Mean Copy Number Research Articles

338: Preeclampsia is associated with placental mitochondrial dysfunction.

We have demonstrated that serum from women with preeclampsia (PE) contains circulating factors which contribute to mitochondrial (Mt) dysfunction and increased reactive oxygen species in cultured human vascular endothelial cells. When Mt are damaged or dysfunctional, there is an increase in Mt DNA copy number, with an associated decreased Electron Transport Chain (ETC) expression, activity and respiration. The objective of this study is to determine if Mt isolated from placentas of women with PE exhibit more of these features compared to Mt from normotensive pregnant (NP) women. Intact placental Mt were isolated from of NP and PE women, real-time PCR was used to determine the relative quantity of Mt DNA compared to nuclear DNA, and complex expression was determined by Western blot with OxPhos, an antibody cocktail targeting the 5 ETC complexes. Finally ETC complex IV activities and respiration rates were measured using Oxygraph-2K. Respiration rates measured include: basal state (no substrates), state 2 (glutamate/malate), state 3 (ADP), state 4 (oligomycin), and maximal state (FCCP). There is significantly increased placental Mt DNA in PE vs NP (159 ±25, n=3 vs 78.04±10, n=3, p=0.042). We noted a trend toward decreased expression of Complex I and II. Complex IV expression is significantly decreased in PE vs NP (0.505±0.09, n=3 vs. 0.912±0.08, n=3 Complex IV/VDAC, p=0.029). Additionally, complex IV activity was drastically reduced in PE vs NP (141.4±18, n=9 vs 238.5±24, n=10 nmol e-/min/mg, P<0.01). In our preliminary data (n=2), we noted a downward trend in Complex I mediated State 3 and maximal state respiration rates in PE. In preeclampsia, there is significantly increased placental Mt DNA mean copy numbers and a simultaneous decrease in ETC Complex IV expression and activity. Impairment of mitochondrial respiration and ETC activities in PE indicate that mitochondrial dysfunction could contribute to oxidative stress and other pathophysiological features of preeclampsia.

Prospective cohort study showing persistent HSV-2 shedding in women with genital herpes 2 years after acquisition

ObjectivesHerpes simplex virus type 2 (HSV-2) is a prevalent infection with great variability in clinical and virological manifestations among individuals. This prospective cohort study aims to evaluate the natural history...

Herpes simplex and human papilloma virus coinfections in oral mucosa of men-A 6-year follow-up study.

Herpes simplex virus (HSV) establishes latency in neurons and recurrent infections in oral mucosa. This prospective study analyzes HSV prevalence in oral mucosal brush samples from men with known human papillomavirus (HPV) status. We hypothesized that HSV-1-infection could facilitate HPV persistence as a cofactor. This study was a part of the Finnish Family HPV study accomplished at the University of Turku/Turku University Hospital, Finland. A total of 139 men (mean age 28.6 ± 4.9 years) were enrolled at 36+-weeks of their partner's pregnancy and thereafter followed-up for 6 years. Altogether, 722 samples, extracted from oral brush samples collected at the enrollment timepoint (baseline) and at 2-, 6-, 12-, 24-, 36-month, and 6 years, were available. HSV DNA was analyzed with quantitative PCR. HSV-1 results were compared with the known HPV data. The prevalence of oral HSV-1 shedding varied between 0-7.2% (mean 2.8%) among the men. Mean copy numbers varied between 4 and 550 genome copies/sample. A total of 18 (12.9%) men were found HSV-1-positive at least once, two of them twice. Neither smoking nor oral sex was associated with the oral HSV-1-DNA finding. HPV/HSV-1 co-infection was found in 6 (4.3%) men, all of them having persistent HPV-infection. In conclusion, HSV-1 and its coinfection with HPV in oral mucosa was rare.

The quasispecies regime for the simple genetic algorithm with roulette wheel selection

Abstract We introduce a new parameter to discuss the behavior of a genetic algorithm. This parameter is the mean number of exact copies of the best-fit chromosomes from one generation to the next. We believe that the genetic algorithm operates best when this parameter is slightly larger than 1 and we prove two results supporting this belief. We consider the case of the simple genetic algorithm with the roulette wheel selection mechanism. We denote by ℓ the length of the chromosomes, m the population size, pC the crossover probability, and pM the mutation probability. Our results suggest that the mutation and crossover probabilities should be tuned so that, at each generation, the maximal fitness multiplied by (1 - pC)(1 - pM)ℓ is greater than the mean fitness.

Abstract P049: Mitochondrial DNA Copy Number and Diabetes in the ARIC Study

Introduction: In the United States, the rising number of adults with type 2 diabetes (T2D) is thought to be associated with the rise in obesity. Obesity may be associated with T2D through biologic pathways where excess weight strains the body’s metabolic machinery, such as mitochondria. Mitochondria are dynamic organelles whose regulation and function respond to cell stress, such as insulin resistance. Previous research reveals that insulin resistance is associated with obesity prior to hyperglycemia. Mitochondrial DNA copy number is a measure of mitochondrial DNA content, and correlates with both the number and size of mitochondria. We assessed the hypothesis that mitochondrial DNA copy number is associated with T2D in a community-based, prospective cohort study. A lower mitochondrial DNA copy number in these analyses represents worse mitochondrial function. Methods: We included 6,633 white ARIC participants without coronary heart disease who had mitochondrial DNA copy number measured from visit 2 (1990-1992). Our sample had a mean age of 57 ± 5.6 years, was 43% male, average BMI of 27 ± 4.9 kg/m2, and 27% with hypertension. Those with diabetes had an average hemoglobin A 1c of 7.2 ± 1.8 (n=681). The mitochondrial DNA copy number value used in analyses represents a sample’s standard deviation (SD) from a mean of zero for age- and sex-adjusted distributions. The mitochondrial DNA copy number data was then divided into quintiles, with the most negative mitochondrial DNA copy number in quintile 1 (Q1), the mean of zero in Q3, and the most positive in Q5. We defined T2D as self-report of doctor diagnosis, current use of glucose-lowering medication, or fasting blood glucose ≥ 126 mg/dL or non-fasting blood glucose ≥ 200 mg/dL measured at study visits. We used logistic regression to estimate the odds of prevalent T2D for each quintile group compared to Q3. Confounders considered in the base model included age at sample collection, sex, education level, and medication use (thyroid and estrogen). All analyses were done in Stata 14.1. Results: The prevalence of T2D was higher in the lower mitochondrial DNA copy number quintiles: 15% in Q1 (mean copy number: -1.40 SD) 11% in Q2 (-0.46 SD), and 9% in Q3-Q5 (0.04 SD, 0.52 SD, 1.33 SD). There was increased odds of T2D in lower mitochondrial DNA copy number quintiles, but with no additional benefit of copy number above the mean. The odds of T2D in each quintile compared to Q3 was 1.9 in Q1 (95% CI 1.49, 2.46), 1.36 in Q2 (95% CI 1.05, 1.77), 0.99 in Q4 (95% CI 0.76, 1.30) and 0.96 in Q5 (95% CI 0.74, 1.26). Conclusion: Examining the association of mitochondrial dysfunction and diabetes in a large community-based cohort connects results from experimental studies to epidemiologic studies and provides the opportunity to characterize the complex pathogenesis of diabetes using cohort data.

Abstract P2-12-01: Comprehensive genomic profiling of 34 cases of breast angiosarcoma

Abstract Background: Angiosarcoma of the breast (BAS) is a rare but lethal neoplasia, either arising de novo or secondary to radiation therapy, with incidence of the latter disease increasing. We queried a database of more than 70,000 advanced cancer patients assayed with comprehensive genomic profiling (CGP) in the course of clinical care to uncover the frequency, type and associated genomic alterations (GA) in BAS and to highlight possible routes to benefit from targeted therapy. Methods: CGP was performed for 34 BAS cases using a hybrid-capture, adaptor ligation based next generation sequencing assay of up to 315 genes to a mean coverage depth of &amp;gt;500X. The results were analyzed for base substitutions, short insertions and deletions, selected rearrangements, and copy number changes. RNA sequencing for 265 genes was also performed for 24 cases. Limited clinical histories from submitted pathology reports were reviewed under IRB permission. Results: Clinical specimens from 34 BAS patients, all females, were assayed. The cases harbored 87 total GA for a mean of 2.59 per case, 25% of which were copy number amplifications. The most commonly altered genes were MYC (41%, 14/34), PIK3CA (26%, 9/34), and KDR (26%, 9/34). All MYC alterations were amplifications with a mean copy number of 39, and alterations in other MYC family members (MYCN and MYCL1) were not observed. KDR was recurrently altered as T771R (7/9) and T771K (1/9) and amplified in one case (1/9). MYC and KDR alterations were mutually exclusive (p&amp;lt;0.0001). 6/14 MYC amplified cases had prior histories of breast carcinoma, with 3/6 noted as being treated with radiation therapy. For the remainder of MYC amplified cases (8/14), no relevant clinical history was available. Two cases harboring gene fusions were identified including CIC-MEGF8 and NTRK1-PEAR1. Two rearrangements of potential functional significance including CIC-DEDD2 and HT-ALK (exon1 HT - exon5-29 ALK including kinase domain) were also observed. The case harboring HT-ALK also had MYC amplification and known prior radiation therapy. Two other MYC amplified cases also harbored targetable kinase alterations, including FLT4 amplification (described as targetable in Ravi et al JNCCN 2016) and FGFR3 S249C, a known activating mutation. Conclusions: MYC amplification defines over 40% (14/34) of advanced BAS cases. Of MYC amplified cases, 28% (4/14) harbored targetable alterations of tyrosine kinases including a potential novel ALK fusion. FLT4 amplification only co-occurred with MYC amplification, but this result was not statistically significant in this small series. KDR and MYC alteration were mutually exclusive, and 45% of non-MYC altered cases (9/20) harbored KDR alterations, which were predominantly mutations of T771. Further clinico-pathologic correlation, particularly history of radiation therapy, will be explored in this series, as well defining BAS that harbor neither MYC nor KDR alterations. Citation Format: Ravi V, Madison R, Schrock AB, Cote G, Millis S, Alvarez R, Choy E, Katz D, Chung J, Gay L, Miller VA, Ross JS, Ali SM, Schnitt S. Comprehensive genomic profiling of 34 cases of breast angiosarcoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-12-01.

Development of a SYBR-GreenⅠ quantitative PCR assay for the detection and genotyping of different hantaviruses.

Hemorrhagic fever with renal syndrome(HFRS) is a severe, viral zoonotic disease which occurs worldwide, particularly in Asia and Europe. In China, the Hantaan virus(HTNV) and the Seoul virus(SEOV) are known to be the most prevalent causative agents of HFRS. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for disease surveillance. In the present study, the viral loads in cell culture supernatant, infected mice blood and clinical serum samples were quantified using the SYBR‑GreenI-based reverse transcription-quantitiative polymerase chain reaction(RT-qPCR) assay, which targeted the Sgene sequence of the HTNV and SEOV genomes. The cRNA of these two viruses were synthesized as a positive control and 10-fold serially diluted from 1x105 to 1x100 copies/µl. Standard curves were generated by plotting the mean cycle threshold(Ct) values versus copy numbers. The standard curve of HTNV had a correlation coefficient(R2) of 0.994, efficiency of amplification(E) of 101.9%, and the slope of -3.278, whereas that of SEOV had an R2of0.993, Eof104.8%, and the slope of -3.212. The minimum detection limit of the RT-qPCR assay for HTNV and SEOV was 101copies/µl. Two qPCR assays were successfully established for the detection of HTNV and SEOV, respectively. Taken together, these findings demonstrate that using the SYBR‑GreenI-based RT-qPCR assay, the HTNV and SEOV may be genotyped precisely without cross-reactivity. Furthermore, viral RNA may be detected and quantified in cells, mice and infected individuals, which may be useful in epidemiological studies as well as for early monitoring and further preventative treatment against SEOV and HTNV-induced diseases.

Infectious agents in congenital cataract in a tertiary care referral center in North India

Congenital cataract has the potential for inhibiting early visual development. Intrauterine infections with Rubella virus, Herpes simplex virus (HSV) and Toxoplasma gondii plays an important role in the development of congenital cataract. The study included 120 children under the age of 6 years presenting with congenital cataract and diagnosed using serology and polymerase chain reaction (PCR). The IgM positivity for rubella, HSV, T. gondii was found to be 5.8%, 1.6% and 8.3% respectively. The overall PCR positivity was found to be 40(33.3%), 25 (20.8%) and 39 (32.5%) for rubella, HSV and T. gondii with mean copy number of 1599 copies/μL; 1716 copies/μL and 1503 copies/μL respectively. Infective etiology significantly contributes to the causation of congenital cataract particularly for rubella virus which is a potentially eradicable disease. This study provides an epidemiological data for rubella, HSV and T. gondii in children with congenital cataract and highlights the need to introduce rubella vaccine in the National Immunization Programme of India.

Partial heterologous protection by low pathogenic H9N2 virus against natural H9N2-PB1 gene reassortant highly pathogenic H5N1 virus in chickens

Low pathogenic avian influenza H9N2 and highly pathogenic avian influenza H5N1 viruses continue to co-circulate in chickens. Prior infection with low pathogenic avian influenza can modulate the outcome of H5N1 infection. In India, low pathogenic H9N2 and highly pathogenic H5N1 avian influenza viruses are co-circulating in poultry. Herein, by using chickens with prior infection of A/chicken/India/04TI05/2012 (H9N2) virus we explored the outcome of infection with H5N1 virus A/turkey/India/10CA03/2012 natural PB1 gene reassortant from H9N2. Four groups (E1-E4) of SPF chickens (n = 6) prior inoculated with 106 EID50 of H9N2 virus were challenged with 106 EID50 of H5N1 natural reassortant (PB1-H9N2) virus at days 1 (group E1); 3 (group E2); 7 (group E3) and 14 (group E4) post H9N2 inoculation. The survival percentage in groups E1-E4 was 0, 100, 66.6 and 50%, respectively. Virus shedding periods for groups E1-E4 were 3, 4, 7 and 9 days, respectively post H5N1 challenge. Birds of group E1 and E2 were shedding both H9N2 and H5N1 viruses and mean viral RNA copy number was higher in oropharyngeal swabs than cloacal swabs. In group, E3 and E4 birds excreted only H5N1 virus and mean viral RNA copy number was higher in most cloacal swabs than oral swabs. These results indicate that prior infection with H9N2 virus could protect from lethal challenge of reassortant H5N1 virus as early as with three days prior H9N2 inoculation and protection decreased in groups E3 and E4 as time elapsed. However, prior infection with H9N2 did not prevent infection with H5N1 virus and birds continue to excrete virus in oropharyngeal and cloacal swabs. Amino acid substitution K368E was found in HA gene of excreted H5N1 virus of group E3. Hence, concurrent infection can also cause emergence of viruses with mutations leading to virus evolution. The results of this study are important for the surveillance and epidemiological data analysis where both H9N2 and H5N1 viruses are co-circulating.

A tremendous expansion of copy number in crossbred bulls ( × ).

Crossbreeding between cattle () and yak () exhibits significant hybrid advantages in milk yield and meat production. By contrast, cattle-yak F hybrid bulls are sterile. Copy number variations (CNV) of multicopy gene families in male-specific regions of the mammalian Y chromosome (MSY) affect human and animal fertility. The present study investigated CNV of (), (), (), and () in 5 yak breed bulls ( = 63), cattle-yak F ( = 22) and F ( = 2) hybrid bulls, and Chinese Yellow (CY) cattle bulls ( = 10) by quantitative real-time PCR. showed restricted amplification in yak bulls in that the average geometric mean copy number (CN) was estimated to be 4 copies. The most compelling finding is that there is a tremendous expansion of CN in F hybrids (385 copies; 95% confidence interval [CI] = 351-421) and F hybrids (356 copies) compared with the male parent breed CY cattle (142 copies; 95% CI = 95-211). Copy numbers of and were also extensively expanded on the Y chromosome in yak and CY cattle bulls. The geometric mean CN of and were estimated to be 123 (95% CI = 114-132) and 250 copies (95% CI = 233-268) in yak bulls and 71 (95% CI = 61-82) and 133 (95% CI = 107-164) copies in CY cattle, respectively. Yak and CY cattle have 2 copies of the gene on the Y chromosome. Similarly to gene, the F and F hybrid bulls have higher CN of , , and than CY cattle ( < 0.01). These results indicated that the MSY of yak and cattle-yak crossbred hybrids was fundamentally different from cattle MSY in the context of genomic organization. Based on the model of cattle-yak F and F hybrid bull sterility, the CNV of may serve as a potential risk factor for crossbred bull ( × ) infertility. To our knowledge, this is the first study to examine differences in multicopy genes in MSY between yak and cattle-yak bulls.

EMA-qPCR to monitor the efficiency of a closed-coupled solar pasteurization system in reducing Legionella contamination of roof-harvested rainwater

Solar pasteurization is effective in reducing the level of indicator organisms in stored rainwater to within drinking water standards. However, Legionella spp. were detected at temperatures exceeding the recommended pasteurization temperatures using polymerase chain reaction assays. The aim of the current study was thus to apply EMA quantitative polymerase chain reaction (EMA-qPCR) to determine whether the Legionella spp. detected were intact cells and therefore possibly viable at pasteurization temperatures >70°C. The BacTiter-Glo™ Microbial Cell Viability Assay was also used to detect the presence of ATP in the tested samples, as ATP indicates the presence of metabolically active cells. Chemical analysis also indicated that all anions and cations were within the respective drinking water guidelines, with the exception of iron (mean: 186.76μg/L) and aluminium (mean: 188.13μg/L), which were detected in the pasteurized tank water samples at levels exceeding recommended guidelines. The BacTiter-Glo™ Microbial Cell Viability Assay indicated the presence of viable cells for all pasteurized temperatures tested, with the percentage of ATP (in the form of relative light units) decreasing with increasing temperature [70–79°C (96.7%); 80‐ 89°C (99.2%); 90‐95°C (99.7%)]. EMA-qPCR then indicated that while solar pasteurization significantly reduced (p<0.05) the genomic copy numbers of intact Legionella cells in the pasteurized tank water (~99%), no significant difference (p>0.05) in the mean copy numbers was detected with an increase in the pasteurization temperature, with 6×103 genomic copies/mL DNA sample obtained at 95°C. As intact Legionella cells were detected in the pasteurized tank water samples, quantitative microbial risk assessment studies need to be conducted to determine the potential health risk associated with using the water for domestic purposes.

Low Mitochondrial DNA Copy Number is Associated With Adverse Clinical Outcomes in Peritoneal Dialysis Patients.

Mitochondrial dysfunction may play an important role in abnormal glucose metabolism and systemic inflammation. We aimed to investigate the relationship between mitochondrial DNA (mtDNA) copy number and clinical outcomes in peritoneal dialysis (PD) patients. We recruited 120 prevalent PD patients and determined mtDNA copy number by PCR. Primary outcome was all-cause mortality, whereas secondary outcomes included cardiovascular events, technical PD failure, and incident malignancy. Cox proportional hazards analysis determined the independent association of mtDNA copy number with outcomes. The mean patient age was 52.3 years; 42.5% were men. The mean log mtDNA copy number was 3.30 ± 0.50. During a follow-up period of 35.4 ± 19.3 months, all-cause mortality and secondary outcomes were observed in 20.0% and 59.2% of patients, respectively. Secondary outcomes were significantly lower in the highest mtDNA copy number group than in the lower groups. In multiple Cox analysis, the mtDNA copy number was not associated with all-cause mortality (lower two vs highest tertile: hazard ratio [HR] = 1.208, 95% confidence interval [CI] = 0.477-3.061). However, the highest tertile group was significantly associated with lower incidences of secondary outcomes (lower two vs highest tertile: HR [95% CI] = 0.494 [0.277-0.882]) after adjusting for confounding factors. The decreased mtDNA copy number was significantly associated with adverse clinical outcomes in PD patients.

Identification of Selective Lead Compounds for Treatment of High-Ploidy Breast Cancer

Increased ploidy is common in tumors but treatments for tumors with excess chromosome sets are not available. Here, we characterize high-ploidy breast cancers and identify potential anticancer compounds selective for the high-ploidy state. Among 354 human breast cancers, 10% have mean chromosome copy number exceeding 3, and this is most common in triple-negative and HER2-positive types. Women with high-ploidy breast cancers have higher risk of recurrence and death in two patient cohorts, demonstrating that it represents an important group for improved treatment. Because high-ploidy cancers are aneuploid, rather than triploid or tetraploid, we devised a two-step screen to identify selective compounds. The screen was designed to assure both external validity on diverse karyotypic backgrounds and specificity for high-ploidy cell types. This screen identified novel therapies specific to high-ploidy cells. First, we discovered 8-azaguanine, an antimetabolite that is activated by hypoxanthine phosphoribosyltransferase 1 (HPRT1), suggesting an elevated gene-dosage of HPRT1 in high-ploidy tumors can control sensitivity to this drug. Second, we discovered a novel compound, 2,3-diphenylbenzo[g]quinoxaline-5,10-dione (DPBQ). DPBQ activates p53 and triggers apoptosis in a polyploid-specific manner, but does not inhibit topoisomerase or bind DNA. Mechanistic analysis demonstrates that DPBQ elicits a hypoxia gene signature and its effect is replicated, in part, by enhancing oxidative stress. Structure-function analysis defines the core benzo[g]quinoxaline-5,10 dione as being necessary for the polyploid-specific effects of DPBQ. We conclude that polyploid breast cancers represent a high-risk subgroup and that DPBQ provides a functional core to develop polyploid-selective therapy. Mol Cancer Ther; 15(1); 48-59. ©2015 AACR.

P-D15 Peripheral blood lymphocyte HIV DNA levels correlate with HIV associated neurocognitive disorders

Introduction: Mononuclear cells play a key role in facilitating the pathogenic mechanisms leading to HIV associated neurocognitive disorders (HAND). We examined the association between levels of HIV DNA within monocytes and lymphocytes and HAND. Method: Blood samples were obtained from 40 antiretroviral naive participants in Nigeria. CD14+ cells and T&B lymphocytes were isolated from peripheral blood mononuclear cells by bead separation (84-98% purity for CD14+ cells). Total HIV DNA was quantified using real-time PCR assay targeting HIV LTR-gag and cell input numbers determined by the number of CCR5 copies/sample. Utilizing a 7-domain neuropsychological test battery and activities of daily living assessment, participants were classified as either unimpaired, having asymptomatic neurocognitive impairment (ANI), minor neurocognitive disorder (MND), or HIV associated dementia (HAD) in line with the Frascati criteria. Results: The mean log10 HIV DNA copies/106 cells were higher for T & B lymphocytes than for CD14+ monocytes (P-value: 0.0002). In a multivariable linear regression adjusting for CD4 count, viral load and lymphocyte count, compared to unimpaired individuals, the mean copy numbers for T & B lymphocytes were higher among those with HAND (P-value: 0.01) and for those with MND [P-value: 0.02 (Tukey adjusted: 0.07)]. In a multivariable logistic regression adjusting for same variables, the odds of cognitive impairment was 6.7 times greater per log increase in HIV DNA within T & B lymphocytes (P-value: 0.02). There was no significant association between cognitive impairment and HIV DNA within CD14+ monocytes. Conclusions: In this cohort we found a strong association between levels of cell-associated HIV DNA within the lymphocyte subset and HAND. Further studies looking at similar association among patients with suppressed viremia are required for inference on integrated DNA.

The Effect of Legionella Pneumophila Contamination in the Surface Dust of the Air Ducts of Central Air Conditioning Systemson Indoor Air Quality

The aim of this study was to determine the concentration of Legionella pneumophila in the surface dust of the air ducts of central air conditioning systems and evaluate its effects on indoor air quality. Thirty dust samples were collected from thirty air ducts and analyzed using real-time TaqMan PCR targeting the mip gene. The concentration of surface dust, PM10 mass concentration of air, total plate count of surface dust, and total plate count of supply air were also investigated. Legionella pneumophila microorganisms were detected in 20 of the 30 samples (66.7%) with a mean copy number of 6212 copies/g surface dust. The Spearman’s rank correlation coefficient between Legionella pneumophila concentration and total plate count in surface dust of the air duct was 0.834 (P=0.000). The pollutants in the air ducts in central air conditioning systems were found to affect indoor air quality.

Quantification of human polyomavirus JC virus load in urine and blood samples of healthy tribal populations of North-Eastern part of West Bengal, India

Background: Human polyomavirus JC (JCV) is a widespread human virus with profound pathogenic potential. A study was undertaken to quantify JCV load in urine and peripheral blood samples of immunocompetent, apparently healthy tribal individuals of North-Eastern part of West Bengal, India for the first time. Materials and Methods: One hundred and thirteen samples of urine or blood were collected from different tribal groups of this region. For the quantitative estimation of the viral load in each sample, real-time polymerase chain reaction method using the SYBR Green dye was employed. Results: The viral load estimated was found in the range between 3.5 × 102 and 2.12 × 106 copies/ml of samples having a mean and median viral copy numbers of 8.67 × 105 and 9.19 × 105 copies/ml of sample respectively. Conclusion: The mean viral DNA load in urine samples of the studied immunocompetent population was found to be higher than that found in a study conducted in the USA, but lower than similar groups of Italy and healthy adult women in the USA. However when compared with median values of viral DNA loads in urine samples of immunocompetent human subjects of Kuwait, Portugal, and Switzerland the observed viral DNA load was found to be substantially higher.

Effective use of the TSPY gene-specific copy number in determining fetal DNA in the maternal blood of cynomolgus monkeys.

Since the available concentration of single-copy fetal genes in maternal blood DNA is sometimes lower than detection limits by PCR methods, the development of specific and quantitative PCR detection methods for fetal DNA in maternal blood is anticipated, which may broaden the methods that can be used to monitor pregnancy. We used the TaqMan qPCR amplification for DYS14 multi-copy sequence and the SRY gene in maternal blood plasma (cell-free DNA) and fractional precipitated blood cells (cellular DNA) from individual cynomolgus monkeys at 22 weeks of pregnancy. The availability of cell-free fetal DNA was higher in maternal blood plasma than that of cellular DNA from fractional precipitated blood cells. There was a significantly higher (P < 0.001) mean copy number of fetal male DYS14 from maternal plasma (4.4 × 10(4) copies/mL) than that of detected fetal cellular DNA from fractional blood cell pellets. The sensitivity of the DYS14 PCR assay was found to be higher than that of the SRY assay for the detection of fetal DNA when its presence was at a minimum. The DYS14 assay is an improved method for quantifying male fetal DNA in circulating maternal blood in the primate model.

Assessment of HER2 status in invasive breast cancers with increased centromere 17 copy number.

This study was designed to evaluate usefulness of additional fluorescence in situ hybridization (FISH) using other reference genes on chromosome 17 for assessment of HER2 status in invasive breast cancers with increased centromere 17 copy number, and to compare this approach with conventional methods based on the 2007 and 2013 ASCO/CAP guidelines. We performed FISH with probes for SMS, RARA, and TP53 on 253 breast cancers with centromeric probe CEP17 copy number ≥ 2.6 using tissue microarrays. If one or more gene had a mean copy number <2.6, the largest number for that gene(s) was chosen as an alternative to CEP17 copy number. Of the 243 cases in which re-grading was possible, only 2 had copy numbers ≥ 2.6 for RARA, SMS, and TP53. Of the 151 breast cancers which were considered HER2 non-amplified by the 2007 ASCO/CAP guidelines using the HER2:CEP17 ratio, 42 (27.8%) were re-graded as amplified and 33 (21.8%) as equivocal after FISH using additional reference genes. Of the 101 HER2-non-amplified cases by the 2013 ASCO/CAP guidelines, 2 (2.0%) were reclassified as amplified and 24 (23.8%) as equivocal. Of 46 equivocal cases, 35 (76.1%) were re-graded as amplified. After re-grading, HER2-amplified cases were significantly increased, and the concordance between HER2 FISH and HER2 immunohistochemistry decreased. And some pathologic features of the cases which were designated to have HER2 amplification after additional FISH were not compatible with those of HER2-amplified breast cancers. The use of additional reference genes has been suggested as an option for accurate assessment of HER2 status in breast cancers with increased CEP17 copy number. However, this has limitations in that it can cause over-grading of HER2 status in tumors that lose the new reference genes. Thus, at present, it seems that additional FISH using other reference gene such as SMS, RARA, and TP53 for the cases with increased CEP17 copy number is not suitable for daily practice.

Abstract 290: Lower Limb Revascularization of Patients with Peripheral Artery Disease Reduces Human Enterovirus Infection of the Gastrocnemius

Introduction: Peripheral artery disease (PAD), caused by obstruction of arteries supplying the lower extremities, is characterized by leg muscle degeneration. Recently, we showed that infectious human enterovirus (HEV), which has been implicated in other myodegenerative diseases, is present in ischemic muscle of PAD patients and that viral copy numbers correlate with disease severity. Hypothesis: We tested the hypothesis that prevalence of infectious HEV and viral copy number in the gastrocnemius of patients with PAD decrease in response to revascularization. Methods: Gastrocnemius biopsies were collected from controls (N=14) and from PAD patients (N=17), before and after revascularization. Biopsies were examined for the presence of HEV RNA, viral capsid protein, viral RNA copy number, and viral infectivity and for viral sub-genotype. Results: HEV RNA was detected in biopsies of 65% (11/17) of PAD patients and in none of the controls. After revascularization, the gastrocnemius of 5 patients who were HEV positive before surgery no longer harbored detectable virus. The prevalence of HEV infection was reduced from 65% (11/17 patients) before surgery to 35% (6/17 patients) after surgery. Positive-strand viral RNA copy numbers were decreased significantly after revascularization. Mean positive-strand copy number (average 10 2.61 copies/mg muscle wet weight) in post-surgical biopsies that were HEV-positive were lower (p&lt;0.001) compared to mean copy number (average 10 5.46 copies/mg muscle wet weight) in pre-surgical biopsies that were HEV-positive. Copy numbers of negative-strand (template) viral RNA were also decreased after revascularization. Viral replication was confirmed by detection of negative-strand viral RNA in all specimens positive for HEV. Cultures of HeLa and human skeletal muscle cells treated with muscle homogenates showed HEV replication and the presence of HEV capsid protein. By sequence analysis, HEV in PAD muscle exhibited 97% homology with Coxsackievirus B1. Conclusion: Our data introduce HEV infection as a potential mechanism for degeneration of ischemic muscle in PAD patients and demonstrate that revascularization of PAD patients reduces both prevalence of HEV infection and viral copy number in the gastrocnemius.

Detection and quantification of Anaplasma phagocytophilum and Babesia spp. in Ixodes ricinus ticks from urban and rural environment, northern Poland, by real-time polymerase chain reaction.

Anaplasma phagocytophilum and Babesia spp. are emerging tick-borne pathogens which can threaten human health. A duplex real-time PCR and qPCRs with primers and probes targeting 97 and 116 bp fragments of 16S rRNA and 18S rRNA genes, respectively, were used for qualitative and quantitative detection of both pathogens in Ixodes ricinus ticks. Altogether 1875 ticks (1084 adults and 791 nymphs) were collected from rural and urban habitats in northern Poland. Of them, at least 0.9 % were found to be infected with A. phagocytophilum while 2.5 % with Babesia spp. A comparison of the infection rates by the tick stage, the type of area, the collection site, habitats of different tick density and by the month of collection was done. The prevalence of pathogens was significantly lower in nymphs than in adult ticks (p = 0.02) and in rural areas than in urban areas (p = 0.007). Four different 16S rRNA gene variants of A. phagocytophilum were determine, however none of them showed 100 % identity with compared sequences isolated from human patients. The dominant Babesia species was B. venatorum. Results of qPCRs with circular and linearized forms of plasmids used as the standards showed significant difference in the pathogen loads (p = 0.001). The copy numbers of A. phagocytophilum and Babesia spp. estimated from the linear plasmids were 28.7 and 5.1 times lower, respectively, when compared with their circular forms, and were accepted as more reliable. The average number of copies of 16S rRNA gene of A. phagocytophilum in the positive I. ricinus samples were 3.39 × 105 ± 6.09 × 105. The mean copy number of 18S rRNA gene of Babesia spp. was ~2.55 × 105 ± 1.04 × 106. We confirmed the presence of A. phagocytophilum and Babesia spp. in I. ricinus in both rural and urban environments. The determined low infection rates suggests, however, that the risk for local population and tourists to acquire infection is also low. Moreover, we confirmed recent findings that serious overestimation by circular plasmid DNA makes it less suitable as a standard and that the linear standards should be recommended for qPCR.