Deoxyribonucleic Acid Copy Research Articles

Association of eating habits with mitochondrial DNA copy number in eveningness chronotypes: origin research

Objective: To determine the relationship between eating habits and mitochondrial deoxyribonucleic acid copy number in adult cases of eveningness chronotypes. Method: The cross-sectional, analytical study was conducted from September 2022 to June 2023 at the Physiology Department of the Islamic International Medical College, Rawalpindi, in collaboration with the Genetic Resource Centre, Rawalpindi, Pakistan, and comprised adult subjects who were assessed using the Morningness-Eveningness Questionnaire. The participants' eating habits were assessed using the Healthy Eating Assessment Questionnaire, and on they were divided into those with healthy eating habits in group A and those with unhealthy eating habits in group B. Deoxyribonucleic acid was extracted using the Chelex method, the mitochondrial deoxyribonucleic acid copy number of all participants was quantified using quantitative polymerase chain reaction. Data was analysed using SPSS 27. Results: Of the 80 subjects, 30(37.5%) were males and 50(62.5%) were females. The overall mean age was 24.27±6.91 years (range: 18-45 years). There were 40(50%) subjects in each group. The mean mitochondrial deoxyribonucleic acid copy number in group A was 2.74±0.14 compared to 2.26±0.25 in group B (p<0.001). Conclusion: Subjects with healthy eating habits exhibited higher mitochondrial deoxyribonucleic acid copy numbers, indicating reduced damage to mitochondrial deoxyribonucleic acid. Key Words: Mitochondria, Mitochondrial DNA, Eating habits, Chronotype.

Mitochondrial Deoxyribonucleic Acid Copy Number Elevation As a Predictor for Extended Survival and Favorable Outcomes in High-Grade Brain Tumor Patients: A Malaysian Study.

Investigating the role of mitochondrial DNA (mtDNA) alterations and their impact on brain tumor progression remains a significant focus in cancer research. The research aimed to explore the specific contributions of mtDNA copy number changes and their correlations with patient survival, large mtDNA deletions, and TFAM mutations in brain tumor patients. A total of 41 patients with confirmed brain tumors underwent DNA extraction from both tumor tissues and blood samples. The relative mtDNA copy number in comparison to the nuclear genome was quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Long-range PCR assessed largescale mtDNA deletions, and Sanger sequencing was applied to detect exon 4 TFAM mutations. Analysis revealed significantly increased mtDNA copy numbers in brain tumor tissues (80.5%) compared to matched blood samples (P < .001). Median delta Ct (∆Ct) values were 7.35 in cancerous tissues and 11.81 in blood (P <.001), with median relative mtDNA content of 0.0123 and 0.0006, respectively (P <.001). Patients with higher mtDNA copy numbers experienced longer overall survival periods (P=.045) and notably favorable outcomes, particularly in high-grade tumor cases (P=.016). Furthermore, a singlenucleotide deletion was identified in exon 4 of TFAM in a patient with glioblastoma IV, while no large-scale mtDNA deletions were found in brain tumor patients. Our study strongly supports the role of increased mtDNA copy numbers as a reliable predictor for improved survival and positive outcomes in high-grade brain tumor patients.

The course of chronic heart failure in patients after COVID-19 against the background of active cytomegalovirus infection

Background. A new coronavirus infection may be complicated in the post-COVID period by the development of adverse cardiovascular events associated with chronic heart failure.&#x0D; Aim. To study the features of the course of chronic heart failure in patients after COVID-19 infection against the background of active cytomegalovirus infection.&#x0D; Material and methods. The study included 102 patients with chronic heart failure with reduced and intermediate left ventricular ejection fractions, who underwent COVID-19 in mild and moderate forms. The control group consisted of 61 patients with chronic heart failure and no COVID-19. Within 6 months after the infection with COVID-19, the features of the course of chronic heart failure were assessed. Quantitative determination of deoxyribonucleic acid (DNA) of cytomegalovirus in blood plasma was carried out by polymerase chain reaction. To assess the risk of adverse events, the odds ratio (OR) with a 95% confidence interval (95% CI) was calculated. Quantitative data were presented as median and interquartile range (25th and 75th percentiles).&#x0D; Results. After coronavirus infection, the risk of an unfavorable course of chronic heart failure (OR=6.237; 95% CI=2.91113.362; p=0.001), hospitalization due to decompensation of chronic heart failure (OR=5.9; 95% CI=1.31326.504; p=0.033), an increase in the functional class of heart failure by 1 class or more (OR=4.19; 95% CI=1.63610.736; p=0.009), development of atrial fibrillation paroxysms (OR=3.832; 95% CI=1.38510.599, p=0.014), significantly increased. The number of copies of cytomegalovirus DNA in patients who underwent COVID-19 was significantly higher in the group of patients with an unfavorable course of chronic heart failure compared to the same group, in which patients did not tolerate COVID-19 2238.5 (1888.5; 2647.5) and 1411.5 (1112.5; 1684.5) copies/ml, respectively (p 0.001).&#x0D; Conclusion. Patients with chronic heart failure after 6 months of COVID-19 infection are at high risk of developing adverse cardiovascular events against the background of active cytomegalovirus infection.

Clinical Differences between Posner-Schlossman Syndrome Patients with Intermittent Intraocular Pressure Elevation and Glaucomatous Damage

Introduction: Although there is abundant evidence that Posner-Schlossman syndrome (PSS) can lead to secondary glaucoma, data on the clinical differences between PSS patients with secondary glaucoma and those with intermittent intraocular pressure (IOP) elevation are sparse. Methods: This retrospective observational study included 52 patients (52 eyes) diagnosed with PSS and admitted to Zhongnan Hospital of Wuhan University between January 2019 and February 2022. Demographic characteristics and clinical features were gathered from admission records. Patients were divided into two groups: 27 cases with intermittent IOP elevation (group A) and 25 cases with secondary glaucoma (group B and C). Of the secondary glaucoma cases, 18 were further divided into the topical IOP-lowering medications group (group B) and 7 into the glaucoma surgery group (group C). Clinical characteristics of different groups were compared. Results: Compared to the intermittent IOP elevation group, PSS patients with secondary glaucoma had a longer course of disease, a higher incidence of iris depigmentation, lower best corrected visual acuity, lower endothelial cell density, and higher interferon-γ (IFN-γ) concentration and cytomegalovirus (CMV) deoxyribonucleic acid (DNA) copy number in the aqueous humor (all p < 0.05). Group C presented a higher CMV DNA copy number in the aqueous humor than groups A and B (p < 0.05). Compound trabeculectomy proved effective in group C, with a functional filter bleb and well-controlled IOP without disease progression after 1 year of follow-up. Conclusion: Distinctive characteristics existed between PSS patients with secondary glaucoma and those with intermittent IOP elevation. Compound trabeculectomy appears to be an effective treatment option when IOP cannot be controlled through topical medications.

Quantitative Analysis of Placental Mitochondrial DNA in Severe Hypertensive Pregnancies: A Cross-sectional Study

Introduction: To address the causes of maternal mortality and morbidity, reproductive health and related disabilities is a motto of World Health Organisation (WHO). Gestational hypertension constitutes as one of the main cause for maternal morbidity and mortality. Aetiology of gestational hypertension- remains a mystery. Mitochondrial dysfunction, particularly in trophoblastic cells, could play an important role in causing gestational hypertension and is postulated to be associated with oxidative stress, impaired differentiation and invasion of trophoblastic tissue. Alterations in peripheral blood mitochondrial Deoxyribonucleic Acid (DNA) copy numbers have been thought as a possible biomarker for mitochondrial dysfunction in disorders induced by oxidative stress. Aim: To estimate, evaluate and compare the mitochondrial copy numbers between placenta of severe gestational hypertensive pregnancies and normotensive pregnancies. Materials and Methods: A cross-sectional study was conducted at Tirunelveli Medical College Hospital, Tirunelveli, Tamil Nadu, India, with 25 clinically proven cases of severe gestational hypertensive pregnancies and 25 normotensive pregnancies, for a period of one year (August 2020 to July 2021). The placenta were collected from the Department of Obstetrics, in containers within two hours of delivery and frozen in liquid nitrogen. Relative quantitative analysis of placental mitochondrial DNA (mtDNA), were done by calculating the copy number value by comparing the levels of two nuclear genes viz., BECN1 and NEB to two mitochondrial genes viz., ND1 and ND6 by using real time quantitative Polymerase Chain Reaction (PCR). Results: The placental mitochondrial DNA copy number was higher in severe gestational hypertensive patients than in normotensive pregnancies. A total of 21 (84%) cases of the severe hypertensive placenta had higher copy numbers 100-110 vs only 13 (52%) cases had copy numbers 100-105 in the normotensive group. The difference in copy number value between them was statistically significant with p-value of 0.03. Conclusion: Present study findings suggest significant alterations at the level of the mitochondria in placenta from women with gestational hypertension, which may well contribute to gestational hypertension pathophysiology. Future research should include direct measure of placental and maternal whole body mitochondrial function, and assays that elucidate the potential modifiers of this association.

IL13 Promoter (-1055) Polymorphism Associated with Leukocyte Mitochondria DNA Copy Number in Chronic Obstructive Pulmonary Disease.

IL13 polymorphism is associated with chronic obstructive pulmonary disease (COPD). Patients with COPD have smaller numbers of mitochondria deoxyribonucleic acid copies (mtDNA-CN) than people without COPD do. However, whether IL13 polymorphism affects the mutation and recombination of mitochondria remains unclear. Data for patients with COPD and non-COPD were collected from Kaohsiung Chang Gung Memorial Hospital to enable a comparison of their leukocyte mtDNA-CN and the association of this information with IL-13 promoter (−1055) polymorphism. This study included 99 patients with COPD and 117 individuals without COPD. The non-COPD individuals included 77 healthy individuals that never smoked and 40 healthy smokers. The patients with COPD exhibited significantly lower mtDNA-CN than non-COPD did (250.34 vs. 440.03; p < 0.001); mtDNA-CN was particularly pronounced in individuals with the IL13 CC and CT genotypes compared with individuals with the TT genotype. When only individuals without COPD were considered and when all participants were considered, the differences in the mtDNA-CNs in individuals with the CC and CT genotypes were more significant than those in individuals with the TT genotype (448.4 and 533.6 vs. 282.8; p < 0.05 in non-COPD group); (368.8 and 362.6 vs. 249.6, p < 0.05 in all participants). The increase mtDNA-CN in the CC and CT genotypes was also more than that in the TT genotype in COPD patients, but showed no significance (260.1 and 230.5 vs. 149.9; p = 0.343). The finding shows that COPD is a mitochondria regulatory disorder and IL-13 promoter (−1055) polymorphism is associated with leukocyte mtDNA-CN. Developing COPD control methods based on mitochondrial regulation will be possible.

Peripheral blood mononuclear cell mitochondrial copy number and adenosine triphosphate inhibition test in NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is associated with mitochondrial dysfunction. This study aims to develop biomarkers for assessing mitochondrial dysfunction in patients with NAFLD. Mitochondrion-associated transcriptome analysis was performed. Peripheral blood mononuclear cells obtained from patients with NAFLD (69) and healthy controls (19) were used to determine the mitochondrial DNA (mtDNA) copy number. A mitochondrial inhibition substrate test (ATP assay) was performed in HepG2 cells using the patient serum. Hepatic mRNA transcriptome analysis showed that the gene expression related to mitochondrial functions (mitochondrial fusion, apoptotic signal, and mitochondrial envelope) increased in patients with steatohepatitis, but not in those with NAFL. Gene set enrichment analysis revealed that the upregulated expression of genes is related to the pathways of the tricarboxylic (TCA) cycle and deoxyribonucleic acid (DNA) replication in patients with steatohepatitis, but not in healthy controls. The mtDNA copy number in the peripheral blood mononuclear cells was 1.28-fold lower in patients with NAFLD than that in healthy controls (P <.0001). The mitochondrial inhibition substrate test showed that the cellular adenosine triphosphate (ATP) concentration was 1.2-fold times less in NAFLD patients than that in healthy controls (P <.0001). The mtDNA copy number and mitochondrial ATP inhibition substrate test demonstrated negative correlations with the degree of hepatic steatosis, whereas the ATP concentration showed a positive correlation with the mtDNA copy number. The mitochondrial copy number of peripheral blood mononuclear cells and mitochondrial ATP inhibition substrate can be used as biomarkers for assessing the mitochondrial dysfunction in patients with NAFLD.

Mitochondrial dysfunction in cumulus cells is related to decreased reproductive capacity in advanced-age women

To reveal the relationship between mitochondrial function in cumulus cells (CCs) and the aging-related decline in ovarian function and reproductive capacity. Retrospective and transcriptome analysis of human tissue. University hospital. A total of 186 infertile women with normal weight and no other diseases, including 86 young women (aged <38 years) with normal ovarian reserve and 100 advanced-age women (aged ≥38 years) with diminished ovarian reserve. None. Embryo development data were analyzed. The mitochondrial ultrastructure of CCs was observed by transmission electron microscopy. Mitochondrial activity was detected by immunofluorescence. The per-CC mitochondrial deoxyribonucleic acid copy numbers and cellular adenosine triphosphate levels were quantified. Unbiased comprehensive genome-wide transcriptomics was performed. The functions of all annotated genes and biologic pathways were analyzed by the Kyoto Encyclopedia of Genes and Genomes. Advanced-age women with diminished ovarian function had significantly fewer retrieved oocytes and lower embryo quality. The normal mitochondrial rate in CCs was significantly lower. In addition to mitochondrial morphology and structural changes, the fluorescence intensity of Fluo-3/AM was significantly higher, and that of MitoTracker was lower than that of young women with normal ovarian reserve, suggesting that age negatively affects the mitochondrial activity of CCs. The per-CC mitochondrial deoxyribonucleic acid copy number and adenosine triphosphate levels significantly decreased in the advanced-age group. The Kyoto Encyclopedia of Genes and Genomes analysis showed that differentially expressed genes were significantly enriched in the oxidative phosphorylation pathway. Additionally, most mitochondrially encoded genes related to oxidative phosphorylation were significantly down-regulated in the advanced-age group. We present current evidence that mitochondrial dysfunction in CCs may play a key role in the age-related decline in ovarian function and reproductive capacity.

Melatonin Supplementation for Six Weeks Had No Effect on Arterial Stiffness and Mitochondrial DNA in Women Aged 55 Years and Older with Insomnia: A Double-Blind Randomized Controlled Study

Melatonin is a hormone produced in the pineal gland that controls sleep and circadian rhythm. Some studies have reported antioxidant and anti-inflammatory effects of melatonin that could benefit cardiometabolic function; however, there is a lack of evidence to support these assertions. The aim of this study was to investigate whether melatonin has beneficial effects on arterial stiffness and mitochondrial deoxyribonucleic acid (DNA) in humans. Methods: This study was designed as a double-blind randomized controlled study. Thirty-eight healthy women aged 55 years and older were enrolled. All had insomnia (Pittsburgh Sleep Quality Index (PSQI) ≥ 5), not treated with any medications, for at least three months before enrollment. Subjects were divided into a melatonin and a placebo group according to melatonin supplementation. The melatonin group took 2 mg melatonin every night for six weeks. The cardio–ankle vascular index (CAVI) was used as an indicator of arterial stiffness. After six weeks, CAVI, mitochondrial DNA (mtDNA) copy number in white blood cells (WBCs), and other metabolic indices, such as homeostasis model assessment of insulin resistance (HOMA-IR), were checked. Results: Sleep quality index using PSQI was improved in the melatonin group from a score of 11 to 8 (p = 0.01), but did not change significantly in the control group. However, there was no significant intergroup difference in PSQI. Systolic blood pressure (SBP) decreased in the melatonin group from 135 to 128 mmHg (p = 0.015), while remaining stable in the placebo group. Right CAVI, mitochondrial DNA copy number, and HOMA-IR were not altered in either group. There were no intergroup differences in CAVI, mtDNA, HOMA-IR, or SBP between baseline and week six. Conclusions: We found no evidence that melatonin supplementation improved cardiometabolic parameters like arterial stiffness, mtDNA, or insulin resistance compared to the placebo between baseline and week six. Sleep quality was improved in the melatonin group. Further research, including longer-term studies with higher doses of melatonin, is warranted.

Cytomegalovirus infection in solid malignancies

Introduction: Cytomegalovirus (CMV) infection is very common, although its manifestation as disease occurs usually in the setting of immunosuppression. It is usually seen in hematological malignancies and in post-transplant recipient patients. Data about CMV reactivation in solid malignancies are limited.Materials and Methods: This was a retrospective analysis of adult patients (from the past 10 years of clinical records) with various solid malignancies, who had CMV deoxyribonucleic acid (DNA) positivity and had varied clinical presentation.Results: Of total 73 adult patients of solid malignancies who had been tested for CMV DNA (by real-time polymerase chain reaction [RT-PCR]) with a detection limit of 150 copies/ml), 30 patients had at least one instance of CMV DNA positivity. Of them, we were able to obtain detailed records of 17 patients, who had CMV DNA positivity with clinical manifestations. Fever was the most common symptom in 88% of patients. CMV DNA copy numbers in these patients ranged from 250 to 1,490,000 copies/ml (by RT-PCR). Twelve of the 17 patients (71%) had positive tests for other microbes (based on culture testing) besides the positive CMV test. Ten of 17 (59%) patients were treated with ganciclovir and 7 patients responded to treatment. Seven patients (3 treated with ganciclovir and 4 remained untreated) died during the hospital course. Ten patients recovered (7 treated with ganciclovir and 3 remained untreated, with only monitoring of CMV DNA levels) of their acute illness.Conclusion: CMV infection and its manifestation in patients with solid malignancies is probably underdiagnosed. Given the high morbidity and mortality in these patients (with or without co-infections), it is very important to suspect and treat CMV reactivation.

The relationship between neutrophil-lymphocyte ratio and onset of lactation among postpartum women: A prospective observational cohort study

BackgroundDelayed onset of lactation is a key factor in the low rate of exclusive breast-feeding in 6 months after caesarean section. The mechanism of delayed onset of lactation is not clear. Milk production depends largely on mitochondrial adenosine triphosphate synthesis, and the neutrophil-lymphocyte ratio is closely related to mitochondrial deoxyribonucleic acid copy number and adenosine triphosphate production. Presently, it is unclear whether a difference in the neutrophil-lymphocyte ratio exists between those undergoing vaginal delivery and those undergoing caesarean delivery and, if so, whether the difference correlates to the time of onset of lactation. ObjectivesTo identify whether the neutrophil-lymphocyte ratio at 24 hours after delivery is different between mothers delivering by caesarean section and those giving birth vaginally and whether the neutrophil-lymphocyte ratio is related to the delayed onset of lactation. DesignThe study adopted a prospective cohort study design. SettingsMaternity units of an Obstetrics & Gynecology Hospital. Participants327 mother-infant pairs who met inclusion/exclusion criteria and were followed up to the time of onset of lactation. MethodsMother-infant pairs were allocated to the vaginal birth group or the caesarean section group according to birth method. The neutrophil-lymphocyte ratio was calculated as the absolute value of neutrophils divided by the absolute value of lymphocytes based on full blood counts. Before delivery, full blood counts were obtained from medical records. After delivery, blood samples were drawn 24 hours postpartum, and blood cells were classified and counted. The onset of lactation was confirmed by the maternal perception of breast fullness. The neutrophil-lymphocyte ratio and its relationship with lactation onset were analyzed by multivariable regression. ResultsThe neutrophil-lymphocyte ratios of both groups were elevated after delivery. Based on the covariance analysis, after adjusting for baseline full blood counts before delivery, the neutrophil-lymphocyte ratio in the caesarean group was higher than the ratio in the vaginal group after delivery (p = .000). In addition, after adjustment for confounding factors, multivariable regression analyses showed that an increased neutrophil-lymphocyte ratio was correlated with delayed onset of lactation (95% confidence interval 0.285–1.646). ConclusionsThe neutrophil-lymphocyte ratio in the caesarean section group was higher than that in the vaginal delivery group and was related to a delayed onset of lactation. Given the decreased mitochondrial copy number in the elevated neutrophil-lymphocyte ratio and therefore the associated reduction in adenosine triphosphate synthesis, these findings may elucidate the mechanism for delayed onset of lactation in caesarean section births.

Molecular Classification of Gastric Adenocarcinoma.

As one of the leading causes of cancer-related deaths, gastric cancer (GC) has gained more and more attention. Although most GCs are adenocarcinomas, they have considerable heterogeneity among patients. Thus, appropriate classification and individualized treatment of GCs is essential. The traditional morphology-based classification systems including the World Health Organization (WHO) classification and the Lauren’s classification have a limited utility in guiding clinical treatment due to the molecular heterogeneity of GC. Classifications based on molecular features become important. Recent years, molecular methods such as next-generation sequencing (NGS) including deoxyribonucleic acid (DNA) sequencing, ribonucleic acid (RNA) sequencing, whole-exome sequencing, copy number variation analysis and DNA methylation arrays have been used to classify the GC into molecular subtypes which can convey more detailed information of tumor than histopathological characteristics. In this review, we described the current molecular classifications of GC including the intrinsic subtypes, Lei subtypes, The Cancer Genome Atlas (TCGA) subtypes, Asian Cancer Research Group (ACRG) subtypes, and some other additional classifications.

Prenatal Household Air Pollution Alters Cord Blood Mononuclear Cell Mitochondrial DNA Copy Number: Sex-Specific Associations.

Background: Associations between prenatal household air pollution (HAP) exposure or cookstove intervention to reduce HAP and cord blood mononuclear cell (CBMC) mitochondrial deoxyribonucleic acid copy number (mtDNAcn), an oxidative stress biomarker, are unknown. Materials and Methods: Pregnant women were recruited and randomized to one of two cookstove interventions, including a clean-burning liquefied petroleum gas (LPG) stove, or control. Prenatal HAP exposure was determined by serial, personal carbon monoxide (CO) measurements. CBMC mtDNAcn was measured by quantitative polymerase chain reaction. Multivariable linear regression determined associations between prenatal CO and cookstove arm on mtDNAcn. Associations between mtDNAcn and birth outcomes and effect modification by infant sex were explored. Results: LPG users had the lowest CO exposures (p = 0.02 by ANOVA). In boys only, average prenatal CO was inversely associated with mtDNAcn (β = -14.84, SE = 6.41, p = 0.03, per 1ppm increase in CO). When examined by study arm, LPG cookstove had the opposite effect in all children (LPG β = 19.34, SE = 9.72, p = 0.049), but especially boys (β = 30.65, SE = 14.46, p = 0.04), as compared to Control. Increased mtDNAcn was associated with improved birth outcomes. Conclusions: Increased prenatal HAP exposure reduces CBMC mtDNAcn, suggesting cumulative prenatal oxidative stress injury. An LPG stove intervention may reverse this effect. Boys appear most susceptible.

Цитомегаловирусная инфекция и функции эндотелия у пациентов с острым инфарктом миокарда

to study elucidate association of active cytomegalovirus (CMV) infection with endothelial dysfunction in patients with acute myocardial infarction (AMI). The study included 42 volunteers without ischemic heart disease (IHD) and 63 patients with AMI. Blood samples for analysis of the deoxyribonucleic acid (DNA) of CMV in plasma by real-time polymerase chain reaction were taken in patients - before coronary angiography, in volunteers - at admission. In addition, in patients with AMI and volunteers without IHD, endothelial function was analyzed using endothelium-dependent vasodilatation (EDVD) test of the brachial artery. We showed that in patients with AMI, the concentration of CMV DNA in plasma was statistically significantly increased when compared with that in volunteers without IHD, which reflects active CMV infection - 1185.7 (0; 3003.0) vs. 0 (0; 910.8) copies of DNA / ml plasma (p=0.011). In comparison with volunteers without IHD, patients with AMI also more often had endothelial dysfunction - 11.5 (7.5, 15.2) % vs. 4.4 (0; 9.6) % of cases (p.

Detection and identification of azithromycin resistance mutations on &lt;em&gt;Treponema pallidum&lt;/em&gt; 23S rRNA gene by nested multiplex polymerase chain reaction

Background: Azithromycin-resistant strains of Treponema pallidum is associated with the mutation of 23S rRNA gene of T. pallidum. Although these strains are now prevalent in many countries, there is no laboratory test kit to detect and identify these mutations. Thus, in this study we developed a nested multiplex polymerase chain reaction (PCR) to detect and identify A2058G and A2059G mutations in 23S rRNA gene.Methods: Three primer sets were designed for nested PCR reactions. To obtain maximum PCR reaction, all parameters were optimized. The specificity of the primer sets was evaluated towards some microorganisms. A sensitivity test was conducted to get detection limit of deoxyribonucleic acid (DNA). Forty-five whole blood specimens were tested by PCR, and positive results were confirmed by the DNA sequencing.Results: The assay could detect at least 4,400 DNA copy number and showed no cross reaction with other microorganisms used in the specificity test. A total 13 of 45 whole blood specimens were PCR positive for T. pallidum, and no single mutations (either A2058G or A2059G) were detected. Two positive specimens were confirmed by the DNA sequencing and showed no mutation.Conclusion: Nested multiplex PCR developed in this study showed a specific and sensitive test for the detection and identification of A2058G and/or A2059G mutations of 23S rRNA T. pallidum gene.

A Molecular Approach to Nested RT-PCR Using a New Set of Primers for the Detection of the Human Immunodeficiency Virus Protease Gene.

BackgroundThe human immunodeficiency virus (HIV-1) is the etiologic agent of AIDS. The disease can be transmitted via blood in the window period prior to the development of antibodies to the disease. Thus, an appropriate method for the detection of HIV-1 during this window period is very important.ObjectivesThis descriptive study proposes a sensitive, efficient, inexpensive, and easy method to detect HIV-1.Patients and MethodsIn this study 25 serum samples of patients under treatment and also 10 positive and 10 negative control samples were studied. Twenty-five blood samples were obtained from HIV-1-infected individuals who were receiving treatment at the acquired immune deficiency syndrome (AIDS) research center of Imam Khomeini hospital in Tehran. The identification of HIV-1-positive samples was done by using reverse transcription to produce copy deoxyribonucleic acid (cDNA) and then optimizing the nested polymerase chain reaction (PCR) method. Two pairs of primers were then designed specifically for the protease gene fragment of the nested real time-PCR (RT-PCR) samples. Electrophoresis was used to examine the PCR products. The results were analyzed using statistical tests, including Fisher’s exact test, and SPSS17 software.ResultsThe 325 bp band of the protease gene was observed in all the positive control samples and in none of the negative control samples. The proposed method correctly identified HIV-1 in 23 of the 25 samples.ConclusionsThese results suggest that, in comparison with viral cultures, antibody detection by enzyme linked immunosorbent assay (ELISAs), and conventional PCR methods, the proposed method has high sensitivity and specificity for the detection of HIV-1.

Nonlinear Joint Latent Variable Models and Integrative Tumor Subtype Discovery.

Integrative analysis has been used to identify clusters by integrating data of disparate types, such as deoxyribonucleic acid (DNA) copy number alterations and DNA methylation changes for discovering novel subtypes of tumors. Most existing integrative analysis methods are based on joint latent variable models, which are generally divided into two classes: joint factor analysis and joint mixture modeling, with continuous and discrete parameterizations of the latent variables respectively. Despite recent progresses, many issues remain. In particular, existing integration methods based on joint factor analysis may be inadequate to model multiple clusters due to the unimodality of the assumed Gaussian distribution, while those based on joint mixture modeling may not have the ability for dimension reduction and/or feature selection. In this paper, we employ a nonlinear joint latent variable model to allow for flexible modeling that can account for multiple clusters as well as conduct dimension reduction and feature selection. We propose a method, called integrative and regularized generative topographic mapping (irGTM), to perform simultaneous dimension reduction across multiple types of data while achieving feature selection separately for each data type. Simulations are performed to examine the operating characteristics of the methods, in which the proposed method compares favorably against the popular iCluster that is based on a linear joint latent variable model. Finally, a glioblastoma multiforme (GBM) dataset is examined.

Analysis of the entire ryanodine receptor type 1 and alpha 1 subunit of the dihydropyridine receptor (CACNA1S) coding regions for variants associated with malignant hyperthermia in Australian families.

Defects in the genes coding for the skeletal muscle ryanodine receptor (RYR1) and alpha 1 subunit of the dihydropyridine receptor (CACNA1S) have been identified as causative for malignant hyperthermia (MH). Sixty-two MH susceptible individuals presenting to the same diagnostic centre had copy deoxyribonucleic acid, derived from muscle ribonucleic acid, sequenced to identify variants with the potential to be responsible for the MH phenotype in both RYR1 and CACNA1S. These genetic findings were combined with clinical episode details and in vitro contracture test results to improve our understanding of the Australian MH cohort. Twelve novel variants were identified in RYR1 and six in CACNA1S. Known RYR1 causative mutations were identified in six persons and novel variants in RYR1 and CACNA1S in a further 17 persons. Trends indicated higher mutation identification in those with more definitive clinical episodes and stronger in vitro contracture test responses.

Genetic Evidence of Vertical Transmission and Cycling of Yersinia ruckeri in Hatchery-Origin Fall Chinook Salmon Oncorhynchus tshawytscha

Abstract Enteric redmouth disease, caused by the bacterium Yersinia ruckeri, can be a severe problem in the culture of salmonid fishes, and can persist as a chronic infection within hatchery facilities for an indefinite amount of time ranging from months to years. Using a nested polymerase chain reaction (nPCR) assay and a real-time quantitative PCR (qPCR) assay, the prevalence of Y. ruckeri deoxyribonucleic acid (DNA) was documented across a single production cycle of fall Chinook salmon Oncorhynchus tshawytscha reared at a hatchery. Ovarian fluid from spawning female adults, unfertilized eggs, eyed eggs, sac fry, and juvenile fish all tested positive for Y. ruckeri DNA using both nPCR and qPCR assays. Presence of Y. ruckeri DNA in ovarian fluids and unfertilized eggs indicates that vertical transmission of the pathogen from females to their progeny is possible. Juvenile fish had the highest level of Y. ruckeri DNA at a log10-transformed level of 4.3 DNA copies per gram of kidney tissue. The limited mortality observed was attributed to handling and hatchery procedures during the study period and indicates that the low levels of the pathogen's DNA can persist without severe detrimental effects over the hatchery rearing cycle.

Clinical utility of recombinant adenoviral human p53 gene therapy: current perspectives.

Gene therapy has promised to be a highly effective antitumor treatment by introducing a tumor suppressor gene or the abrogation of an oncogene. Among the potential therapeutic transgenes, the tumor suppressor gene p53 serves as an attractive target. Restoration of wild-type p53 function in tumors can be achieved by introduction of an intact complementary deoxyribonucleic acid copy of the p53 gene using a suitable viral vector, in most cases an adenoviral vector (Adp53). Preclinical in vitro and in vivo studies have shown that Adp53 triggers a dramatic tumor regression response in various cancers. These viruses are engineered to lack certain early proteins and are thus replication defective, including Gendicine, SCH-58500, and Advexin. Several types of tumor-specific p53-expressing conditionally replicating adenovirus vectors (known as replication-competent CRAdp53 vectors) have been developed, such as ONYX 015, AdDelta24-p53, SG600-p53, OBP-702, and H101. Various clinical trials have been conducted to investigate the safety and efficiency of these adenoviral vectors. In this review we will talk about the biological mechanisms, clinical utility, and therapeutic potentials of the replication-deficient Adp53-based and replication-competent CRAdp53-based gene therapy.