythemia vera (PV) and other secondary erythrocytosis; JAK2, VHL and EPOR mutational screen were previously excluded in the Centro Infantil Boldrini (Campinas, State of São Paulo, Brazil), where the children are followed. No hemoglobin or globin variants were detected by standard methods: alkaline electrophoresis in cellulose acetate (pH 8.9), acidic electrophoresis in agar gel (pH 5.8), cation-exchange HPLC (Variant II, Bio-Rad) and RP-HPLC (Alliance, Waters). Direct genome sequencing of HBA and HBB performed in automated sequencer (ABI PRISM™ 377 DNA Automated Sequencer, Applied BioSystems) after DNA amplification obtained from peripheral leukocytes (QIAamp DNA Blood, Qiagen) did not reveal any mutation. Familial study also did not show any alteration in blood count, hemoglobin or even globin gene mutation in their relatives. Deletional and non-deletional alpha thalassemia was also excluded by Multiplex-gap-PCR and restriction enzyme reaction (Hph and NcoI). Despite of normal results for Hb characterization, functional tests performed with Hb purified by exclusion (Sephadex G-25, Sigma-Aldrich) and ion exchange chromatography (Amberlite MB-3, Sigma-Aldrich) in Hepes buffer indicated some alteration. O2 affinity was evaluated by determining P50 with stripped lysates ([Hb]=70 μM/Heme) in pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, for Bohr effect calculations. Heme-heme cooperativity was determined by Hill coefficient (n) and the activity of allosteric effector in the Hb-O2 ligation was also tested, using inositol hexaphosphate ([IHP]=1mM), a mimetic compound of the intraerythrocytic 2,3-BPG. P50 was slightly decreased (both patients, stripped Hb samples) at extreme acidic and alkaline pH (pH 6.5 and 8.5) leading to altered Bohr effect, in comparison to healthy and erythrocytosis control (caused by cardiopathy, with no Hb variant). P50 was also found decreased at pH 8.5 in the presence of IHP. Heme-heme cooperativity was very decreased at pHs 6.5, 7.0, 8.0 and 8.5, in stripped state as well as in the presence of IHP. In order to investigate the unexpected results mass spectrometry of the purified intraerythrocytic content was performed. RBC lysate was separated from stroma by ultracentrifugation (14.000rpm) and purified by molecular exclusion filtration (Amicon filter - Millipore, 10KDa cutoff) and ion exchange chromatography (Amberlite MB-3, Sigma-Aldrich) for desalinization. Quadrupole Mass spectrometry (MS) with 0.8 ppm MS and MS/MS accuracy (Impact High DefinitionTM, Bruker), preceded by UPLC (Ultra Pressure Liquid Chromatography) system (Shimadzu Nexera) with C18 Polymer RP-column (Supelco apHera C18 Polymer 150 x 2 mm, 5um) was used to separate and identify and compare the compounds to control sample (secondary erythrocytosis with no Hb function alteration). No alteration in peptide, hemoglobin or heme were found. Four small compounds, two derived of sphingomyelin and two derived of phosphatidylserine were identified in both patient samples (C39H79N2O6P, C42H80NO8P, C42H82NO8P, C44H84NO8P) - Figure 1. Sphingomyelin and phosphatidylserine are RBC membrane compounds rich in PO4-, which theoretically could bind b1b2 allosteric 2,3-BPG site in Hb. There is no evidence that these molecules were effectively bound to Hb except that were found in purified Hb samples with some function alteration, particularly with decreased heme-heme cooperativity. Some evidence suggests that analog compounds could be found in higher concentrations in PV or secondary erythrocytosis. To our knowledge it is the first-time that alteration in Hb function was observed in samples with these molecules. These preliminary results should be further validated and at least 10 other similar cases should be better characterized. (Financial support: Fapesp, Faepex-Unicamp, Educorp-Unicamp). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Blood Cell Parameters Combined with Inflammatory Markers in the Early Diagnosis of Pulmonary Embolism

Venous thrombosis is a semi-solid formation of blood components that coalesce in the venous system, and the pathological process of its formation is called venous thrombosis. The deep veins of the lower extremities are a common site of prevalence, and the clinical diagnosis of lower extremity deep vein thrombosis can occur independently or as a complication of other diseases. There is a clear link between inflammation and coagulation/anticoagulation, with inflammatory mechanisms upregulating pro-inflammatory factors, downregulating natural anticoagulant substances, and inhibiting fibrinolytic activity; systemic inflammation is a strong pro-thrombotic stimulus; and in vivo, natural anticoagulant substances not only prevent thrombosis, but also deter inflammatory processes. The interconnection between inflammation and coagulation plays an important role in venous thrombosis. In this study, we analyzed the relationship between inflammatory markers CRP and Fg, FVIII:C and FIX:C by measuring plasma CRP concentration, Fg level, FVIII:C and FIX:C levels in patients with DVT diagnosed by ultrasound, and explored the role and mechanism of inflammatory response and coagulation factor abnormalities and the interaction between them in the development of DVT. In this paper, human blood DNA was extracted by phenol-chloroform-isoamyl alcohol extraction, and CRP 1059G/C gene polymorphism was detected by polymerase chain reaction-restriction enzyme segment length polymorphism (PCR-RFLP) nucleotide typing technique, and the genotypes of each subject were distinguished according to the bands seen by gel electrophoresis, and the frequency of each genotype was counted. Plasma CRP concentrations were measured by immunoturbidimetric assay, FVIII:C and FIX:C levels were measured by phase I assay, and plasma Fg levels were measured by coagulation assay in 59 cases (38 males and 21 females, aged 21-82 years, mean 49.67±11.12 years) and 26 controls (17 males and 9 females, aged 32-67 years, mean 50.13±8.96 years). The above indexes were compared between the two groups, and the correlation between CRP and FVIII:C, FIX:C and Fg was analyzed. Polymerase chain reaction-restriction enzyme segment length polymorphism nucleotide typing technique was used to detect the relationship between CRP 1059G/C gene polymorphism and DVT, to further search for risk factors of venous thrombosis, thus providing new ideas for the future prevention and treatment of this disease in clinical practice.

Molecular Identification, and Characterization of Mycobacterium kansasii Strains Isolated from Four Tuberculosis Regional Reference Laboratories in Iran During 2016-2018.

BackgroundNon-tuberculous mycobacterial (NTM) infections are growing concern in many countries around the globe including Iran. Among them, Mycobacterium kansasii (M. kansasii) causes both pulmonary and extra-pulmonary infections. Despite the high prevalence of M. kansasii isolates in Iran, unfortunately little is known about the epidemiological aspects of M. kansasii infection. Hence, the aim of the present study was to investigate the molecular identification, determination of subtypes variation and geographic distribution of clinical isolates of M. kansasii isolates.MethodsIn the present study, 108 clinical pulmonary isolates suspected to NTM were collected from four Tuberculosis Regional Reference Laboratories in Iran during 2016–2018. The isolates were confirmed as NTM using conventional and molecular methods. Among them, M. kansasii isolates were subjected to rpoB gene sequencing. For determination of subtyping of M. kansasii isolates, polymerase chain reaction-restriction enzyme analysis (PCR-REA) based on the hsp65 gene was performed.ResultsBased on the rpoB gene sequence analysis, 33 (30.5%) isolates were identified as M. kansasii species, compared to 31 (28.7%) isolates using phenotypic methods. The subtype I was the most frequent subtype (n=24; 72.7%), followed by subtype II (n=8; 24.2%).ConclusionWe indicated that the rate of M. kansasii isolation with clinical significance appears to be increasing in Iran, especially in highly industrialized cities. The high rate of M. kansasii subtype I may suggest that this genotype has a particular potency for colonization, and a higher epidemiological potential for causing infection in humans. More studies are needed to provide a better understanding of the biology and pathogenicity of M. kansasii subtype I.

Genetic Analysis of EGLN1 C127S Variant in Taiwanese Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder related to nigrostriatal dopaminergic neuron degeneration and iron accumulation. As a cellular oxygen sensor, prolyl hydroxylase domain containing protein 2 (PHD2, encoded by egl-9 family hypoxia inducible factor 1, EGLN1) modifies hypoxia-inducible factor alpha (HIF-α) protein for proteasomal destruction under normoxic condition. In addition, 2-oxoglutarate- (OG-) dependent dioxygenase activity of PHD2 is involved in the oxygen and iron regulation of iron-responsive element binding protein 2 (IRP2) stability. Previously increased expression of EGLN1 was found in the substantia nigra of the parkinsonian brain. We investigated the possible role of c.380 G > C (p.C127S) of EGLN1 gene in Taiwanese patients with PD. 479 patients and 435 healthy controls were recruited. Polymerase chain reaction and BsmAI restriction enzyme analysis were applied for analysis. An association between CC genotype and reduced PD risk in the recessive model (CC vs. GG + GC) was found. Our study provides a link between EGLN1 c.380 G > C SNP and the development of PD.

Cloning and molecular characterization of thiol-specific antioxidant gene of Leishmania tropica Turkey isolate

Background/aimThiol-specific antioxidant (TSA) protein is one of the most promising molecules among candidates for vaccine against cutaneous leishmaniasis. It was found to be significantly protective against different Leishmania species. In this study, cloning and molecular characterization of thiol-specific antioxidant gene of L. tropica Turkey isolate (LtTSA) were aimed. Materials and methods LtTSA was amplified by PCR using the specific primers of TSA gene and cloned into the pcDNA3.1 vector. The cloning was confirmed by PCR screening, restriction enzyme reactions, and DNA sequence analysis. Finally, three-dimensional structure and antigenic properties of the protein encoded by the LtTSA were determined.ResultsSix hundred base pair bands belonging to LtTSA were shown with electrophoresis. It was found that LtTSA and its encoded protein have high similarity with different Leishmania species. LtTSA protein consisting of 199 amino acids was found to have 7 different antigenic regions. ConclusionLtTSA and its encoded TSA protein were found to be highly immunogenic and similar to TSA proteins previously tested as a vaccine candidate.

Association of ICAM-1 gene variant rs5498 (1462A>G) with non-obstructive azoospermia

The blood testicular barrier (BTB) is a barrier protecting the testes from damage. It also plays an important role in spermatogenesis. The intracellular adhesion molecule-1 (ICAM-1) is a member of the immunoglobulin cell adhesion molecule family and is a constituent component of the BTB. This study was carried out to investigate the association between the single nucleotide polymorphism (SNP) rs5498 in the ICAM-1 gene and non-obstructive azoospermia. A total of 100 male patients with non-obstructive azoospermia and 100 fertile males (healthy control) were included in this study. Genotyping of ICAM-1 gene rs5498 was performed using polymerase chain reaction-restriction enzyme fragment length polymorphism (PCR-RFLP). Both AG and GG genotypes were significantly more prevalent among the infertile men compared to the control group (p < 0.001). The odds ratio (OR) for the polymorphic allele (G) was 2.4 with 95% confidence interval from 1.38 to 3.19 (p < 0.001). The study shows that ICAM-1 rs5498 (1462A>G) SNP is positively associated with an increased risk for obstructive azoospermia, with the genotype AG heterozygotes showing a significantly higher frequency among infertile men.

Mitochondrial DNA G15927A and G15928A variations in patients with multiple sclerosis

BackgroundModern genetics has offered a fresh perspective on the pathology of Multiple Sclerosis (MS). As mitochondrial DNA (mtDNA) variations are held to be potential contributors to the complex pathobiology of MS, the present study tests the claim that mtDNA G15927A or G15928A variations, or both, are associated with MS in an Iranian population. Materials and methodsFollowing DNA extraction from blood samples of 100 subjects with relapsing-remitting MS, and 100 healthy unrelated control subjects, PCR-RFLP analyses was carried out by HpaII restriction enzyme reaction. Electrophoresis was then performed with 3% Agarose gel. As the restriction enzyme did not differentiate between two neighboring nucleotide positions (G15927A and G15928A), all PCR products with a variant allele were sequenced to determine the exact position of the variation. ResultsThe MtDNA G15927A or G15928A variations were observed in 11 of all 100 cases of MS (11%) and in 7 of 100 healthy control subjects (7%) (P = 0.3, OR = 1.6, 95% CI = 0.5–5.2). Having sequenced all the PCR products with the variant allele (11 cases and 7 controls), the mtDNA G15927A variation was found in one of the 100 cases (1%) and 3 of 100 controls (3%) (P = 0.3, OR = 0.3, 95% CI = 0.0–4.1). Therefore, the mtDNA G15928A variation was present in 10 of the 100 cases (10%) and in 4 of 100 controls (4%) (P = 0.09, OR = 2.6, 95% CI = 0.7–12.0). ConclusionNeither mtDNA variation, G15927A or G15928A, was associated with MS in the studied Iranian population. There was a non-significant association of the G15927A and the G15928A variations separately with MS.

MOLECULAR SCREENING OF FOUR CANINE HEREDITARY DISEASES IN TAIWAN

Animals that have inherited a mutant gene from their parents (father and/or mother) may acquire hereditary diseases. Most mutations are autosomal recessive. Usually, hereditary diseases go unnoticed until symptoms arise. The objective of this study is to screen four canine hereditary diseases in Taiwan. The diseases include persistent Müllerian duct syndrome (PMDS) and myotonia congenita (MC) in Miniature Schnauzers, Golden Retriever muscular dystrophy (GRMD) in Golden Retrievers and glycogen storage disease type Ia (GSD Ia) in Maltese. We collected 98 samples from Miniature Schnauzers, 57 samples from Golden Retrievers and 54 samples from Maltese. All molecular tests are based on restriction fragment length polymorphism (RFLP), which consists of a genomic polymerase chain reaction (PCR) plus specific restriction enzyme digestion. Results showed that the 98 samples, which were collected from Miniature Schnauzers, were all MC wild type (WT), with 78 being PMDS WT and 20 (20%) being PMDS heterozygous type. None of the samples were MC mutant type (MT) or PMDS MT; on the other hand, no GRMD mutant gene or GSD Ia mutant gene was found in Golden Retrievers and Maltese samples.

Identification of the Infection Source of an Outbreak of Mycobacterium Chelonae Keratitis After Laser in Situ Keratomileusis

Nontuberculous mycobacteria keratitis is a rare but challenging complication of laser in situ keratomileusis (LASIK). This study was conducted to determine the source(s) of infection in a cluster of cases of keratitis after LASIK and to describe this outbreak and patients' outcomes. In this retrospective, case series, single-center study, 86 patients were included who underwent LASIK or photorefractive keratectomy between December 2011 and February 2012. Corneal scrapes from the affected eyes, samples of tap and distilled water, water from the reservoir of the distilling equipment, steamer, and autoclave cassette; antiseptic and anesthetic solutions and surgical instrument imprints were cultivated in liquid and on solid media. Gram-negative bacteria and yeasts were identified using automated systems and mycobacteria by polymerase chain reaction-restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and DNA sequencing. Mycobacterial isolates were typed by pulsed-field gel electrophoresis. The cases and outcomes are described. The main outcome measure was identification of the source(s) of the mycobacterial infections. Eight (15 eyes) of 86 patients (172 eyes) who underwent LASIK developed infections postoperatively; no patients who underwent photorefractive keratectomy developed infections. Mycobacterium chelonae was isolated from 4 eyes. The distilled water collected in the surgical facility contained the same M. chelonae strain isolated from the patients' eyes. Different gram-negative bacteria and yeasts were isolated from samples collected at the clinic but not from the patients' eyes. Tap water distilled locally in surgical facilities may be a source of infection after ocular surgery and its use should be avoided.

Cutaneous infection due to Mycobacterium immunogenum: an European case report and review of the literature

(no more than 200 words): In the last few years, the incidence of cutaneous infections caused by nontuberculous mycobacteria is increasing. Since Mycobacterium immunogenum was first described in 2001, few case reports have been described, all of them in the American continent. We report a case with cutaneous infection caused by this newly discovered NTB in Europe.A 65-year-old woman presented with a 3-months history of pruritic lesions on abdomen. Examination revealed erythematous inflammatory papules, pustules, and crusts. Three weeks later, mycobacteria were cultured from the biopsy specimen. Mycobacterium immunogenum was identified based on susceptibility test results and polymerase chain reaction (PCR) restriction enzyme analysis. Treatment with clarithromycin was started. M. immunogenum is a nontuberculous mycobacterium that was first described by Wilson et al. in 2001 as a rapidly growing variety and new species in the Mycobacterium chelonae-Mycobacterium abscessus group. PCR-restriction analysis of a 439-bp segment of the hsp65 gene and/or sequencing the species-specific region of the 16S rDNA can confirm this new species. Since the description of M. immunogenum, 8 clinical case reports have been published, most involving cutaneous infections and all of them in the American continent. We present a case of cutaneous infection caused by M. immunogenum in a Spanish woman.

Site accessibility tailors DNA cleavage by restriction enzymes in DNA confined monolayers.

Density-tunable nanografted monolayers (NAMs) of short oligonucleotide sequences on gold surfaces show novel properties that make them suitable for advanced biosensing applications, and in particular to study the effects of crowding and confinement on biomolecular interactions. Here, combining atomic force microscopy nanolithography, topography measurements and coarse-grained molecular dynamics simulations, we investigated restriction enzyme reaction mechanisms within confined DNA brushes highlighting the role played by the DNA sequence conformation and restriction site position along the chain, respectively, in determining the accessibility of the enzyme, and its consequent cleavage efficiency.

Effect of MTHFR Gene Polymorphism Impact on Atherosclerosis via Genome-Wide Methylation.

BackgroundAtherosclerosis seriously threats human health. Homocysteine is an independent risk factor closely related to DNA methylation. MTHFR C667T loci polymorphism is closely associated with homocysteine level. This study aimed to investigate the relationship among MTHFR C667T loci polymorphism, genome-wide methylation, and atherosclerosis.Material/MethodsBlood sample was collected from 105 patients with coronary atherosclerosis and 105 healthy controls. Pyrosequencing methylation was used to detect LINE-1 methylation level. Polymerase chain reaction-restriction enzyme fragment length polymorphism (PCR-RFLP) was used to test MTHFR.ResultsLINE-1 methylation level in the patient group was significantly lower than in the controls (t=5.007, P<0.001). MTHFR C667T genotype distribution presented marked differences in the 2 groups. TT genotype carriers had significantly increased risk of atherosclerosis (OR=3.56, P=0.009). Three different genotypes of MTHFR C667T loci showed different LINE-1 methylation level between the 2 groups (P<0.01). LINE-1 methylation level in TT and CT genotype carriers was obviously lower than in CC genotype carriers (P<0.05).ConclusionsMTHFR C667T loci polymorphism may affect atherosclerosis by regulating genome methylation level.

Development of SNP-based dCAPS markers for identifying male sterile gene tms5 in two-line hybrid rice.

Molecular markers can increase both the efficiency and speed of breeding programs. Functional markers that detect the functional mutations causing phenotypic changes offer a precise method for genetic identification. In this study, we used newly derived cleaved amplified polymorphic sequence markers to detect the functional mutations of tms5, which is a male sterile gene that is widely used in rice production in China. In addition, restriction cutting sites were designed to specifically digest amplicons of tms5 but not wild type (TMS5), in order to avoid the risk of false positive results. By optimizing the condition of the polymerase chain reaction amplifications and restriction enzyme digestions, the newly designed markers could accurately distinguish between tms5 and TMS5. These markers can be applied in marker-assisted selection for breeding novel thermo-sensitive genic male sterile (TGMS) lines, as well as to rapidly identify the TGMS hybrid seed purity.

Determination of genotypic varieties and genotyping of multiple drug-resistant tuberculosis by the RFLP and spoligotyping methods.

The purpose of the present study was to determine the distribution and epidemiological features of mycobacteria with molecular methods. Fifty-five culture-positive samples were analyzed by polymerase chain reaction-restriction enzyme length polymorphism (PCR-RFLP) at species level, and their molecular typing was performed with spoligotyping. The IS6110 region and the locus of gene coding for Hsp65 were amplified. RFLP profiles were obtained by cutting the Hsp65 region with the Hae III and BstE II (Eco91I) enzymes. Spoligotyping was carried out by commercial kit. The H37Rv strain was used as the control. All samples showed the same cutting pattern with the H37Rv strain. The RFLP profiles of 9 strains identified as "mycobacteria other than tuberculosis" were compatible with the M. tuberculosis complex. Spoligotyping of 55 isolates detected 13 different genetic profiles. The Beijing genotype was not detected. One isolate was described as an orphan strain according to the SpolDB4 database. The most frequently detected family was T1 with 32 strains (64%), followed by 9 isolates (18%) belonging to the LAM7 TUR family. PCR-RFLP is a specific, rapid, and effective method in routine diagnosis of mycobacteria. Spoligotyping is an ideal method in the determination of genotypic varieties of mycobacteria.

Cloning and expression of Pteropine Orthoreovirus sigma C protein in bacterial cells

Introduction: Pteropine Orthoreovirus (PRV) was first cloned in 2011 to represent a collective of orthoreoviruses isolated from bat species of the genus Pteropus . Humans, especially infants or children, infected with Mammalian Orthoreovirus (MRV), a genetically closely related ‘ relative’ to PRV, commonly present with mild upper respiratory tract illness or enteritis. However, this does not hold true for PRV, as acute respiratory syndrome is commonly seen in patients infected with PRV. Objectives: The focus of this study was to clone the sigma C gene in two vectors (pMal-C5X and pMal-P5X) and express it in different bacterial cells (ER2523 and BL21). However, the targeted receptor will be identified in a future study. Materials and methods: The sigma C gene of PRV1K (Kampar virus), PRV2P (Pulau virus) and PRV3M (Melaka virus) were amplified and cloned into (pMal-C5X and pMal-P5X) vectors. Colony polymerase chain reaction (PCR) was preformed to screen for positive clones prior to being expressed in BL21 and ER2523 bacteria. Periplasmic and total lysis extraction was performed on both bacterial cells prior to separation by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and visualization with Coomassie Blue stain. Results: The PCR of sigma C gene for three different viruses, namely Pulau, Melaka and Kampar, were performed. PCR of Pulau and Kampar viruses were successful but Melaka virus was unsuccessful because it did not reach the desired band and was excluded. The protein receptors were confirmed by a restriction enzyme reaction. Conclusions: Sigma C of PRV2P was expressed in bacterial cells without any lethal effect on the bacteria. The protein will be purified and targeted receptor identified in a future study. Acknowledgements: We would like to acknowledge our University and our Dean, Dr Khalid Al Qumaizy, for giving us this great opportunity, and we acknowledge Dr Keeny Voon from International Medical University of Malaysia for helping us conduct this research.

Genetic Variant of the C-reactive Protein Gene and Prevalence of Peripheral Arterial Disease in Patients with Type 2 Diabetes Mellitus

Objective: Diabetes mellitus is strongly associated with inflammatory procedures, obesity and macrovascular complications, while genetic factors are involved in the disease pathogenesis and its complications. The aim of this study was to examine the impact of A3872G polymorphism on C-reactive protein (CRP) gene on the prevalence of peripheral arterial disease (PAD) in Greek Caucasian subjects with type 2 diabetes mellitus (T2DM). Methods: The study population consisted of 423 patients with T2DM. The A3872G polymorphism was detected by polymerase chain reaction and appropriate restriction enzyme digestion (HpyCH4III). High sensitivity CRP was assayed by particle-enhanced immunonephelometry. PAD was diagnosed using clinical or ultrasonography criteria and/or ankle brachial index <0.9. Results: The genotype distribution (%) in PAD patients (GG: 48.1, AG: 29.1, AA: 22.8) did not differ significantly compared to non-PAD disease subjects (GG: 53.8, AG: 26.7, AA: 19.5), (P=0.258). After adjustment for classical risk factors, carriers of “A” allele (AG+AA) compared with GG homozygotes had higher odds (OR) for PAD, (AG+AA): 82 (40.0) vs. GG: 76 (34.2), (OR 1.622, 95% confidence intervals 1.029-2.536, P=0.037). Conclusions: In Caucasian subjects with T2DM, the GG homozygotes of the A3872G genetic polymorphism on the C-reactive protein gene may be protected from the risk of peripheral arterial disease compared to carriers of the “A” allele. These results indicate a potential genetic role of inflammation in atherosclerosis in patients with type 2 diabetes mellitus.

Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products.

In this study, we optimized a restriction-ligation-free (RLF) method to save time and cost of constructing multiple plasmids with the same gene insert, and examined the efficacy of RLF on high-throughput multi-plasmid cloning. This method utilizes the precise DNA repair and recombination systems within Escherichia coli, which allows to bypass the in vitro restriction and ligation enzyme reactions commonly included in routine cloning procedures. A homologous arm is linked to the 5'-end of the forward primer used to amplify both the target gene and vector. A different homologous arm is linked to the 5'-end of the reverse primer. Therefore, genes can be cloned into the vectors by homologous recombination after co-transformation of the amplified target gene and the linearized vector, which bear the same homologous arm on either end. More than twenty-four different plasmids were generated by this method, which uses two simple polymerase chain reaction steps. This method is highly efficient in cloning any gene of interest into any vector at any site without sequence constraints, as no restriction and ligation reactions are required.

Relationship between a lipoprotein lipase gene polymorphism in placental tissue and insulin resistance in patients with gestational diabetes mellitus

The aim of this study was to investigate the relationship between a lipoprotein lipase (LPL) gene polymorphism in placental tissue and insulin resistance (IR) in patients with gestational diabetes mellitus. Using polymerase chain reaction-restriction enzyme fragment length polymorphism (PCR-RFLP) analysis, the LPL HindIII RFLP was examined in the placental tissue of 110 patients with gestational diabetes mellitus (observation group) and 110 women with normal gestation (control group). The relationships between fasting plasma glucose (FPG), postprandial plasma glucose (PPG), fasting insulin (FINS), cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL), body mass index (BMI), and IR indices and the LPL polymorphism in the two study groups and their offspring were determined. The frequency of the H+ allele was significantly higher in the observation group than in the controls (P < 0.05). There were statistically significant differences in the observation group between the FPG, PPG, LDL, TC, TG, HDL, BMI, FINS, and IR indices of the H+H+ group and those of the non H+H+ type patients (P < 0.05). Correlation analysis showed that the LPL gene polymorphism was positively related to IR. There were statistically significant differences between HDL, BMI, and IR indices between the two study groups (P < 0.05). In conclusion, the LPL gene polymorphism was determined to be the main factor related to IR in women with gestational diabetes, and was also found to be related to the IR of their offspring.

A study of the association between the connexin 40 rs35594137 polymorphism and atrial fibrillation in Xinjiang Chinese Han and Uygur populations.

Atrial fibrillation (AF) occurrence has a known genetic component. Many reports have revealed a correlation between gene mutation and AF, involving genes related to ion channels, connexin (Cx), and those within the angiotensin system. In this study, the correlation between the Cx 40 polymorphism (rs35594137) and AF was investigated in patients with AF in the Xinjiang, Turpan, and Kashi regions and in controls. The AF cohort included 122 patients (58 Han and 64 Uygur). The control subjects were recruited according to the 1:1 corresponding method and matched for age and gender. Polymerase chain reaction-restriction enzyme fragment length polymorphism was used to analyze Cx 40 (rs35594137) genotype and allele distributions. Compared with the control group, A allele and A allele frequency were higher in the promoter region of Cx 40 (rs35594137) site (P < 0.05). In the Uygur population, the distribution of rs35594137 genotype and allele frequencies was not significantly different (P > 0.05) from the AF and control groups of Chinese Han origin. The confounding factors, including coronary heart disease, hypertension, smoking, and drinking, were evaluated by multivariate conditional logistic regression analysis. Cx 40 (rs35594137) differences between AF and control groups of Han origin were not significant (P > 0.05), but were statistically significant in the Uygur population (P < 0.05). These results demonstrated that Cx 40 (rs35594137) was associated with AF. In the Uygur population, Cx 40 (rs35594137) should be considered as an independent risk factor for patients with AF, who might have racial differences in rs35594137 variant frequencies.