Gonadotropin suppression in men leads to a reduction in claudin-11 at the Sertoli cell tight junction.

STUDY QUESTION: Are Sertoli cell tight junctions (TJs) disrupted in men undergoing hormonal contraception? SUMMARY ANSWER: Localization of the key Sertoli cell TJ protein, claudin-11, was markedly disrupted by 8 weeks of gonadotropin suppression, the degree of which was related to the extent of adluminal germ cell suppression. WHAT IS KNOWN ALREADY: Sertoli cell TJs are vital components of the blood-testis barrier (BTB) that sequester developing adluminal meiotic germ cells and spermatids from the vascular compartment. Claudin-11 knockout mice are infertile; additionally claudin-11 is spatially disrupted in chronically gonadotropin-suppressed rats coincident with a loss of BTB function, and claudin-11 is disorganized in various human testicular disorders. These data support the Sertoli cell TJ as a potential site of hormonal contraceptive action. STUDY DESIGN, SIZE, DURATION: BTB proteins were assessed by immunohistochemistry (n = 16 samples) and mRNA (n = 18 samples) expression levels in available archived testis tissue from a previous study of 22 men who had undergone 8 weeks of gonadotropin suppression and for whom meiotic and post-meiotic germ cell numbers were available. The gonadotropin suppression regimens were (i) testosterone enanthate (TE) plus the GnRH antagonist, acyline (A); (ii) TE + the progestin, levonorgestrel, (LNG); (iii) TE + LNG + A or (iv) TE + LNG + the 5α-reductase inhibitor, dutasteride (D). A control group consisted of seven additional men, with three archived samples available for this study. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Immunohistochemical localization of claudin-11 (TJ) and other junctional type markers [ZO-1 (cytoplasmic plaque), ß-catenin (adherens junction), connexin-43 (gap junction), vinculin (ectoplasmic specialization) and ß-actin (cytoskeleton)] and quantitative PCR was conducted using matched frozen testis tissue. MAIN RESULTS AND THE ROLE OF CHANCE: Claudin-11 formed a continuous staining pattern at the BTB in control men. Regardless of gonadotropin suppression treatment, claudin-11 localization was markedly disrupted and was broadly associated with the extent of meiotic/post-meiotic germ cell suppression; claudin-11 staining was (i) punctate (i.e. 'spotty' appearance) at the basal aspect of tubules when the average numbers of adluminal germ cells were <15% of control, (ii) presented as short fragments with cytoplasmic extensions when numbers were 15-25% of control or (iii) remained continuous when numbers were >40% of control. Changes in localization of connexin-43 and vinculin were also observed (smaller effects than for claudin-11) but ZO-1, ß-catenin and ß-actin did not differ, compared with control. LIMITATIONS, REASONS FOR CAUTION: Claudin-11 was the only Sertoli cell TJ protein investigated, but it is considered to be the most pivotal of constituent proteins given its known implication in infertility and BTB function. We were limited to testis samples which had been gonadotropin-suppressed for 8 weeks, shorter than the 74-day spermatogenic wave, which may account for the heterogeneity in claudin-11 and germ cell response observed among the men. Longer suppression (12-24 weeks) is known to suppress germ cells further and claudin-11 disruption may be more uniform, although we could not access such samples. WIDER IMPLICATIONS OF THE FINDINGS: These findings are important for our understanding of the sites of action of male hormonal contraception, because they suggest that BTB function could be ablated following long-term hormone suppression treatment. STUDY FUNDING/COMPETING INTERESTS: National Health and Medical Research Council (Australia) Program Grants 241000 and 494802; Research Fellowship 1022327 (to R.I.M.) and the Victorian Government's Operational Infrastructure Support Program. None of the authors have any conflicts to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Long noncoding RNAs and the genetics of cancer.

Cancer is a disease of aberrant gene expression. While the genetic causes of cancer have been intensively studied, it is becoming evident that a large proportion of cancer susceptibility cannot be attributed to variation in protein-coding sequences. This is highlighted by genome-wide association studies in cancer that reveal that more than 80% of cancer-associated SNPs occur in noncoding regions of the genome. In this review, we posit that a significant fraction of the genetic aetiology of cancer is exacted by noncoding regulatory sequences, particularly by long noncoding RNAs (lncRNAs). Recent studies indicate that several cancer risk loci are transcribed into lncRNAs and these transcripts play key roles in tumorigenesis. We discuss the epigenetic and other mechanisms through which lncRNAs function and how they contribute to each stage of cancer progression, understanding of which will be crucial for realising new opportunities in cancer diagnosis and treatment. Long noncoding RNAs play important roles in almost every aspect of cell biology from nuclear organisation and epigenetic regulation to post-transcriptional regulation and splicing, and we link these processes to the hallmarks and genetics of cancer. Finally, we highlight recent progress and future potential in the application of lncRNAs as therapeutic targets and diagnostic markers.

Intergenic disease-associated regions are abundant in novel transcripts.

BACKGROUND: Genotyping of large populations through genome-wide association studies (GWAS) has successfully identified many genomic variants associated with traits or disease risk. Unexpectedly, a large proportion of GWAS single nucleotide polymorphisms (SNPs) and associated haplotype blocks are in intronic and intergenic regions, hindering their functional evaluation. While some of these risk-susceptibility regions encompass cis-regulatory sites, their transcriptional potential has never been systematically explored. RESULTS: To detect rare tissue-specific expression, we employed the transcript-enrichment method CaptureSeq on 21 human tissues to identify 1775 multi-exonic transcripts from 561 intronic and intergenic haploblocks associated with 392 traits and diseases, covering 73.9 Mb (2.2%) of the human genome. We show that a large proportion (85%) of disease-associated haploblocks express novel multi-exonic non-coding transcripts that are tissue-specific and enriched for GWAS SNPs as well as epigenetic markers of active transcription and enhancer activity. Similarly, we captured transcriptomes from 13 melanomas, targeting nine melanoma-associated haploblocks, and characterized 31 novel melanoma-specific transcripts that include fusion proteins, novel exons and non-coding RNAs, one-third of which showed allelically imbalanced expression. CONCLUSIONS: This resource of previously unreported transcripts in disease-associated regions ( http://gwas-captureseq.dingerlab.org ) should provide an important starting point for the translational community in search of novel biomarkers, disease mechanisms, and drug targets.

Identification of archaeal genes encoding a novel stationary phase-response protein.

A novel stationary phase-response protein has been identified in the acid-soluble protein extract of the thermophilic archaeon, Thermococcus zilligii. N-Terminal sequencing data were used to identify likely genes for homologues of the protein in the complete genome sequences of various archaeal species. The corresponding genes were identified and analysed. The genes encode a protein ranging from 83 to 92 amino acids in length, with a calculated pI ranging from 4.6 to 9.7. The amino acid sequences of the genes were highly conserved, even between members belonging to the different archaeal kingdoms. The computed secondary structure of the protein indicates it consists of four large helical regions separated by short coiled regions. We propose this protein as a candidate regulator of gene expression in stationary phase.

Placement of cardiac electrodes: written, simulated, and actual accuracy.

BACKGROUND: Research has shown that critical care nurses show low accuracy on written tests of placement of electrodes, yet it is unknown how this low accuracy translates into placement of electrodes on actual patients. OBJECTIVES: To determine if accuracy scores differ between three methods (written knowledge, simulated clinical practice, actual clinical practice) of evaluating placement of continuous ECG electrodes by a group of cardiac care nurses. METHODS: A standardized scoring diagram was used with three different methods of measuring the accuracy of 44 nurses who worked on a telemetry unit or medical ICU in placing continuous ECG electrodes. The three methods were (1) written knowledge--placement of stickers on a diagram of a torso, (2) simulated clinical practice--placement of electrodes on a human model, and (3) actual clinical practice--placement of electrodes on an assigned patient. RESULTS: For the total diagram score (maximum score = 11), no significant differences among groups were found. For the V1 subscale score (maximum score = 4), a significant difference among groups was found: Scheffé's test showed that the significant difference was between simulated and actual clinical practice. Percentages of nurses achieving the maximum, or accurate, score were 18% for written knowledge, 25% for simulated clinical practice, and 9% for actual clinical practice. CONCLUSIONS: Although total scores did not differ among groups, the mean scores indicate that placement of electrodes was not accurate by any method. This finding has implications for how electrode placement is taught to nurses and for the accountability of nurses for placement of electrodes on their patients.

Associations between number of lifetime partners and other health behaviors.

OBJECTIVE: To examine associations between health behaviors and lifetime sexual partners among college students. METHODS: Data from the 1995 National College Health Risk Behavior Survey were analyzed. RESULTS: After adjusting for age, sex, and race, having 2 or more lifetime sexual partners was associated with infrequently using seat belts, driving after drinking, having a physical fight, considering suicide, and using chewing tobacco and marijuana. Significant sex interactions occurred with cigarette smoking and fruit and vegetable consumption, and significant age interactions occurred with binge drinking. CONCLUSIONS: Having multiple lifetime sexual partners (> or = 2) was associated with several negative health behaviors.

Factors related to publication productivity in a sample of female health educators.

OBJECTIVE: To examine factors related to success in academic publishing for women in health education and promotion. METHODS: Ten participants, identified as productive authors, submitted a copy of their curriculum vita and completed a questionnaire related to publishing productivity. RESULTS: Factors that contributed to successful publication included personal attributes, such as self-discipline and effective time management, and situational factors, such as talented collaborators, access to mentoring, and grant funding. Tips for maximizing productivity and enhancing collaborative efforts with colleagues are also presented. CONCLUSION: Findings support correlates of productivity in faculty members that have been reported in other academic disciplines.

Stability and convergent validity of the Physical Activity Scale for the Elderly (PASE).

AIM: The purpose of this study was to assess the stability and convergent validity of the Physical Activity Scale for the Elderly (PASE) among rural, community dwelling elderly persons using Computer Science and Applications, Inc. Actigraph Monitors (Actigraph) as the direct criterion measure. EXPERIMENTAL DESIGN: a correlational design was employed. SETTING: rural community in the United States. PARTICIPANTS: 56 subjects (age=75.7+/-7.9 years) who were living independently and volunteered to participate in the study. MEASURES: subjects wore an Actigraph monitor during all waking hours for 7 consecutive days. At the conclusion of the 7 days, each subject met with a trained interviewer to complete the PASE questionnaire. Three days later the subjects met with the same interviewer to complete the PASE a 2nd time recalling their physical activity for the same 7-day period. RESULTS: Actigraph data indicated that subjects averaged 168.1+/-76.3 counts x minute(-1) during the 7-day period. A high intraclass correlation coefficient (r=0.91) was calculated between the 1st interview total PASE score (115.97+/-59.91) and the 2nd interview total PASE score (115.71+/-50.97). In addition, there was a statistically significant Spearman correlation coefficient of 0.43 (p<0.01) between Actigraph mean counts x minute(-1) and 1st interview total PASE scores. CONCLUSION: In this rural elderly sample, the PASE was a stable instrument with validity indices similar to those previously reported in younger, more active, populations.

Assessment of health knowledge after "a healthy adventure".

This study evaluated the general health education program for third graders at Hult Health Education Center (Hult HEC). A quasi-experimental, nonequivalent pretest-posttest control group design with four groups was employed to collect pretest and posttest data from 168 third graders. The four groups included a control group, a traditional classroom health education group, a group that visited Hult HEC, and a group that visited Hult HEC and incorporated center curricular materials in the classroom. Analysis of covariance (ANCOVA) was calculated to test for posttest differences among the four groups, while reducing the effects of initial group pretest score differences. A statistically significant difference occurred in posttest scores among the four groups (F = 120.62, df = 4, 163, p = .0001). Post hoc testing revealed the best results were obtained when combining curricular materials with a visit to the Hult HEC.

Development of an instrument to measure community acceptance of nurse practitioners and physician assistants.

The purpose of this study was to identify the significant dimensions of the concept of community acceptance of nurse practitioners/physician's assistants and to construct a reliable and valid instrument which would reflect these dimensions. The methodological approach included: conceptualization of categories, development of items for each category, development of the tool, administration of the tool, and psychometric analysis of results. Community input through focus-group interviews and post-administration questions provided qualitative data. The survey tool, consisting of items in four conceptualized categories (knowledge, access, competence, and trust), was administered in five rural communities. The responses of 967 residents were analyzed through factor analysis. The criterion, eigenvalue > or = 1.0, resulted in seven factors. Oblique rotation was applied to the seven factors and marker variables (loadings > .70) facilitated the identification of the underlying dimensions of each factor. Overall, 98% of the items assigned to the original categories were maintained after factor analysis. The identification of these dimensions helped to simplify the description and understanding of community acceptance of nurse practitioners and physicians' assistants. Community acceptance of these advanced health care providers is a necessary precursor to use of services.

Alkali salts of C3-symmetric, linked aryloxides: selective binding of substrates with metal aggregates.

The lithium, sodium, and potassium salts of tris(3,5-dialkyl-2-hydroxyphenyl)methanes (tert-butyl, tert-pentyl, methyl) have been prepared by reaction of the triarylmethane with n-butyllithium, sodium hydride, and potassium hydride, respectively. These compounds are all hexanuclear aggregates composed of two triarylmethane units. Whereas the lithium salt is compact and cannot bind oxygen-donor solvent molecules, the sodium and potassium systems have vacant coordination sites that can interact with solvents. For the sodium compounds, the solvent can be subsequently removed, and the resulting coordinatively unsaturated compounds have been shown to selectively bind oxygen-donor substrates (ethers, aldehydes, and ketones) of suitable size and shape. The paper reports the synthesis and characterization of these novel compounds, including thirteen crystal structures of the salts and their adducts.

Growth phase-dependent expression and degradation of histones in the thermophilic archaeon Thermococcus zilligii.

HTz is a member of the archaeal histone family. The archaeal histones have primary sequences and structural similarity to the eukaryal histone fold domain, and are thought to resemble the archetypal ancestor of the eukaryal nucleosome core histones. The effects of growth phase on the total soluble proteins from Thermococcus zilligii, isolated after various stages of growth from mid-logarithmic to late stationary phase, were examined by denaturing polyacrylamide gel electrophoresis. On entry into stationary phase, at least 11 proteins were detected that changed considerably in level. One of these proteins was identified by Western hybridization as HTz. The level of HTz decreased dramatically as cells entered stationary phase, and it could not be detected by late stationary phase. Unexpectedly, the Western hybridization detected a second protein, with an estimated molecular mass of approximately 14 kDa, which paralleled the decrease in level of HTz. Native purified HTz was shown to retain complete activity after prolonged incubation at the growth temperature of the organism, suggesting that the decrease in HTz was a specific cell-regulated process. Analysis of native purified HTz by electrospray ionization mass spectrometry revealed the molecular masses of HTz1 and HTz2 to be 7204 +/- 3 Da and 7016 +/- 3 Da respectively. The only non-covalent species that was detected corresponded to the molecular mass of an HTz1-HTz2 heterodimer. Northern analyses of T. zilligii total RNA with an htz1 gene probe indicated a rapid decrease in expression of htz1 with progression of the growth phase, and complete repression of htz1 transcript synthesis by late logarithmic phase. Three proteins that changed in level with growth phase were identified by N-terminal sequence analysis. The first was homologous to a hypothetical protein conserved in all Archaea sequenced to date, the second to the Sac10b family of archaeal DNA-binding proteins and the third to the C-terminal region of the leucine-responsive regulatory family of DNA-binding proteins (LRPs).

Novel analogues of neuropeptide Y with a preference for the Y1-receptor.

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and acts in humans via at least three receptor subtypes: Y1, Y2, and Y5. Whereas selective agonists and antagonists are known for the Y2- and Y5-receptors, the Y1-receptor still lacks a highly selective agonist. This work presents the first NPY-based analogues with Y1-receptor preference and agonistic properties. Furthermore, the importance of specific amino acids of NPY for binding to the Y-receptor subtypes is presented. Amongst the analogues tested, [Phe7,Pro34]pNPY (where pNPY is porcine neuropeptide Y) showed the most significant Y1-receptor preference (> 1 : 3000-fold), with subnanomolar affinity to the Y1-receptor, and Ki values of approximately 30 nM for the Y2- and Y5-subtype, respectively. Variations of position 6, especially [Arg6,Pro34]pNPY and variations within positions 20-23 of NPY were found to result in further analogues with significant Y1-receptor preference (1 : 400-1 : 2000). In contrast, cyclo S-S [Cys20,Cys24]pNPY was found to be a highly selective ligand at the Y2-receptor, binding only threefold less efficiently than NPY. Analogues containing variations of positions 31 and 32 showed highly reduced affinity to the Y1-receptor, while binding to the Y5-receptor was affected less. Inhibition of cAMP-accumulation of selected peptides with replacements within position 20-23 of NPY showed preserved agonistic properties. The NPY analogues tested give insights into ligand-receptor interaction of NPY at the Y1-, Y2- and Y5-receptor and contribute to our understanding of subtype selectivity. Furthermore, the Y1-receptor-preferring peptides are novel tools that will provide insight into the physiological role of the Y1-receptor.

Accessibility and perceived value of health services in five western Illinois rural communities.

The purpose of this study was to determine the accessibility and perceived value of health services in five selected rural communities in South Fulton County, Illinois. The Health Services Accessibility and Value Scale (HSAVS) component of the larger Fulton County Health Care Survey was used in the investigation. The 12 items comprising the HSAVS surveyed participant perceptions relative to medical, dental, nursing, and public health department services available to them as rural residents. The HSAVS was completed by 1709 subjects. The reliability of the HSAVS was assessed by computing coefficient alpha. The scale had acceptable internal consistency reliability (alpha = .7884). To examine the construct validity of the scale, a principle component factor analysis was completed. This analysis resulted in a four factor solution which accounted for 66.6% of the cumulative total variance. Item means were calculated and were used to rank the HSAVS statements. Emergency and primary medical services were valued the most by the survey participants. Relatively high in importance to the rural residents surveyed were access to pharmacy, eye care, dental care, and immunization services. Availability of home health care, transportation to and from health care facilities, and access to mental health services were considered to be of lesser value to persons living in the rural area studied. Of least importance were alcohol and other drug counseling services, prenatal care/well baby services, and family planning services. HSAVS total scores and individual statements were also analyzed by sex, age, place of residence, and income and the results were reported.

Human ATP-binding cassette transporter 1 (ABC1): genomic organization and identification of the genetic defect in the original Tangier disease kindred.

Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.