Separate Polymerase Chain Reaction Research Articles

Detection of Virulence Genes in Pseudomonas aeruginosa Isolates by Polymerase Chain Reaction and Investigation of High-Risk Clones by Matrix Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometer Method

Pseudomonas aeruginosa is a non-fermentative gram-negative bacillus. Many virulence factors play a role in the pathogenesis of P.aeruginosa. The aim of this study was to early detection of ST111, ST175, ST235, ST253, ST395 which are named high-risk clones with increased epidemic potential due to multidrug resistance in P.aeruginosa isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method and to evaluate the relationship between high-risk clones and the presence of P.aeruginosa virulence factors and carbapenemase production genes.P.aeruginosa isolates (n= 100) found to be resistant to at least imipenem or meropenem antibiotics isolated from the various clinical samples in the medical microbiology laboratory between 01.01.2021 and 07.06.2022 were included in the study. For the detection of virulence genes uniplex polymerase chain reaction (PCR) for toxA and multiplex PCR for algD, plcN, lasB, plcH were performed in P.aeruginosa isolates. In the detection of carbapenemase genes, two separate multiplex PCRs used for blaKPC , blaNDM , blaVIM , blaOXA-48 and for blaIMP , blaSPM , blaSIM , blaGIM , blaGES . Investigation of the peaks specific to high-risk clones was performed by using VITEK®-MS (bioMérieux, France) system. P.aeruginosa isolates were mostly isolated from intensive care units (45%) and respiratory tract samples (46%). The antibiotic to which the isolates were found to be most susceptible was amikacin, while highest resistance was detected for piperacillin. In PCR results, toxA, lasB, plcH, plcN and algD were detected as 89%, 99%, 98%, 100%, 100%, respectively. When the presence of characteristic peaks belonging to high-risk clones was evaluated with MALDI-TOF MS, ST253 (7%) and ST175 (2%) were detected. The peaks specific to ST235 and ST395 clones were not detected in our study. blaVIM was detected in two isolates and blaGES-5 carbapenemase was detected in two isolates. Virulence factors were detected at high rates in both high-risk clones and other strains and no significant relationship was found between high-risk clones and virulence factors. Early detection of high-risk clones, identification of antimicrobial resistance mechanisms will help to develop strategic treatment options and prevent their worldwide spread.

Interleukin-21 receptor gene polymorphism (rs2285452 A/G) is associated with susceptibility to Behçet's disease.

Behçet's disease (BD) is a chronic auto inflammatory disorder of unknown aetiology. Recently, the dysregulation of interleukin-21 receptor (IL-21R) has been incriminated in different autoimmune and auto-inflammatory diseases, such as systemic lupus erythematous, rheumatoid arthritis, and type 1 diabetes. Herein, we aimed to investigate the association of two Il-21R gene polymorphisms with BD. IL-21R rs2214537 and IL-21R rs2285452 genotypings were investigated in a cohort of 110 adult patients with BD and 116 age and gender unmatched healthy controls. Genotyping was performed by mutagenically separated polymerase chain reaction with newly designed primers. IL-21R rs2285452 genotypes and alleles distribution were statistically different between patients with BD and controls. GA and AA genotypes carrying the minor A allele were more frequent in patients with BD than in healthy controls (37.3% and 11.8% vs. 23.3% and 3.4%, respectively). The minor A allele was associated with an increased BD risk (odds ratios =2.42, 95% confidence interval =1.214.87, p =.005). IL-21R rs2214537 GG genotype was found to be associated with susceptibility to BD in the recessive model (GG vs. CC + CG; p =.046, OR = 1.91, 95% CI = 1.003.650. IL-21R rs2285452 and IL-21R rs2214537 were not in linkage disequilibrium (D' =0.42). The AG haplotype was more frequently observed in patients with BD than in controls (0.247 vs. 0.056, p = .0001). This study for the first time reports the association of IL-21R rs2285452 and IL-21R rs2214537 with BD. Functional studies are required to elucidate the exact role of these genetic variants.

Clinical data-driven approach to identifying COVID-19 and influenza from a gradient-boosting model

Corona Virus Disease 2019 (COVID-19) and influenza are both caused by viruses, seriously affect human health, and are highly infectious. However, because the clinical manifestations of these two groups of diseases have almost identical symptoms, separate Polymerase Chain Reaction (PCR) tests must be used for patients in each disease group. This study proposes an automatic data-processing model based on artificial intelligence and gradient boosting to identifying COVID-19 and influenza. The model can learn directly from raw data without the need for human input to delete empty data. Methodology and techniques operate in two stages: first, it evaluates and processes data to reduce the dataset’s complexity using the light gradient boosting machine (LightGBM); then, in the second stage, it builds a classification model for each disease group based on the extreme gradient boosting (XGBoost) method. The research tools showed that combining two gradient-boosting models both LightGBM and XGBoost to generate automatic COVID-19 and influenza classifiers from clinical data produced strong results and a superior performance versus one model alone, with an overall accuracy of over 99.96%. In the future, the developed model will enable patients to be diagnosed simply and accurately and thereby reduce countries’ testing costs for COVID-19 and similar pandemics that may arise.

Conjunctivitis with Monkeypox Virus Positive Conjunctival Swabs

A 39-year-old man consulted for unilateral red eye and itchiness (Fig A, C) 5 days after positive monkeypox virus (MPOX) cutaneous polymerase chain reaction (PCR) swab from chin and lip lesions (Fig B). Slit-lap examination showed conjunctival follicular reaction and the presence of small white vesicles on the nasal bulbar conjunctiva (Fig C, arrow). The rest of the anterior and posterior segment were normal, and the fellow eye remained uninvolved during follow up. Two separate conjunctival PCR swabs were positive for MPOX, confirming indirectly similar loads of the virus on conjunctival and eye secretions compared with cutaneous lesions (26.7 vs 24.8 [cycle threshold] respectively), raising the possibility of transmission via eye contact, i.e., during ophthalmologic examination.

Gene polymorphisms of Interleukin 6 (\u2212174 G/C) and transforming growth factor \u03b2-1(+915 G/C) in ovarian cancer patients

BackgroundIn the study on hand, we investigated the effect of IL-6 (−174 G/C; rs 1800795) and TGF-β1 (+915G/C; rs 1800471) gene polymorphisms on the susceptibility to Ovarian Cancer and their effect on plasma levels. IL-6 (−174 G/C) SNP was analyzed using mutagenically separated polymerase chain reaction (MS-PCR) while TGF-β1 +915G/C (codon 25) SNP was investigated by the sequence-specific primer polymerase chain reaction (SSP-PCR). An enzyme-linked immunosorbent assay (ELISA) was used to quantify IL-6 and TGF-β1 plasma levels in 48 ovarian cancer patients and 48 normal controls.ResultsRegarding IL 6 (−174 G/C), a significant increase in CC and GC+CC genotypes parallel with the C allele was considered as risk factors for ovarian cancer; on the other hand, the G allele was considered as a protective factor for ovarian cancer. TGF-β1 (+915G/C) investigations showed a significant elevation in GC and GC+CC genotypes which can be considered as a risk factor for ovarian cancer. Plasma IL-6 and TGF-β1 were higher in ovarian cancer patients compared with controls. No specific genotype or allele could be responsible for the elevation of TGF-β1 in ovarian cancer patients’ plasma, while the highest significant value for IL6 in subjects carrying GG and CC genotypes in comparison with GC genotype.ConclusionsThis study supports an association of IL6 (−174G/C) and TGF-β1 (+915G/C) gene polymorphisms with the susceptibility to ovarian cancer.

Acute Myocarditis Secondary to Paediatric Inflammatory Multisystem Syndrome Temporally Associated With COVID-19 Infection

A 17-year-old female, who was previously fit and well with no preexisting health conditions, presented with a four-day history of worsening shortness of breath and diarrhoea. She had recent close contact with a relative diagnosed with COVID-19. On clinical examination, she was drowsy, hypotensive, tachycardic, tachypnoeic, and pyrexial. Her blood tests showed elevated inflammatory markers and lymphopenia. She underwent a transthoracic echocardiogram, which confirmed a severely impaired left ventricular (LV) systolic function with an ejection fraction of 35%. An initial impression of acute viral myocarditis was made. Three separate polymerase chain reaction (PCR) tests for SARS-CoV-2 RNA were performed, but they all returned negative. The patient was not responding to initial therapy. Therefore, the regional paediatrics hospital was consulted, and a diagnosis of paediatric inflammatory multisystem syndrome temporally associated with COVID-19 (PIMS-TS) was made, based on similar regional presentations. The patient was administered IV immunoglobulin therapy, to which she responded very well. Following a five-day hospital stay, the patient was discharged home as medically stable. A repeat transthoracic echocardiogram (TTE) showed recovery of the LV systolic function to 62%. Few cases have been reported on myocardial involvement in young patients with PIMS-TS. This case report highlights the initial presentation, medical care, and clinical course of this patient.

Search for Genetic Markers of Tick-Borne Encephalitis Virus for Its Specific Indication, Genomic Analysis

Objective of the study is to analyze the genetic markers of tick-borne encephalitis virus, which can be used to specifically indicate the maximum number of virus strains and isolates.Materials and methods. Plasmid DNA and nucleic acids of the tick, the genus Ixodes and Dermacentor, were used as amplified material. Polymerase chain reaction (PCR) was conducted on C1000 amplifier with a CFX96 optical unit (BioRad). The species (strain) variety of detected organisms, using the analyzed genetic markers, was determined in the nBLAST software utility. The design of the nucleotide sequences of primers and probes was performed using “Vector NTI 9.1.0” (Invitrogen Corporation). Nucleic acids were isolated by magnetic sorption with a reagent panel MAGNO-sorb. Primers and probes were synthesized at “Evrogen” company, Moscow, Russia. The reagents for PCR were manufactured by “Syntol” company, Moscow, Russia.Results and discussion. When indicating the genome of the tick-borne encephalitis virus, the main criterion for choosing a marker sequence is the specific detection of nucleic acids of only the desired microorganism. The genomewide nucleotide sequences of various strains and isolates of tick-borne encephalitis virus were analyzed to search for a specific marker nucleotide sequence. Mutual comparison of all the above mentioned genomes made it possible to determine 7 conventionally conservative loci characterized by minimal nucleotide variability. The further work was based on the results of alignment of the nucleotide sequences of tick-borne encephalitis virus isolates, including the nucleotide sequences of heterogeneous microorganisms; primers and probes were designed to amplify each of the marker loci; the most analytically significant oligonucleotides were developed based on loci 4 and 7. Amplification with oligonucleotide primers to indicate tick-borne encephalitis virus was effective, both in a separate PCR with a positive control, and in combination with tick DNA.

Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Verification of Co-Integrates and Selection of Resolved BAC Clones.

Successful modification of the bacterial artificial chromosome (BAC) after two-step BAC engineering is confirmed in two separate polymerase chain reactions (PCRs). The first reaction (5' co-integrate PCR) uses a forward 5' co-integrate primer (a sequence located upstream of the 5' end of the A-box) and a reverse 3' primer on the vector (175PA+50AT) or within the reporter sequence or mutated region as appropriate. The second reaction (3' co-integrate PCR) uses a forward 5' primer on the recA gene (RecA1300S) and a reverse 3' co-integrate primer (a sequence located downstream from the 3' end of the B-box). Those colonies shown to be positive in PCR analysis are further tested for sensitivity to UV light. After the resolution, colonies that have lost the excised recombination vector including sacB and recA genes become UV light sensitive.

Use of real-time multiplex PCR, malaria rapid diagnostic test and microscopy to investigate the prevalence of Plasmodium species among febrile hospital patients in Sierra Leone

BackgroundMalaria continues to affect over 200 million individuals every year, especially children in Africa. Rapid and sensitive detection and identification of Plasmodium parasites is crucial for treating patients and monitoring of control efforts. Compared to traditional diagnostic methods such as microscopy and rapid diagnostic tests (RDTs), DNA based methods, such as polymerase chain reaction (PCR) offer significantly higher sensitivity, definitive discrimination of Plasmodium species, and detection of mixed infections. While PCR is not currently optimized for routine diagnostics, its role in epidemiological studies is increasing as the world moves closer toward regional and eventually global malaria elimination. This study demonstrates the field use of a novel, ambient temperature-stabilized, multiplexed PCR assay in a small hospital setting in Sierra Leone.MethodsBlood samples from 534 febrile individuals reporting to a hospital in Bo, Sierra Leone, were tested using three methods: a commercial RDT, microscopy, and a Multiplex Malaria Sample Ready (MMSR) PCR designed to detect a universal malaria marker and species-specific markers for Plasmodium falciparum and Plasmodium vivax. A separate PCR assay was used to identify species of Plasmodium in samples in which MMSR detected malaria, but was unable to identify the species.ResultsMMSR detected the presence of any malaria marker in 50.2% of all tested samples with P. falciparum identified in 48.7% of the samples. Plasmodium vivax was not detected. Testing of MMSR P. falciparum-negative/universal malaria-positive specimens with a panel of species-specific PCRs revealed the presence of Plasmodium malariae (n = 2) and Plasmodium ovale (n = 2). The commercial RDT detected P. falciparum in 24.6% of all samples while microscopy was able to detect malaria in 12.8% of tested specimens.ConclusionsWider application of PCR for detection of malaria parasites may help to fill gaps existing as a result of use of microscopy and RDTs. Due to its high sensitivity and specificity, species coverage, room temperature stability and relative low complexity, the MMSR assay may be useful for detection of malaria and epidemiological studies especially in low-resource settings.

A novel simple genotyping assay for detection of the ‘Gait keeper’ mutation in DMRT3 and allele frequencies in Azteca and Costa Rican Saddle Horse breeds

The ‘Gait keeper’ mutation in the DMRT3 gene alters locomotion and gait patterns in horses. This mutation (C>A) has been found in all gaited breeds of horses analyzed but is absent in most non-gaited breeds. We developed a new mutagenically separated polymerase chain reaction (MS-PCR) based method for simple detection of horse DMRT3 genotype. Our method was applied in a preliminary study to determine DMRT3 allele frequencies in 78 Azteca horses (AZ) and 53 Costa Rican Saddle Horses (CRSH). We found a wild-type C allele frequency of 100% in the AZ horses. For the CRSH, the wild-type C frequency and mutant A allele frequency were 88.7% and 11.3%, respectively.

IRAK2 is associated with systemic lupus erythematosus risk.

Interleukin-1 receptor-associated kinases (IRAKs) are serine-threonine kinases involved in toll-like receptor and interleukin-1 signaling pathways. They play a key role in inflammation and innate immunity. IRAKs have been previously incriminated in autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis and inhibition of IRAKs has been recently regarded as a potential therapeutic strategy for SLE. The aim of the present study was to test the association between IRAK2 rs708035 and rs3844283 with SLE. IRAK2 rs708035 and rs3844283 were genotyped by mutagenically separated polymerase chain reaction (MS-PCR) in 142 SLE patients and 149 age- and gender-matched controls. The hyperfunctional IRAK2 rs708035 A allele was more frequent among SLE patients than controls (62.9% versus 54.7%, p = 0.046). IRAK2 rs3844283 C allele was present in 66.5% of patients and 75.5% of controls. The CC genotype was the most frequently exhibited genotype. It was carried by 45.1% of patients with SLE and 57.7% of controls. The G allele was associated with an increased risk of SLE (OR = 1.54, 95%, CI = 1.07-2.22, p = 0.017). IRAK2 rs708035 and IRAK2 rs3844283 were in linkage disequilibrium (D' = 0.64). The AG haplotype was more frequently observed in SLE patients than in controls (0.292 versus 0.194, p = 0.008). This study for the first time ever reveals the association of IRAK2 rs708035 and IRAK2 rs3844283 and the corresponding haplotypes with SLE. Our findings give additional rationale to target IRAKs in the treatment of SLE.Key Points• IRAK2 rs708035 A allele is more frequent in SLE patients than in controls and IRAK2 rs3844283 G allele is associated with SLE susceptibility.• These two alleles are in linkage disequilibrium.• The AG haplotype is associated with SLE.

Determination of Arylamine N-Acetyltransferase 2 Acetylation Genotype by PCR and Phenotyping Using Dapsone Through High-Pressure Liquid Chromatography Assay: A Gender Wise Study.

The main aim of the study was to establish the acetylation status of local population of Pakistan by N-acetyltransferase 2 (NAT2) enzyme and to find out the concordance between phenotypic and genotypic methods for the determination of NAT2 acetylation. Gender-wise comparison of selected healthy male and female volunteers aged greater than 18 years was also conducted to see the effect of sex on NAT2 acetylation. Phenotypically, the rate of acetylation was determined by high-pressure liquid chromatography with dapsone (DDS) probe drug, while genotypically, NAT2 acetylation was determined by using specific primers for NAT2 variant alleles (M1, M2, and M3) amplified in separate polymerase chain reactions. High-pressure liquid chromatography results indicated 64% of the male volunteers to be fast acetylators while 36% were slow acetylators, while ratio of fast and slow acetylators for female was found to be 66% and 34%, respectively. Genotypically, the ratio of fast and slow for male was 60% and 40% and for female was 66% and 34%, respectively. The distribution of 3 NAT2 variant alleles was found in invariable number. For male volunteers, the highest frequency distribution showed by M2 allele was 56%, while for M1 and M3 the frequency was 32% and 12%, respectively, and for female volunteers highest frequency (51%) was shown by the M2 variant allele while lowest frequency (18%) was shown by M3 allele. There was the 94% concordance between the DDS phenotype and genotype. Gender effect on the acetylation was found to be nonsignificant (P > .05). Therefore, it is concluded that NAT2 acetylation rate can be used to check in vivo acetylation status with dapsone as probe drug. It is concluded that NAT2 acetylation rate was unaffected by gender and can be used to check in vivo acetylation status with dapsone as probe drug, which is inexpensive and less time-consuming.

Interleukin-6 promotor gene polymorphisms and susceptibility to chronic hepatitis B virus in Egyptians

AimTo investigate the association between IL-6 polymorphisms (−174G/C, −572G/C and −597G/A) and susceptibility to chronic hepatitis B virus (CHB) infection. MethodTotal 108 subjects with CHB infection and 102 healthy controls were enrolled in this study. IL-6 (−174G/C) was genotyped using Mutagenically separated Polymerase Chain Reaction (MS-PCR) while sequence specific primers-PCR (SSP-PCR) was used for studying −572G/C and −597G/A. IL-6 plasma level was measured using Enzyme-linked immunosorbent assay (ELISA). ResultsA significant increase (P < 0.01, P < 0.01, P < 0.001) in −174GG, −572GC and −597GA; respectively in the CHB group compared to control group, while −572GG genotype was significantly decreased (P < 0.01) in CHB patients. A significant increase (p < 0.01, p < 0.01) in −174 G and −597A alleles was observed in the CHB patient group; respectively. GGA haplotype is significantly increased (P < 0.05) while GCA haplotype is significantly decreased (P < 0.001) in the patient group. A moderate linkage disequilibrium (LD) (D′ = 0.719, r2 = 0.474; P < 0.001) between IL-6 (−572G/C and −597G/A) was observed. A significant reduction (P < 0.01) in IL-6 plasma level in CHB patients compared to healthy controls (22.28 ± 1.93 versus 32.08 ± 2.41), which was negatively correlated (r = −0.216; P < 0.01) with HBV infection. ConclusionsThis study pointed to the potential role of IL-6 (−174G/C, −572 G/C and −597G/A) gene polymorphisms in the susceptibility to HBV infection. Our results allow for only preliminary conclusions due to relatively small sample size. There is a need for further larger scale studies to fully examine the possible relationship between these cytokine gene polymorphisms and the development of CHB.

Association of Asian mitochondrial DNA haplogroup B with new development of knee osteoarthritis in Koreans.

The aim of this study was to determine which mitochondrial DNA (mtDNA) haplogroup is associated with new development of knee osteoarthritis (OA). Epidemiologic data and knee radiographs with the Kellgren-Lawrence (K/L) score of the Ansung cohort study were prospectively obtained from the 2nd (2005-2006) and 6th follow-up periods (2013-2014). The mtDNA was analyzed by multiplex mutagenetically separated polymerase chain reaction to determine Asian mtDNA haplogroups (M, G, D, D4, D5, M7, M8, M9, M10, N, A, N9, R, F and B). The frequency of the mtDNA haplogroup was compared between participants with knee OA (K/L score ≥2 or total knee replacement arthroplasty [OA group]) and those without knee OA (K/L<2 [control group]) at the 6th follow-up. Multiple logistic regression was used to determine the relative risk (RR) of mtDNA haplogroups for OA by adjusting for sex, age, body mass index, smoking and metabolic syndrome. There were 1115 participants with epidemiological data, knee radiographs and DNA samples. Of these, 572 participants had a K/L score of 0 at the 2nd follow up, and 438 underwent knee radiography at the 6th follow up. Among the 438 participants, 160 were classified as having knee OA, and 278 were classified as the control group. The haplogroup B showed a significantly higher frequency in the OA group than in the control group (unadjusted RR=1.794, P=0.030; adjusted RR=2.389, P=0.004). Our data suggest that mtDNA haplogroup B contributed to the new development of knee OA in Koreans.

Tumor Suppressor Gene Mutations in Iraqi Women with Breast Cancer and Their Relatives

Background: Breast cancer is characterized by various malignant tumors arise from breast tissues,age, genetic factors,diet and environmental factors,reproductive and hormonal factors, some types of benign breast disease andsocio-economic factors are factors correlated to an enlarged risk of breast cancer.Tumor suppressor genes are genes whose loss of roleresults in the elevation of malignancy. Aim of the Study: The present study was conducted to verify the tumor suppressor gene mutations in breast cancer women and their relatives. Material and Methods: Three groups of samples were included. The first consisted of 100 blocks of formalinfixed, paraffinembedded(FFPE) breast cancer tissuesof women(group 1). The second contained blood samples of 46 breast cancerwomen(group 2). The third comprised blood samples of46 apparently healthy women who were relatives to the breast cancer patients of group two(group 3). The ages of patients of samples tissue were 46.78±11.5 years, while for samples of blood patients were 47.34±11.18 years. The ages of the healthy relatives were 40.79±9.84 years. Five tumor suppressor gene mutations were examined.BRCA1 185delAG, BRCA1 5382insC and BRCA2 6174delT mutations were evaluated by mutagenically separated polymerase chain reaction, while CHEK2 1100delC and Tp53 exon 7 mutations were analyzed by RFLP. Results: The amplification of BRCA1gene for185 delAG mutation revealed amplicons of 335 bp for the wild type and 354 bp for the mutant gene, whereas for 5382 insC mutation exhibited 2 amplicons of sizes 271 bp for the wild type and 295 bp for the mutant gene. The analysis of BRCA26174 delT mutation indicated 2 amplicons of 151 bp for the wild type and 171 bp for the mutant gene. Tp53 exon 7 mutation analysis highlighted an amplicon of 125 bp size. Digestion of thisamplicon with the Hae III enzyme resulted in 2 fragments with sizes of 83 bp and 42 bp for the wild type. CHEK2 1100 delC mutation evaluation showed an amplicon of 116 bp size. Digestion of this amplicon with Sca I enzyme exhibited 2 fragments with sizes of 92 bp and 24 bpfor the wild type. Data analysis demonstrated that 27 (27%) out of the 100 enrolled breast cancer patients of tissue samples have mutations in tumor suppressor genes, 11 (23.9%) out of 46enrolled breast cancer patients of blood samples have such mutations, while 8 (17.4%) out of 46 relatives have these mutations. Nine (9%) patients of group1 were indicated to have BRCA1 185delAGmutation, while 4 (8.7%) and 3 (6.5%)women from groups 2 and3 exhibited the same mutationrespectively. BRCA15382insC mutation was identified in 7 (7%), 3 (6.5%) and 1 (2.2%) women of groups 1, 2 and 3 respectively. The BRCA2 6174delT mutation was observed in 5 (6%), 2 (4.3%) and 2 (4.3%)women of groups 1,2 and 3 respectively. Three (3%) patients of group1, 1 (2.2%) and 1 (2.2%) women from groups 2 and 3 were found to have CHEK2 1100delC respectively. Tp53exon 7 mutation was evident in 2 (2%), 1 (2.2%) and 1 (2.2%) women of groups 1, 2 and 3 respectively. No patient had more than one mutation. Conclusions: About one quarter of the investigated breast cancer patients have mutations in their tumor suppressor genes and 17% of their relatives have such mutations. Most of breast cancer patients enrolled in this study are sporadic not familial breast cancer.

Separation of Small DNAs by Gel Electrophoresis in a Fused Silica Capillary Coated with a Negatively Charged Copolymer

Active development of compact analytical instruments suitable for point-of-care testing (POCT) requires optimization of existing methods. To aid the development of capillary gel electrophoresis instruments for POCT, we attempted to separate polymerase chain reaction products (small DNAs) using a short, fused silica capillary coated with an acrylamide (AM)/acrylic acid (AA) copolymer (poly(AM-co-AA)). To realize the high capability of this capillary to separate small DNAs, the magnitude of electroosmotic flow (EOF) was controlled by varying the content of negatively charged AA in the copolymer, which significantly affected the separation ability. At an AA content ≥3.75 mol %, sample DNAs could not be injected into the copolymer-coated capillary owing to strong EOF, whereas a 100 bp DNA ladder sample was successfully separated at an AA content of ≤3.5 mol %, showing that even slight AA content variations impact DNA flow. EOF values measured using a neutral coumarin 334 solution suddenly decreased at an AA content of 3.5 mol % relative to those at an AA content of ≥3.75 mol %. Theoretical plate values revealed that an AA content of 2.75 mol % was optimal for separating ladder DNAs with sizes ≥600 bp. Hence, EOF control achieved by varying the amount of negatively charged AA in the poly(AM-co-AA) coating can promote further development of short capillaries for POCT applications.

Prevalence of two BRCA1 mutations, 5382insC and 300T > G, in ovarian cancer patients from Ukraine.

Ovarian cancer is the seventh most common cancer in women worldwide and the leading cause of gynecological malignant diseases-related deaths in women. The most significant risk factor for ovarian cancer is an inherited genetic mutation in one of two genes: breast cancer gene 1 (BRCA1) or breast cancer gene 2 (BRCA2). The germline mutation c.5266dupC (also known as 5382insC or 5385insC) is the most common mutation among Slavic patients with breast and/or ovarian cancer. Missense mutation c.181T > G (also known as 300T > G or p.C61G) is regarded as the founder change in many Central European countries. We screened 306 ovarian cancer patients diagnosed at different ages by mutagenically separated polymerase chain reaction (PCR) and real-time PCR. A total of 25 BRCA1 mutations were detected (18 cases of 5382insC and 7 cases of 300T > G). The frequency of the BRCA1 5382insC mutation is similar in breast and ovarian cancer patients from Ukraine, but the frequency of 300T > G was estimated in Ukraine at first time.

Micro RNA-146a But Not IRAK1 is Associated with Rheumatoid Arthritis in the Tunisian Population.

Rheumatoid arthritis (RA) is characterized by the production of an array of proinflammatory cytokines through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. Interleukin-1 receptor (IL-1R) and Toll-like receptors contain a common cytoplasmic motif the Toll/IL-1R (TIR) homology domain. This motif is required for NF-κB activation. IL-1R-associated kinase 1 (IRAK1) is a key adapter molecule recruited during the signaling cascade of the TIR. Its gene expression is regulated by the micro-RNA (miR)-146a. We investigated the role of IRAK1 single-nucleotide polymorphism (SNP) rs3027898 (IRAK1 rs3027898) and miR-146a SNP rs2910164 (miR-146a rs2910164) in Tunisian patients with RA and their association with C reactive protein (CRP), rheumatoid factor (RF), anticyclic citrullinated peptide (anti-CCP) antibodies, and erosion. In a cohort of 172 adult RA patients and 224 matched controls, IRAK1 rs3027898 genotyping was determined by mutagenically separated polymerase chain reaction (MS-PCR) with newly designed primers, and miR-146a rs2910164 genotyping was determined by fragment length polymorphism PCR-restriction (RFLP-PCR). The IRAK1 rs3027898 A allele was detected in 67% of RA patients and 70% of controls indicating that it is not associated with RA in codominant, dominant, or recessive models even after stratification by age and gender. The miR-146a rs2910164 G allele was detected in 76% of RA patients and 68% of controls, thus the C allele confers some protection based on a dominant model [CC+GC (odds ratio (95% confidence interval) = 0.6 (0.3-0.9), p = 0.03)]. No association with CRP, RF, anti-CCP, or erosion was found for either SNPs. The IRAK1 rs3027898 was not associated with RA, whereas C allele of miR-146a rs2910164 was found to be protective. Functional studies are required to investigate the exact role of miR-146a rs2910164 during RA.

New test for endothelin receptor type B (EDNRB) mutation genotyping in horses

Lethal white foal syndrome (LWFS) is an autosomal recessive disease of neonatal foals characterized by a white hair coat and a functional intestinal obstruction. Traditional techniques for identifying the dinucleotide mutation (TC→AG) of the endothelin receptor B gene (EDNRB) associated with LWFS are time-consuming. We developed a new technique based on mutagenically separated polymerase chain reaction (MS-PCR) for simple detection of the EDNRB genotype in horses.

Novel Association of WNK4 Gene, Ala589Ser Polymorphism in Essential Hypertension, and Type 2 Diabetes Mellitus in Malaysia.

With-no-lysine (K) Kinase-4 (WNK4) consisted of unique serine and threonine protein kinases, genetically associated with an autosomal dominant form of hypertension. Argumentative consequences have lately arisen on the association of specific single nucleotide polymorphisms of WNK4 gene and essential hypertension (EHT). The aim of this study was to determine the association of Ala589Ser polymorphism of WNK4 gene with essential hypertensive patients in Malaysia. WNK4 gene polymorphism was specified utilizing mutagenically separated polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) method in 320 subjects including 163 cases and 157 controls. Close relation between Ala589Ser polymorphism and elevated systolic and diastolic blood pressure (SBP and DBP) was recognized. Sociodemographic factors including body mass index (BMI), age, the level of fasting blood sugar (FBS), low density lipoprotein (LDL), and triglyceride (TG) in the cases and healthy subjects exhibited strong differences (p < 0.05). The distribution of allele frequency and genotype of WNK4 gene Ala589Ser polymorphism showed significant differences (p < 0.05) between EHT subjects with or without type 2 diabetes mellitus (T2DM) and normotensive subjects, statistically. The WNK4 gene variation influences significantly blood pressure increase. Ala589Ser probably has effects on the enzymic activity leading to enhanced predisposition to the disorder.