Sub-nucleosomal Genome Structure Reveals Distinct Nucleosome Folding Motifs.

Elucidating the global and local rules that govern genome-wide, hierarchical chromatin architecture remains a critical challenge. Current high-throughput chromosome conformation capture (Hi-C) technologies have identified large-scale chromatin structural motifs, such as topologically associating domains and looping. However, structural rules at the smallest or nucleosome scale remain poorly understood. Here, we coupled nucleosome-resolved Hi-C technology with simulated annealing-molecular dynamics (SA-MD) simulation to reveal 3D spatial distributions of nucleosomes and their genome-wide orientation in chromatin. Our method, called Hi-CO, revealed distinct nucleosome folding motifs across the yeast genome. Our results uncovered two types of basic secondary structural motifs in nucleosome folding: α-tetrahedron and ß-rhombus analogous to α helix and ß sheet motifs in protein folding. Using mutants and cell-cycle-synchronized cells, we further uncovered motifs with specific nucleosome positioning and orientation coupled to epigenetic features at individual loci. By illuminating molecular-level structure-function relationships in eukaryotic chromatin, our findings establish organizational principles of nucleosome folding.

Effectiveness of Oral Vancomycin for Prevention of Healthcare Facility-Onset Clostridioides difficile Infection in Targeted Patients During Systemic Antibiotic Exposure.

BACKGROUND: Limited retrospective data suggest prophylactic oral vancomycin may prevent Clostridioides difficile infection (CDI). We sought to evaluate the effectiveness of oral vancomycin for the prevention of healthcare facility-onset CDI (HCFO-CDI) in targeted patients. METHODS: We conducted a randomized, prospective, open-label study at Novant Health Forsyth Medical Center in Winston-Salem, North Carolina, between October 2018 and April 2019. Included patients were randomized 1:1 to either oral vancomycin (dosed at 125 mg once daily while receiving systemic antibiotics and continued for 5 days postcompletion of systemic antibiotics [OVP]) or no prophylaxis. The primary endpoint was incidence of HCFO-CDI. Secondary endpoints included incidence of community-onset healthcare facility-associated CDI (CO-HCFA-CDI), incidence of vancomycin-resistant Enterococci (VRE) colonization after receiving OVP, adverse effects, and cost of OVP. RESULTS: A total of 100 patients were evaluated, 50 patients in each arm. Baseline and hospitalization characteristics were similar, except antibiotic exposure. No events of HCFO-CDI were noted in the OVP group compared with 6 (12%) in the no-prophylaxis group (P = .03). CO-HCFA-CDI was identified in 2 patients who were previously diagnosed with HCFO-CDI. No patients developed new VRE colonization, with only 1 patient reporting mild gastrointestinal side effects to OVP. A total of 600 doses of OVP were given during the study, with each patient receiving an average of 12 doses. Total acquisition cost of OVP was $1302, $26.04 per patient. CONCLUSION: OVP appears to protect against HCFO-CDI during in-patient stay in targeted patients during systemic antibiotic exposure. Further prospective investigation is warranted.

Fluorescence fluctuation spectroscopy: an invaluable microscopy tool for uncovering the biophysical rules for navigating the nuclear landscape.

Nuclear architecture is fundamental to the manner by which molecules traverse the nucleus. The nucleoplasm is a crowded environment where dynamic rearrangements in local chromatin compaction locally redefine the space accessible toward nuclear protein diffusion. Here, we review a suite of methods based on fluorescence fluctuation spectroscopy (FFS) and how they have been employed to interrogate chromatin organization, as well as the impact this structural framework has on nuclear protein target search. From first focusing on a set of studies that apply FFS to an inert fluorescent tracer diffusing inside the nucleus of a living cell, we demonstrate the capacity of this technology to measure the accessibility of the nucleoplasm. Then with a baseline understanding of the exploration volume available to nuclear proteins during target search, we review direct applications of FFS to fluorescently labeled transcription factors (TFs). FFS can detect changes in TF mobility due to DNA binding, as well as the formation of TF complexes via changes in brightness due to oligomerization. Collectively, we find that FFS-based methods can uncover how nuclear proteins in general navigate the nuclear landscape.

Kissing may be an important and neglected risk factor for oropharyngeal gonorrhoea: a cross-sectional study in men who have sex with men.

OBJECTIVES: A mathematical model suggested that a significant proportion of oropharyngeal gonorrhoea cases are acquired via oropharynx-to-oropharynx transmission (ie, tongue-kissing), but to date, no empirical study has investigated this. This study aimed to examine the association between kissing and oropharyngeal gonorrhoea among gay and bisexual men who have sex with men (MSM). METHODS: MSM attending a public sexual health centre in Melbourne, Australia, between March 2016 and February 2017 were invited to participate in a brief survey that collected data on their number of male partners in the last 3 months, in three distinct categories: kissing-only (ie, no sex including no oral and/or anal sex), sex-only (ie, any sex without kissing), and kissing-with-sex (ie, kissing with any sex). Univariable and multivariable logistic regression analyses were performed to examine associations between oropharyngeal gonorrhoea positivity by nucleic acid amplification tests and the three distinct partner categories. RESULTS: A total of 3677 men completed the survey and were tested for oropharyngeal gonorrhoea. Their median age was 30 (IQR 25-37) and 6.2% (n=229) had oropharyngeal gonorrhoea. Men had a mean number of 4.3 kissing-only, 1.4 sex-only, and 5.0 kissing-with-sex partners in the last 3 months. Kissing-only and kissing-with-sex were associated with oropharyngeal gonorrhoea, but sex-only was not. The adjusted odds for having oropharyngeal gonorrhoea were 1.46-fold (95% CI 1.04 to 2.06) for men with ≥4 kissing-only partners and 1.81-fold (95% CI 1.17 to 2.79) for men with ≥4 kissing-with-sex partners. CONCLUSIONS: These data suggest that kissing may be associated with transmission of oropharyngeal gonorrhoea in MSM, irrespective of whether sex also occurs.

Nucleosome-level 3D organization of the genome.

Nucleosomes are the unitary structures of chromosome folding, and their arrangements are intimately coupled to the regulation of genome activities. Conventionally, structural analyses using electron microscopy and X-ray crystallography have been used to study such spatial nucleosome arrangements. In contrast, recent improvements in the resolution of sequencing-based methods allowed investigation of nucleosome arrangements separately at each genomic locus, enabling exploration of gene-dependent regulation mechanisms. Here, we review recent studies on nucleosome folding in chromosomes from these two methodological perspectives: conventional structural analyses and DNA sequencing, and discuss their implications for future research.

Only recent sexual partners contribute to oropharyngeal gonorrhoea positivity: the number of sexual partners over different time periods as an indicator of gonorrhoea and chlamydia infection duration among men who have sex with men.

Background Mathematical models have demonstrated that the majority of gonococcal transmission is from oropharynx to oropharynx (i.e. kissing) among men who have sex with men (MSM). The aim of this study is to investigate the association between the number of partners within specific time periods and gonorrhoea and chlamydia positivity. METHODS: This was a retrospective data analysis of MSM attending the Melbourne Sexual Health Centre between 2007 and 2016. Univariable and multivariable logistic regression analyses, with generalised estimating equations (GEE), were performed to determine if the number of partners within specified time periods was associated with site-specific gonorrhoea and chlamydia positivity. RESULTS: There were 45933 consultations which included 15197 MSM. Oropharyngeal gonorrhoea positivity was associated with the number of partners in the past 3 months, but not the number of partners 4-12 months ago; men who had ≥6 partners in the past 3 months had significantly higher odds of acquiring oropharyngeal gonorrhoea (aOR 1.93; 95% CI 1.61-2.31), but this was not the case for men who had ≥6 partners 4-12 months ago. Anorectal gonorrhoea and chlamydia and urethral chlamydia were associated with the number of partners in both time periods after adjusting for age and condom use. CONCLUSIONS: The association of oropharyngeal gonorrhoea with the number of recent partners, but not partners from an earlier period, unlike anorectal gonorrhoea and anorectal and urethral chlamydia, could be explained by a shorter duration of oropharyngeal gonococcal infection. Annual screening for gonorrhoea may be insufficient to materially reduce oropharyngeal prevalence.

Loiasis in US Traveler Returning from Bioko Island, Equatorial Guinea, 2016.

The filarial parasite Loa loa overlaps geographically with Onchocera volvulus and Wuchereria bancrofti filariae in central Africa. Accurate information regarding this overlap is critical to elimination programs targeting O. volvulus and W. bancrofti. We describe a case of loiasis in a traveler returning from Bioko Island, Equatorial Guinea, a location heretofore unknown for L. loa transmission.

Neisseria gonorrhoeae DNA bacterial load in men with symptomatic and asymptomatic gonococcal urethritis.

OBJECTIVE: Previous studies have quantified bacterial loads of Neisseria gonorrhoeae in the pharynx and rectum of men but not the urethra. We quantified the bacterial load of N. gonorrhoeae in men with symptomatic and asymptomatic urethral gonorrhoea infections. METHODS: Consecutive men diagnosed with urethral gonorrhoea by Aptima Combo 2 testing of urine at the Melbourne Sexual Health Centre between March and July 2016 were eligible for the study: symptomatic men with purulent urethral discharge and asymptomatic men with no urethral symptoms. The gonococcal bacterial load in both groups was measured by urethral swab using a standardised collection method and real-time quantitative PCR targeting the opa gene. RESULTS: Twenty men were recruited into the study: 16 had purulent urethral discharge and 4 had asymptomatic urethral gonorrhoea. The median gonococcal bacterial load was significantly higher among symptomatic men (3.7×106 copies per swab, IQR 2.5×106-4.7×106) compared with asymptomatic men (2.0×105 copies per swab, IQR 2.7×104-4.5×105) (p=0.002). CONCLUSIONS: Gonococcal loads in men with urethral discharge were higher than loads seen with asymptomatic urethral gonorrhoea and loads seen in asymptomatic pharyngeal and rectal gonorrhoea infections in previous studies.

Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells.

Eukaryotic gene regulation involves complex patterns of long-range DNA-looping interactions between enhancers and promoters, but how these specific interactions are achieved is poorly understood. Models that posit other DNA loops--that aid or inhibit enhancer-promoter contact--are difficult to test or quantitate rigorously in eukaryotic cells. Here, we use the well-characterized DNA-looping proteins Lac repressor and phage λ CI to measure interactions between pairs of long DNA loops in E. coli cells in the three possible topological arrangements. We find that side-by-side loops do not affect each other. Nested loops assist each other's formation consistent with their distance-shortening effect. In contrast, alternating loops, where one looping element is placed within the other DNA loop, inhibit each other's formation, thus providing clear support for the loop domain model for insulation. Modeling shows that combining loop assistance and loop interference can provide strong specificity in long-range interactions.

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors.

Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to long-range DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250- to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5- to 10-kb distances, was able to increase contact probability 10- to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5- to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells.

Promoter activation by CII, a potent transcriptional activator from bacteriophage 186.

The lysogeny promoting protein CII from bacteriophage 186 is a potent transcriptional activator, capable of mediating at least a 400-fold increase in transcription over basal activity. Despite being functionally similar to its counterpart in phage λ, it shows no homology at the level of protein sequence and does not belong to any known family of transcriptional activators. It also has the unusual property of binding DNA half-sites that are separated by 20 base pairs, center to center. Here we investigate the structural and functional properties of CII using a combination of genetics, in vitro assays, and mutational analysis. We find that 186 CII possesses two functional domains, with an independent activation epitope in each. 186 CII owes its potent activity to activation mechanisms that are dependent on both the σ(70) and α C-terminal domain (αCTD) components of RNA polymerase, contacting different functional domains. We also present evidence that like λ CII, 186 CII is proteolytically degraded in vivo, but unlike λ CII, 186 CII proteolysis results in a specific, transcriptionally inactive, degradation product with altered self-association properties.

Screening for hepatitis C as part of an opioid stewardship quality improvement initiative: Identifying infected patients and analyzing linkage to care.

Screening patients with opioid use disorder (OUD) for HCV can potentially decrease morbidity and mortality if HCV-infected individuals are linked to care. We describe a quality improvement initiative focused on patients with OUD, incorporating an electronic health record decision-support tool for HCV screening across multiple health care venues, and examining the linkage to HCV care. Of 5829 patients with OUD, 4631 were tested for HCV (79.4%), (compared to a baseline of 8%) and 1614 (27.7%) tested positive. Two hundred and thirty patients had died at the study onset. Patients tested in the acute care and emergency department settings were more likely to test positive than those in the ambulatory setting (OR = 2.21 and 2.49, p < 0.001). Before patient outreach, 279 (18.2%) HCV-positive patients were linked to care. After patient outreach, 326 (23.0%) total patients were linked to care. Secondary end points included mortality and the number of patients who were HCV-positive who achieved a cure. The mortality rate in patients who were HCV-positive (12.2%) was higher than that in patients who were HCV-negative (7.4%) (OR = 1.72, p < 0.001) or untested patients (6.2%) (OR = 2.10, p<0.001). Of the 326 with successful linkage to care, 113 (34.7%) had a documented cure. An additional 55 (16.9%) patients had a possible cure, defined as direct acting antiviral ordered but no follow-up documented, known treatment in the absence of documented sustained viral response lab draw, or documentation of cure noted in outside medical records but unavailable laboratory results. A strategy utilizing electronic health record decision-support tools for testing patients with OUD for HCV was highly effective; however, linking patients with HCV to care was less successful.

The BASES Expert Statement on use of music in exercise.

The use of music during exercise has become ubiquitous over the past two decades and is now supported by a burgeoning body of research detailing its effects and the contingencies surrounding its use. The purpose of this statement is to present a synopsis of the body of knowledge, with selected references, and to provide practical recommendations for exercise practitioners regarding music selection. Following the identification of methodological shortcomings in early studies, researchers have been guided by new conceptual frameworks, and have produced more consistent findings as a consequence. The use of music has been found to yield ergogenic effects in the exercise domain while also promoting psychological (e.g. enhanced affect) and psychophysical (reduced ratings of perceived exertion) benefits. There is a paucity of research examining the longitudinal effects of music on key outcome variables such as exercise adherence.

Live cell dynamics of the NF-Y transcription factor.

Transcription factors (TFs) are core players in the control of gene expression, evolutionarily selected to recognise a subset of specific DNA sequences and nucleate the recruitment of the transcriptional machinery. How TFs assemble and move in the nucleus to locate and bind their DNA targets and cause a transcriptional response, remains mostly unclear. NF-Y is a highly conserved, heterotrimeric TF with important roles in both housekeeping and lineage-specific gene expression, functioning as a promoter organiser. Despite a large number of biochemical, structural and genomic studies of NF-Y, there is a lack of experiments in single living cells; therefore, basic assumptions of NF-Y biology remain unproven in vivo. Here we employ a series of dynamic fluorescence microscopy methods (FLIM-FRET, NB, RICS and FRAP) to study NF-Y dynamics and complex formation in live cells. Specifically, we provide quantitative measurement of NF-Y subunit association and diffusion kinetics in the nucleus that collectively suggest NF-Y to move and bind chromatin as a trimeric complex in vivo.

Hi-CO: 3D genome structure analysis with nucleosome resolution.

The nucleosome is the basic organizational unit of the genome. The folding structure of nucleosomes is closely related to genome functions, and has been reported to be in dynamic interplay with binding of various nuclear proteins to genomic loci. Here, we describe our high-throughput chromosome conformation capture with nucleosome orientation (Hi-CO) technology to derive 3D nucleosome positions with their orientations at every genomic locus in the nucleus. This technology consists of an experimental procedure for nucleosome proximity analysis and a computational procedure for 3D modeling. The experimental procedure is based on an improved method of high-throughput chromosome conformation capture (Hi-C) analysis. Whereas conventional Hi-C allows spatial proximity analysis among genomic loci with 1-10 kbp resolution, our Hi-CO allows proximity analysis among DNA entry or exit points at every nucleosome locus. This analysis is realized by carrying out ligations among the entry/exit points in every nucleosome in a micrococcal-nuclease-fragmented genome, and by quantifying frequencies of ligation products with next-generation sequencing. Our protocol has enabled this analysis by cleanly excluding unwanted non-ligation products that are abundant owing to the frequent genome fragmentation by micrococcal nuclease. The computational procedure is based on simulated annealing-molecular dynamics, which allows determination of optimized 3D positions and orientations of every nucleosome that satisfies the proximity ligation data sufficiently well. Typically, examination of the Saccharomyces cerevisiae genome with 130 million sequencing reads facilitates analysis of a total of 66,360 nucleosome loci with 6.8 nm resolution. The technique requires 2-3 weeks for sequencing library preparation and 2 weeks for simulation.

Nascent mutant Huntingtin exon 1 chains do not stall on ribosomes during translation but aggregates do recruit machinery involved in ribosome quality control and RNA.

Mutations that cause Huntington's Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntington's disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model. We found mHttex1 did not appear to stall translation of its own nascent chain, or at best was marginal. We also found the inclusions appeared to recruit low levels of RNA but there was no difference in enrichment between early formed and mature inclusions. Proteins involved in translation or ribosome quality control were co-recruited to the inclusions (Ltn1 Rack1) compared to a protein not anticipated to be involved (NACAD), but there was no major specificity of enrichment in the early formed inclusions compared to mature inclusions. Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions also contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation. Collectively our findings show little evidence that inclusion assembly arises by a discrete clustering of stalled nascent chains and associated quality control machinery. Instead, the machinery appear to be recruited continuously, or secondarily, to the nucleation of inclusion formation.

Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break.

Tumor suppressor p53-binding protein 1 (53BP1) is a DNA repair protein essential for the detection, assessment, and resolution of DNA double strand breaks (DSBs). The presence of a DSB is signaled to 53BP1 via a local histone modification cascade that triggers the binding of 53BP1 dimers to chromatin flanking this type of lesion. While biochemical studies have established that 53BP1 exists as a dimer, it has never been shown in a living cell when or where 53BP1 dimerizes upon recruitment to a DSB site, or upon arrival at this nuclear location, how the DSB histone code to which 53BP1 dimers bind regulates retention and self-association into higher-order oligomers. Thus, here in live-cell nuclear architecture we quantify the spatiotemporal dynamics of 53BP1 oligomerization during a DSB DNA damage response by coupling fluorescence fluctuation spectroscopy (FFS) with the DSB inducible via AsiSI cell system (DIvA). From adopting this multiplexed approach, we find that preformed 53BP1 dimers relocate from the nucleoplasm to DSB sites, where consecutive recognition of ubiquitinated lysine 15 of histone 2A (H2AK15ub) and di-methylated lysine 20 of histone 4 (H4K20me2), leads to the assembly of 53BP1 oligomers and a mature 53BP1 foci structure.

The frequency of kissing as part of sexual activity differs depending on how men meet their male casual sexual partners.

Previous studies have shown that men who have sex with men (MSM) who use smartphone dating applications (apps) are at higher risk of gonorrhoea, but not HIV. We have hypothesised that kissing may be a risk factor for oropharyngeal gonorrhoea. We measured differences in kissing practices among MSM who use different methods to find male casual sexual partners (CSPs). If MSM who use apps kiss more CSPs, then this may help to explain why these men are at increased risk of gonorrhoea but not HIV. This was a cross-sectional questionnaire-based study of MSM attending Melbourne Sexual Health Centre, Australia, between March and September 2015. We measured differences in kissing practices among MSM who use different methods to find male casual sexual partners (CSPs). The questionnaire included questions about numbers of CSPs, numbers of CSPs kissed, and how men found CSPs. We surveyed 753 MSM with a median age of 29 years (interquartile range 25-36). Six hundred and one men (79.8%) reported using apps to find CSPs in the last three months. Users of apps had a higher number of CSPs than non-users (5.0 vs. 3.2; p < 0.001). Users of apps kissed a higher number (4.6 vs. 2.2; p < 0.001), and a higher proportion (90.4% vs. 71.0%; p < 0.001) of CSPs compared to non-users. We are currently investigating whether kissing is a significant mode of transmission of gonorrhoea, and if this proves correct then this study suggests that users of apps would particularly benefit from health promotion that addresses this mode of transmission.