Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens.

BACKGROUND: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens. RESULTS: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%. CONCLUSION: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.

Inherited and noninherited sources of variation in metastasis and the growth rate of tumors: Implications for tumor evolution and therapy

I have undertaken measurements of the genetic (or inherited) and nongenetic (or noninherited) components of the variability of metastasis formation and tumor diameter doubling time in more than 100 metastatic lines from each of three murine tumors (sarcoma SANH, sarcoma SA4020, and hepatocarcinoma HCA-I) syngeneic to C3Hf/Kam mice. These lines were isolated twice from lung metastases and analysed immediately thereafter to obtain the variance to spontaneous lung metastasis and tumor diameter doubling time. Additional studies utilized cells obtained from within 4 passages of isolation. Under the assumption that no genetic differences in metastasis formation or diameter doubling time existed among the cells of a given line, the variance within a line would estimate nongenetic variation. The variability derived from differences between lines would represent genetic origin. The estimates of the genetic contribution to the variation of metastasis and tumor diameter doubling time were significantly greater than zero, but only in the metastatic lines of tumor SANH was genetic variation the major source of metastatic variability (contributing 53% of the variability). In the tumor cell lines of SA4020 and HCA-I, however, the contribution of nongenetic factors predominated over genetic factors in the variability of the number of metastasis and tumor diameter doubling time. A number of other parameters examined, such as DNA content, karyotype, and selection and variance analysis with passage in vivo, indicated that genetic differences existed within the cell lines and that these differences were probably created by genetic instability. The mean metastatic propensity of the lines may have increased somewhat during their isolation and isotransplantation, but the variance was only slightly affected, if at all. Analysis of the DNA profiles of the metastatic lines of SA4020 and HCA-I revealed differences between these lines and their primary parent tumors, but not among the SANH lines and their parent tumor. Furthermore, there was a direct correlation between the extent of genetic influence on metastasis formation and the ability of the tumor cells to develop resistance to cisplatinum. Thus although nongenetic factors might predominate in contributing to metastasis formation, it is probably genetic variation and genetic instability that cause the progression of tumor cells to a more metastatic phenotype and leads to the emergence of drug resistance. ^

Sexual orientation and sexual behavior patterns: Evaluation of agreement and variation in a cohort of male, African American drug users in Houston, TX

Background. Sexual orientation and sexual behavior among men have shown disagreement in past studies. The term "on the down low" has been adopted by many to describe "straight" identifying men who have sex with men but do not inform their primary female partner. Methods. This secondary analysis of data collected from the "DASH Project---A Hepatitis B Vaccine Model for HIV Vaccine Trial in Drug Users," assessed sexual behavior patterns among African American drug-using men over time. Using a screener questionnaire to determine sexual orientation and sexual behavior of the men, the study specifically evaluated "straight" identified men who have sex with women only (MSW) to determine what factors were associated with sexual behavior variation to include men during follow-up. The Fisher's Exact Test was used to evaluate the factors. Results. Variation of sexual behavior was highest among "bisexual" identified men followed by "gay" identified men. Fifteen of the original 593 "straight" and MSW men had sexual behavior variation to include men. In the analysis of "straight" and MSW men with variation in sexual behavior compared to those who did not, living on the streets, greater number of sexual partners, trading sex for drugs, and trading sex for money were associated with sexual behavior variation (all p-values <0.01). Conclusions. The factors were only associated when considering the interview when the variation occurred. The same factors at screening were not predictive of sexual behavior variation in the future. Environmental factors, such as living situation, appear to play a role in sexual behavior variations in "straight" and MSW men. ^ Keywords. sexual behavior, sexual orientation, Fisher's Exact Test^

Variation of genotype and prevalence of Noro virus in U.S. students in Mexico with Traveler's Diarrhea, summer of 2004 and 2005

Noro virus, a positive single stranded RNA virus has been identified as a major etiologic agent in food borne gastroenteritis and diarrheal diseases. The emergence of this organism as a major non-bacterial cause in such outbreaks is partly due to the improved diagnostic tools like Reverse Transcription Polymerase chain reaction (RTPCR) that enable its detection. Noro virus accounts for nearly 96% of non-bacterial gastroenteritis outbreaks in US (1). Travelers' Diarrhea (TD) has remained a constant public health risk in the developed nations for decades and bacteria like Entero toxigenic Escherichia coli, Entero aggregative Escherichia coli have been described as the main etiologic agents for TD (2-4). A possible viral contribution to TD has been discovered in two studies (5, 6). The current study was designed to determine the prevalence of Noro virus in a population of 107 US students with TD acquired in Mexico in 2005 and to compare the prevalence to the prevalence of Noro virus in a similar study done in 2004. This study involved the testing of clinical stool specimens from 107 subjects in 2005 for the presence of Noro virus using RTPCR. The prevalence of Noro virus in 2004 used for comparison to 2005 data was obtained from published data (5). All subjects were recruited as TD subjects in a randomized, double-blinded clinical trial comparing a standard three day dosing of Rifaximin with and without an anti motility drug Loperamide. The prevalence of Noro virus geno group I was similar in both years, but geno group II prevalence differed across the two years (p = 0.003). This study finding suggests that the prevalence of Noro virus geno groups varies with time even within a specific geographic location. This study emphasizes the need for further systematic epidemiologic studies to determine the molecular epidemiology and the prevalence patterns of different geno groups of this virus. These are essential to planning and implementation of public health measures to lessen the burden of TD due to Noro virus infection among US travelers. ^

Clinical relevance of polymorphic DNA copy number variation (CNV) in African American women with stage I-II breast cancer

Objectives. The chief goal of this study was to analyze copy number variation (CNV) in breast cancer tumors from 25 African American women with early stage breast cancer (BC) using molecular inversion probes (MIP) in order to: (1) compare the degree of CNV in tumors compared to normal lymph nodes, and (2) determine whether gains and/or losses of genes in specific chromosomes differ between pathologic subtypes of breast cancer defined by known prognostic markers, (3) determine whether gains/losses in CN are associated with known oncogenes or tumor suppressor genes, and (4) determine whether increased gains/losses in CN for specific chromosomes were associated with differences in breast cancer recurrence. ^ Methods. Twenty to 37 nanograms of DNA extracted from 25 formalin-fixed paraffin embedded (FFPE) tumor samples and matched normal lymph nodes were added to individual tubes. Oligonucleotide probes with recognition sequences at each terminus were hybridized with a genomic target sequence to form a circular structure. Probes are released from genomic DNA obtained from FFPE samples, and those which have been correctly "circularized" in the proper allele/nucleotide reaction combination are amplified using polymerase chain reaction (PCR) primers. Amplicons were fluorescently labeled and the tag sequences released from the genome homology regions by treatment with uracil-N-glycosylase to cleave the probe at the site where uracils are present, and detected using a complementary tag array developed by Affymetrix. ^ Results. Analysis of CN gains and losses from tumors and normal tissues showed marked differences in tumors with numerous chromosomes affected. Similar changes were not observed in normal lymph nodes. When tumors were stratified into four groups based on expression or lack of expression of the estrogen receptor and HER2/neu, distinct patterns of CNV for different chromosomes were observed. Gains or losses in CN for specific chromosomes correlated with amplifications/deletions of particular oncogenes or tumor suppressor genes (i.e. such as found on chromosome 17) known to be associated with aggressive tumor phenotype and poor prognosis. There was a trend for increases in CN observed for chromosome 17 to correlate inversely with time to recurrence of BC (p=0.14 for trend). CNV was also observed for chromosomes 5, 8, 10, 11, and 16, which are known sites for several breast cancer susceptibility alleles. ^ Conclusions. This study is the first to validate the MIP technique, to correlate differences in gene expression with known prognostic tumor markers, and to correlate significant increases/decreases in CN with known tumor markers associated with prognosis. The results of this study may have far reaching public health implications towards identifying new high-risk groups based on genomic differences in CNP, both with respect to prognosis and response to therapy, and to eventually identify new therapeutic targets for prevention and treatment of this disease. ^

Geographic variation of the etiology of travelers' diarrhea

Introduction. Traveler's Diarrhea is an important public health program in travelers from industrialized nations to the developing world with a prevalence rate of between 13 and 60%. Although studies are found on the etiology of traveler's diarrhea, these studies have not described the etiology over different regions of the world. The objective of this study was to identify the frequency of specific etiology of traveler's diarrhea by geographic area of the world. In addition to this, it was also examined whether there are any regional differences in the isolation rate of ETEC and conventional pathogens and variation, if any, in frequency of these pathogens in different regions over time.^ Material and methods. This is a systematic review of the studies on the etiology of traveler's diarrhea by geographic regions. The search databases used were Medline Pubmed and Medline Ovid and key words used for the search were Etiology of traveler's diarrhea, travelers' diarrhea and acute diarrhea of travelers. The articles were selected according to the inclusion and exclusion criteria and relevant data was extracted which was statistically analyzed.^ Results. Out of 110 studies from 1970 to 2004, 52 studies were included and 58 were excluded from the review. All the 52 studies were grouped according to the geographic regions of interest. Latin America (25 studies), Asia (7 studies), Africa (9 studies), and others/Mixed (11 studies), were the 4 major groups of regions studied. The overall most common pathogen was ETEC (29.10%) in this study and other common pathogens were EAEC (14.42%), norovirus (10.95%), EPEC (6%) and rotavirus (5.23%). ETEC and Shigella show a decreasing trend in Latin America & Caribbean but increasing trend in Asia.^ Conclusion. ETEC is the single most common cause of travelers' diarrhea in the world. Potent vaccines against ETEC are required to prevent travelers' diarrhea and thus reduce the attack rate. Also, PCR based studies are required to identify the causes of pathogen negative diarrhea. ^

The relationship between food preparation time, time spent grocery shopping, and BMI

Recent data have shown that the percentage of time spent preparing food has decreased during the past few years, and little information is know about how much time people spend grocery shopping. Food that is pre-prepared is often higher in calories and fat compared to foods prepared at home from scratch. It has been suggested that, because of the higher energy and total fat levels, increased consumption of pre-prepared foods compared to home-cooked meals can lead to weight gain, which in turn can lead to obesity. Nevertheless, to date no study has examined this relationship. The purpose of this study is to determine (i) the association between adult body mass index (BMI) and the time spent preparing meals, and (ii) the association between adult BMI and time spent shopping for food. Data on food habits and body size were collected with a self-report survey of ethnically diverse adults between the ages of 17 and 70 at a large university. The survey was used to recruit people to participate in nutrition or appetite studies. Among other data, the survey collected demographic data (gender, race/ethnicity), minutes per week spent in preparing meals and minutes per week spent grocery shopping. Height and weight were self-reported and used to calculate BMI. The study population consisted of 689 subjects, of which 276 were male and 413 were female. The mean age was 23.5 years, with a median age of 21 years. The fraction of subjects with BMI less than 24.9 was 65%, between 25 and 29.9 was 26%, and 30 or greater was 9%. Analysis of variation was used to examine associations between food preparation time and BMI. ^ The results of the study showed that there were no significant statistical association between adult healthy weight, overweight and obesity with either food preparation time and grocery shopping time. Of those in the sample who reported preparing food, the mean food preparation time per week for the healthy weight, overweight, and obese groups were 12.8 minutes, 12.3 minutes, and 11.6 minutes respectively. Similarly, the mean weekly grocery shopping for healthy, overweight, and obese groups were 60.3 minutes per week (8.6min./day), 61.4 minutes (8.8min./day), and 57.3 minutes (8.2min./day), respectively. Since this study was conducted through a University campus, it is assumed that most of the sample was students, and a percentage might have been utilizing meal plans on campus, and thus, would have reported little meal preparation or grocery shopping time. Further research should examine the relationships between meal preparation time and time spent shopping for food in a sample that is more representative of the general public. In addition, most people spent very little time preparing food, and thus, health promotion programs for this population need to focus on strategies for preparing quick meals or eating in restaurants/cafeterias. ^

TEMPORAL VARIATION OF ACUTE AND LUNG TUMORIGENIC EFFECTS OF URETHAN ON STRAIN A MICE (CHRONOBIOLOGY, CARCINOGENICITY, ANESTHETIC, DNA SYNTHESIS)

The effect of circadian variation on susceptibility to the chemical induction of cancer was assessed utilizing the mouse pulmonary adenoma bioassay. Different groups of male A/Jax mice (standardized for rhythm analysis with light from 0600-1800 and darkness from 1800-0600) each received a single timed i.p. injection of urethan (Bioassay I: 0.25, 0.5 or 1.0 mg/g body weight; Bioassay II: 0.75, 1.0, 1.25 mg/g body weight; Bioassay III: 1.0 mg/g body weight) at the following times, 0100, 0500, 0900, 1300, 1700 or 2100. Mice were sacrificed 16 weeks after treatment. The tumorigenic effect of urethan on the lungs (lung surface pulmonary adenomas) was assessed. In addition, mortality, body weight changes and the anesthetic effect of urethan were determined. The rhythmic pattern of DNA synthesis in the lung and the comparative rhythmic pattern in the liver were assessed using a tritiated thymidine incorporation assay.^ In the first adenoma bioassay, the lung tumorigenic response in mice given the highest dose of urethan exhibited a 12-hour rhythm with a major peak in tumor yield at 0100 and a secondary peak at 1300; reduced yields occurred at 0500-0900 and 2100. The second adenoma bioassay, studied at a 6-month seasonal divergence in time from the first study showed a peak at 1300 but not at 0100. The mice from the third adenoma bioassay, studied at an 11-month seasonal divergence in time from the 2nd study showed an increase in tumor yield during the rest cycle (0900-1700).^ This study found a definite suggestion of a low amplitude rhythm in susceptibility to urethan induced effects. The acute toxic and pharmacological effects correlated to exhibit a maximal effect during dark hours (activity span). This rhythmicity might be explained by an alteration in the amplitude of hepatic metabolism. The chronic carcinogenic response exhibited an opposite pattern. Urethan induced tumor response was greater during daylight hours (rest cycle). This correlated with the slight elevation in DNA synthetic activity found in the lung and liver which might be responsible for the increase in carcinogenic response. (Abstract shortened with permission of author.) ^

Genetic underpinnings of variation in brain ventricular volume: A genome-wide association study

The ventricular system is a critical component of the central nervous system (CNS) that is formed early in the developmental stages and remains functional through the lifetime. Changes in the ventricular system can be easily discerned via neuroimaging procedures and most of the time it reflects changes in the physiology of the CNS. In this study we attempted to identify specific genes associated with variation in ventricular volume in humans. Methods. We conducted a genome wide association (GWA) analysis of the volume of the lateral ventricles among 1605 individuals of European ancestry from two community based cohorts, the Genetics of Microangiopathic Brain Injury (GMBI; N=814) and Atherosclerosis Risk in Communities (ARIC; N=791). Significant findings from the analysis were tested for replication in both the cohorts and then meta-analyzed to get an estimate of overall significance. Results. In our GWA analyses, no single nucleotide polymorphism (SNP) reached a genome-wide significance of p<10−8. There were 25 SNPs in GMBI and 9 SNPs in ARIC that reached a threshold of p<10 −5. However, none of the top SNPs from each cohort were replicated in the other. In the meta-analysis, no SNP reached the genome-wide threshold of 5×10−8, but we identified five novel SNPs associated with variation in ventricular volume at the p<10 −5 level. Strongest association was for rs2112536 in an intergenic region on chromosome 5q33 (Pmeta= 8.46×10−7 ). The remaining four SNPs were located on chromosome 3q23 encompassing the gene for Calsyntenin-2 (CLSTN2). The SNPs with strongest association in this region were rs17338555 (Pmeta= 5.28×10 −6), rs9812091 (Pmeta= 5.89×10−6 ), rs9812283 (Pmeta= 5.97×10−6) and rs9833213 (Pmeta= 6.96×10−6). Conclusions. This GWA study of ventricular volumes in the community-based cohorts of European descent identifies potential locus on chromosomes 3 and 5. Further characterization of these loci may provide insights into pathophysiology of ventricular involvement in various neurological diseases.^

Description and analysis of variation in dairy product and concomitant nutrient purchases between Hispanic and non -Hispanic households using a panel of shoppers' purchase records

The purpose of this research was development of a method of estimating nutrient availability in populations as approximated by supermarket purchase records. Demographic information describing 12,516 panel households was obtained from a marketing and advertising program operated by H. E. Butt Grocery Company of San Antonio, Texas. A non-probability sample of 2,161 households meeting expenditure criteria was selected and all purchases of dairy products for this sample of households were organized into a database constructed to facilitate the retrieval, aggregation, and analysis of dairy product purchases and their nutrient contents. Two hypotheses were tested: (1) no difference would be found between Hispanic and non-Hispanic purchases of dairy product categories during the study period and (2) no difference would be found between Hispanic and non-Hispanic purchases of nutrients contained in those dairy products during the thirteen-week study period.^ Food purchase records were used to estimate nutrient exposure on a weekly, per capita basis for Hispanic and non-Hispanic households by linking some 40,000 dairy purchase Universal Product code (UPC) numbers with food composition values contained in USDA Handbook 8-1. Results of this study suggest Hispanic sample households consistently purchased fewer dairy products than did non-Hispanic sample households and consequently had fewer nutrients available from dairy purchases. While weekly expenditures for dairy products among the sample households remained relatively constant during the study period, shifts in the types of dairy products purchased were observed. The effect of ethnicity on dairy product and nutrient purchases was significant over the thirteen-week period. A database consisting of customer, household, and purchase information can be developed to successfully associate food item UPC numbers with a standard reference of food composition to estimate nutrient availability in a population over extended periods of time. ^