Regions of very low H3K27me3 partition the Drosophila genome into topological domains.

It is now well established that eukaryote genomes have a common architectural organization into topologically associated domains (TADs) and evidence is accumulating that this organization plays an important role in gene regulation. However, the mechanisms that partition the genome into TADs and the nature of domain boundaries are still poorly understood. We have investigated boundary regions in the Drosophila genome and find that they can be identified as domains of very low H3K27me3. The genome-wide H3K27me3 profile partitions into two states; very low H3K27me3 identifies Depleted (D) domains that contain housekeeping genes and their regulators such as the histone acetyltransferase-containing NSL complex, whereas domains containing moderate-to-high levels of H3K27me3 (Enriched or E domains) are associated with regulated genes, irrespective of whether they are active or inactive. The D domains correlate with the boundaries of TADs and are enriched in a subset of architectural proteins, particularly Chromator, BEAF-32, and Z4/Putzig. However, rather than being clustered at the borders of these domains, these proteins bind throughout the H3K27me3-depleted regions and are much more strongly associated with the transcription start sites of housekeeping genes than with the H3K27me3 domain boundaries. While we have not demonstrated causality, we suggest that the D domain chromatin state, characterised by very low or absent H3K27me3 and established by housekeeping gene regulators, acts to separate topological domains thereby setting up the domain architecture of the genome.

The boundary paradox in the Bithorax complex.

The parasegment-specific expression of the three Drosophila Bithorax complex homeotic genes is orchestrated by nine functionally autonomous regulatory domains. Functional autonomy depends upon special elements called boundaries or insulators that are located between each domain. The boundaries ensure the independent activity of each domain by blocking adventitious interactions with initiators, enhancers and silencers in the neighboring domains. However, this blocking activity poses a regulatory paradox--the Bithorax boundaries are also able to insulate promoters from regulatory interactions with enhancers and silencers and six of the nine Bithorax regulatory domains are separated from their target genes by at least one boundary element. Here we consider several mechanisms that have been suggested for how the Bithorax regulatory domains are able to bypass intervening boundary elements and direct the appropriate parasegment-specific temporal and spatial expression of their target gene.

A variably occupied CTCF binding site in the ultrabithorax gene in the Drosophila bithorax complex.

Although the majority of genomic binding sites for the insulator protein CCCTC-binding factor (CTCF) are constitutively occupied, a subset show variable occupancy. Such variable sites provide an opportunity to assess context-specific CTCF functions in gene regulation. Here, we have identified a variably occupied CTCF site in the Drosophila Ultrabithorax (Ubx) gene. This site is occupied in tissues where Ubx is active (third thoracic leg imaginal disc) but is not bound in tissues where the Ubx gene is repressed (first thoracic leg imaginal disc). Using chromatin conformation capture, we show that this site preferentially interacts with the Ubx promoter region in the active state. The site lies close to Ubx enhancer elements and is also close to the locations of several gypsy transposon insertions that disrupt Ubx expression, leading to the bx mutant phenotype. gypsy insertions carry the Su(Hw)-dependent gypsy insulator and were found to affect both CTCF binding at the variable site and the chromatin topology. This suggests that insertion of the gypsy insulator in this region interferes with CTCF function and supports a model for the normal function of the variable CTCF site as a chromatin loop facilitator, promoting interaction between Ubx enhancers and the Ubx transcription start site.

Performance evaluation of a new rapid urine test for chlamydia in men: prospective cohort study.

OBJECTIVE: To evaluate the performance of a rapid test for chlamydia with first void male urine samples as a potential tool for diagnosis and screening of chlamydial infection in men. DESIGN: Evaluation of test performance in prospective cohort study. Settings A young people's sexual health centre (site 1) and a genitourinary medicine clinic (site 2) in the United Kingdom. PARTICIPANTS: 1211 men aged 16-73 attending either of the two sites. MAIN OUTCOME MEASURES: Sensitivity, specificity, positive predictive value, and negative predictive value of the Chlamydia Rapid Test versus polymerase chain reaction assay. Relation between the visual signal of the Chlamydia Rapid Test and organism load. RESULTS: Detection rates for Chlamydia trachomatis infection with polymerase chain reaction were 4.4% (20/454) at site 1 and 11.9% (90/757) at site 2. Compared with polymerase chain reaction assay, the resolved sensitivity, specificity, positive predictive value, and negative predictive value of the Chlamydia Rapid Test was 82.6% (90/109), 98.5% (1085/1102), 84.1% (90/107), and 98.3% (1085/1104), respectively. The organism load in first void urine samples that were positive for chlamydia ranged from 7.28x10(2) to 6.93x10(6) plasmids/ml and correlated significantly with the visual signal of the Chlamydia Rapid Test (r=0.7897, P<0.001). CONCLUSIONS: The performance of the new Chlamydia Rapid Test with first void male urine samples indicates that it would be an effective diagnostic tool for chlamydial infection in men. The availability of test results within an hour allows for immediate treatment and contact tracing, potentially reducing the risks of persistent infection and onward transmission. The test could also provide a simple and reliable alternative to nucleic acid amplification assays for testing of male urine in chlamydial screening programmes in high prevalence settings.

Optimal method of collection of first-void urine for diagnosis of Chlamydia trachomatis infection in men.

First-void urine (FVU) is the preferred specimen for the diagnosis of urogenital Chlamydia trachomatis infection in men. We have developed FirstBurst, a urine collection device that collects the first 4 to 5 ml of FVU and yields a specimen with a sixfold higher C. trachomatis organism load than the regular urine cup by quantitative PCR (32,533 versus 5,271 plasmids/ml; P < 0.0001). Consequently, the use of FirstBurst to collect a urine sample improved the sensitivity of a rapid test for Chlamydia over testing of samples collected with a urine cup (82 versus 47% sensitivity using PCR as a reference; P < 0.0015).

Prevalence of Chlamydia trachomatis infection among low- and high-risk Filipino women and performance of Chlamydia rapid tests in resource-limited settings.

The prevalence of urogenital Chlamydia trachomatis infection was determined with a PCR-based test of women from low- and high-risk populations in Iloilo City, Philippines, between August 2002 and March 2006. Two rapid tests for C. trachomatis, Clearview Chlamydia MF and the Chlamydia Rapid Test (CRT), were also evaluated in these resource-limited settings. Specimens were obtained from female sex workers (FSWs; n = 1,484) attending a social hygiene clinic (SHC) and from women (n = 838) attending an obstetrics-gynecology (OB-GYN) clinic. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the rapid tests were determined, with PCR as the gold standard. The PCR positivity rate for SHC participants (72% asymptomatic) ranged from 17.9 to 32.0% during the study period. Compared with those of PCR, the sensitivities and specificities of the Clearview test were 53.5 and 99.1%, respectively, with endocervical swab specimens (CS; n = 822) from the FSWs and 31.1 and 95.2%, respectively, with vaginal swab specimens (VS; n = 333) from these women. The sensitivity, specificity, PPV, and NPV of the CRT with VS from the FSWs were 71.0, 99.0, 97.1, and 87.9%, respectively. At the OB-GYN site, the PCR positivity rate with VS was 6.3%. The sensitivity, specificity, PPV, and NPV of the CRT with these specimens were 86.8, 99.6, 93.9, and 99.1%, respectively. The performance of the Clearview test at the SHC was thus markedly lower with VS than with CS, whereas the CRT performed well with VS from both populations.

Elevated expression of genes under the control of stress response element (STRE) and Msn2p in an ethanol-tolerance sake yeast Kyokai no. 11.

The sake yeast strain Kyokai no. 11 (K11) is an ethanol-tolerant mutant of strain Kyokai no. 7 (K7), which shows higher viability in an ethanol solution than strain K7. To clarify the mechanism underlying the ethanol tolerance of this strain, the gene expression profiles of K7 and K11 were analyzed using DNA microarrays. The results indicate that many genes induced by stresses were highly expressed in strain K11 not exposed to stresses. Analysis of HSP12, one of the most highly expressed genes in strain K11 compared with strain K7, revealed that a trans-acting factor of strain K11 was involved in the elevated expression of HSP12. Many of the highly expressed genes in strain K11 including HSP12 were under the control of a cis-acting factor called the stress response element (STRE). The addition of STRE sequences to a promoter region of a reporter gene resulted in constitutive high-level expression in strain K11. It was reported that transcription factors Msn2p and Msn4p bind to STRE sequences. DNA sequence analyses of MSN2 and MSN4 of strains K7 and K11 revealed that only Msn2p was functional in these strains. When two copies of MSN2 in strain K11 were disrupted, the expression level of the reporter gene under the control of STRE decreased to the level of strain K7, indicating that Msn2p is required for the elevated expression of the STRE-controlled genes in K11.

Chlamydia trachomatis variant not detected by plasmid based nucleic acid amplification tests: molecular characterisation and failure of single dose azithromycin.

OBJECTIVE: To characterise a Chlamydia trachomatis variant strain from a patient with non-gonococcal urethritis (NGU) whose first void urine (FVU) displayed discrepant Ctrachomatis test results and describe the clinical response to treatment. METHODS: The FVU specimen was assayed with an immune based Chlamydia Rapid Test (CRT) and various nucleic acid amplification tests (NAATs) to establish C trachomatis infection. Sequencing of the major outer membrane protein gene (omp1 also known as ompA) was undertaken to identify the serovar of the variant strain. Polymerase chain reaction (PCR) analysis was also conducted to determine whether the strain harboured deletions in the cryptic plasmid or was plasmid free. RESULTS: The FVU specimen was strongly reactive in CRT but negative with the plasmid based Amplicor PCR (Roche) and ProbeTec ET (Becton-Dickinson) assays. However, NAATs for 16S RNA (Aptima Combo 2, GenProbe), omp1 (RealArt CT PCR, Artus and in-house NAATs) or the outer membrane complex B protein gene (omcB) established C trachomatis infection. Sequencing of omp1 showed that the variant belonged to serovar I. PCR analysis indicated that the variant was plasmid free. The patient did not respond to single dose azithromycin treatment but subsequently responded to a course of doxycycline. CONCLUSIONS: A pathogenic plasmid free C trachomatis variant was identified. Clinicians should be alerted to the possibility of undetected C trachomatis infection caused by such variants and the potential of azithromycin failure in patients with recurrent chlamydial NGU. The occurrence of this variant is rare and should not form the basis for judgment of the performance or usefulness of plasmid based NAATs for C trachomatis detection.

Chlamydia trachomatis load at matched anatomic sites: implications for screening strategies.

Urethral and endocervical swabs and self-collected vaginal swabs (SCVSs) and urine specimens are all used as samples for diagnosis of urogenital infection with Chlamydia trachomatis. We have now determined chlamydial organism load in matched specimens from different anatomic sites and examined its relation to clinical signs and symptoms in men and women. Organism load was measured with assays based on the ligase chain reaction or real-time PCR analysis. The mean organism loads in 58 infected men were 1,200 and 821 elementary bodies (EBs) per 100 microl of sample for first-void urine (FVU) and urethral swabs, respectively (P>0.05). Organism load in FVU samples or urethral swabs was positively associated with symptoms (P<0.01) and clinical signs (P<0.01) in men. The mean organism loads in 73 infected women were 2,231, 773, 162, and 47 EBs/100 microl for endocervical swabs, SCVSs, urethral swabs, and FVU samples, respectively (P<0.001 for each comparison). Only the presence of multiple symptoms or clinical signs was associated with organism load in women. These results show that FVU is a suitable noninvasive sample type for men, given the fact that its chlamydial load did not differ significantly from that of urethral swabs. Given their higher organism load compared with FVU, SCVSs are the preferred noninvasive sample type for women.