Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats

R-loops have been described at immunoglobulin class switch sequences, prokaryotic and mitochondrial replication origins, and disease-associated (CAG)n and (GAA)n trinucleotide repeats. The determinants of trinucleotide R-loop formation are unclear. Trinucleotide repeat expansions cause diseases including DM1 (CTG)n, SCA1 (CAG)n, FRAXA (CGG)n, FRAXE (CCG)n and FRDA (GAA)n. Bidirectional convergent transcription across these disease repeats can occur. We find R-loops formed when CTG or CGG and their complementary strands CAG or CCG were transcribed; GAA transcription, but not TTC, yielded R-loops. R-loop formation was sensitive to DNA supercoiling, repeat length, insensitive to repeat interruptions, and formed by extension of RNA:DNA hybrids in the RNA polymerase. R-loops arose by transcription in one direction followed by transcription in the opposite direction, and during simultaneous convergent bidirectional transcription of the same repeat forming double R-loop structures. Since each transcribed disease repeat formed R-loops suggests they may have biological functions

The World Starts With Me: A multilevel evaluation of a comprehensive sex education programme targeting adolescents in Uganda

<p>Abstract</p> <p>Background</p> <p>This paper evaluates the effect of the World Starts With Me (WSWM), a comprehensive sex education programme in secondary schools in Uganda. The aim of the present study was to assess the effects of WSWM on socio-cognitive determinants of safe sex behaviour (delay; condom use and non-coercive sex).</p> <p>Methods</p> <p>A survey was conducted both before and immediately after the intervention among students in intervention (<it>N </it>= 853) and comparison (<it>N </it>= 1011) groups. A mixed model repeated measures analysis was performed to assess the effectiveness of the WSWM programme on the main socio-cognitive determinants of safe sex behaviour at post-test. A similar post-hoc comparison was made between schools based on completeness and fidelity of implementation of WSWM.</p> <p>Results</p> <p>Significant positive effects of WSMW were found on beliefs regarding what could or could not prevent pregnancy, the perceived social norm towards delaying sexual intercourse, and the intention to delay sexual intercourse. Furthermore, significant positive effects of WSWM were found on attitudes, self-efficacy and intention towards condom use and on self-efficacy in dealing with sexual violence (pressure and force for unwanted sex). A reversed effect of intervention was found on knowledge scores relating to non-causes of HIV (petting, fondling and deep kissing). A follow-up comparison between intervention schools based on completeness of the programme implementation revealed that almost all significant positive effects disappeared for those schools that only implemented up to 7 out of 14 lessons. Another follow-up analysis on the basis of implementation fidelity showed that schools with a "partial" fidelity score yielded more significant positive effects than schools with a "full" fidelity of implementation score.</p> <p>Conclusions</p> <p>The study showed an intervention effect on a number of socio-cognitive determinants. However, the effectiveness of WSWM could be improved by giving more systematic attention to the context in which such a programme is to be implemented. Implications for the systematic development and implementation of school-based safe sex interventions in Uganda will be discussed.</p

Friedreich's Ataxia (GAA)n•(TTC)n Repeats Strongly Stimulate Mitotic Crossovers in Saccharomyces cerevisae

Expansions of trinucleotide GAA•TTC tracts are associated with the human disease Friedreich's ataxia, and long GAA•TTC tracts elevate genome instability in yeast. We show that tracts of (GAA)230•(TTC)230 stimulate mitotic crossovers in yeast about 10,000-fold relative to a “normal” DNA sequence; (GAA)n•(TTC)n tracts, however, do not significantly elevate meiotic recombination. Most of the mitotic crossovers are associated with a region of non-reciprocal transfer of information (gene conversion). The major class of recombination events stimulated by (GAA)n•(TTC)n tracts is a tract-associated double-strand break (DSB) that occurs in unreplicated chromosomes, likely in G1 of the cell cycle. These findings indicate that (GAA)n•(TTC)n tracts can be a potent source of loss of heterozygosity in yeast

Mycoplasma genitalium: An Emerging Cause of Sexually Transmitted Disease in Women

Mycoplasma genitalium is an emerging sexually transmitted pathogen implicated in urethritis in men and several inflammatory reproductive tract syndromes in women including cervicitis, pelvic inflammatory disease (PID), and infertility. This comprehensive review critically examines epidemiologic studies of M. genitalium infections in women with the goal of assessing the associations with reproductive tract disease and enhancing awareness of this emerging pathogen. Over 27,000 women from 48 published reports have been screened for M. genitalium urogenital infection in high- or low-risk populations worldwide with an overall prevalence of 7.3% and 2.0%, respectively. M. genitalium was present in the general population at rates between those of Chlamydia trachomatis and Neisseria gonorrhoeae. Considering more than 20 studies of lower tract inflammation, M. genitalium has been positively associated with urethritis, vaginal discharge, and microscopic signs of cervicitis and/or mucopurulent cervical discharge in seven of 14 studies. A consistent case definition of cervicitis is lacking and will be required for comprehensive understanding of these associations. Importantly, evidence for M. genitalium PID and infertility are quite convincing and indicate that a significant proportion of upper tract inflammation may be attributed to this elusive pathogen. Collectively, M. genitalium is highly prevalent in high- and low-risk populations, and should be considered an etiologic agent of select reproductive tract disease syndromes in women

How to build a bone: PHOSPHO1, biomineralization and beyond

Since its characterization two decades ago, the phosphatase PHOSPHO1 has been the subject of an increasing focus of research. This work has elucidated PHOSPHO1’s central role in the biomineralization of bone and other hard tissues, but has also implicated the enzyme in other biological processes in health and disease. During mineralization PHOSPHO1 liberates inorganic phosphate (Pi) to be incorporated into the mineral phase through hydrolysis of its substrates phosphocholine (PCho) and phosphoethanolamine (PEA). Localization of PHOSPHO1 within matrix vesicles allows accumulation of Pi within a protected environment where mineral crystals may nucleate and subsequently invade the organic collagenous scaffold. Here, we examine the evidence for this process, first discussing the discovery and characterization of PHOSPHO1, before considering experimental evidence for its canonical role in matrix vesicle-mediated biomineralization. We also contemplate roles for PHOSPHO1 in disorders of dysregulated mineralization such as vascular calcification, along with emerging evidence of its activity in other systems including choline synthesis and homeostasis, and energy metabolism

New insights into repeat instability: Role of RNA•DNA hybrids

Expansion of tandem repeat sequences is responsible for more than 20 human diseases. Several cis elements and trans factors involved in repeat instability (expansion and contraction) have been identified. However no comprehensive model explaining large intergenerational or somatic changes of the length of the repeating sequences exists. Several lines of evidence, accumulated from different model studies, indicate that transcription through repeat sequences is an important factor promoting their instability. The persistent interaction between transcription template DNA and nascent RNA (RNA•DNA hybrids, R loops) was shown to stimulate genomic instability. Recently, we demonstrated that cotranscriptional RNA•DNA hybrids are preferentially formed at GC-rich trinucleotide and tetranucleotide repeat sequences in vitro as well as in human genomic DNA. Additionally, we showed that cotranscriptional formation of RNA•DNA hybrids at CTG•CAG and GAA•TTC repeats stimulate instability of these sequences in both E. coli and human cells. Our results suggest that persistent RNA•DNA hybrids may also be responsible for other downstream effects of expanded trinucleotide repeats, including gene silencing. Considering the extent of transcription through the human genome as well as the abundance of GC-rich and/or non-canonical DNA structure forming tandem repeats, RNA•DNA hybrids may represent a common mutagenic conformation. Hence, R loops are potentially attractive therapeutic targets in diseases associated with genomic instability