Performing a genetic screen to identify factors that promote lncRNA-dependent gene repression

Long non-coding RNAs (lncRNAs) were once thought not to have useful functions in organisms but rather to be products of aberrant transcription. However, roles are being found for lncRNAs in beneficial processes such as controlling gene expression. In some of these cases, lncRNAs form R-loops in vivo. R-loops are nucleic acid structures consisting of hybridized strands of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA) as well as the displaced strand of ssDNA. Formation of these R-loops is important for gene regulation by the lncRNAs. However, factors that promote formation of lncRNA R-loops are not known. The gene PHO84 is being used to study this matter. PHO84 encodes a phosphate transporter in Saccharomyces cerevisiae. Two lncRNAs are transcribed in the antisense direction across PHO84 and repress PHO84 gene expression. This phenomenon may be due to the formation of R-loops. A genetic screen is being performed in S. cerevisiae to identify genes that suppress gene expression on PHO84 when overexpressed. The effects of these genes will then be studied in more detail. Then, more work will be done to determine how these genes may interact with lncRNAs

Lipid peroxidation biomarkers for evaluating oxidative stress in equine neuroaxonal dystrophy.

BackgroundEquine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is a neurodegenerative disorder affecting genetically predisposed foals maintained on an α-tocopherol (α-TOH) deficient diet. Currently no antemortem diagnostic test for eNAD/EDM is available.HypothesisBecause α-TOH deficiency is associated with increased lipid peroxidation, it was hypothesized that F2 -isoprostanes (F2 IsoP), F4 -neuroprostanes (F4 NP) and oxysterols derived from free radical oxidation would be increased in the cerebrospinal fluid (CSF) and neural tissue of eNAD/EDM affected horses and could serve as potential biomarkers for disease.AnimalsIsoprostane Study A: 14 Quarter horse foals (10 healthy foals and 4 eNAD/EDM affected foals) at 1 and 6 months of age. Isoprostane Study B: 17 eNAD/EDM affected and 10 unaffected horses ≥ 1-4 years of age. Oxysterol study: eNAD/EDM affected (n = 14, serum; n = 11, CSF; n = 10, spinal cord [SC]) and unaffected horses 1-4 years of age (n = 12, serum; n = 10, CSF; n = 7, SC).ProceduresCerebrospinal fluid [F2 IsoP] and [F4 NP] were assessed using gas chromatography-negative ion chemical ionization mass spectrometry. Serum, CSF, and cervical SC [oxysterols] were quantified using high performance liquid chromatography mass spectrometry. Results were compared with respective α-TOH concentrations.ResultsSpinal cord [7-ketocholesterol], [7-hydroxycholesterol], and [7-keto-27-hydrocholesterol] were higher in eNAD/EDM horses whereas [24-ketocholesterol] was lower. No significant difference was found in CSF [F2 IsoP] and [F4 NP], serum [oxysterols] and CSF [oxysterols] between eNAD/EDM affected and unaffected horses. No correlation was found between [F2 IsoP], [F4 NP], or [oxysterols] and respective [α-TOH].Conclusions and clinical importanceIn the SC, targeted markers of cholesterol oxidation were significantly increased in horses with eNAD/EDM

Metabolic Costs of a 58-minute Multi-Intensity Exercise Session with and Without Music and Cueing

International Journal of Exercise Science 13(2): 358-365, 2020. It is unclear if the presence or absence of music and cueing influence total energy expenditure (TEE) during a multi-intensity exercise program. The purpose of this study was to determine the difference between TEE with or without music and cueing during a 58-minute exercise session using heart rate estimation (HRe) and indirect calorimetry (IC). Using a randomized crossover design, 22 participants (6 males; 16 females; 27.64 ± 10.33 yrs.) were randomized into two groups (Group A = 11; Group B = 11). All participants performed the same 58-minute exercise session under two conditions: with music (WM) and without music and cueing (WOM). TEE was obtained through the Activio heart rate system for all 22 participants. TEE and excess post-exercise oxygen consumption (EPOC) were also obtained in a subset of eight participants (4 males; 4 females; 28.25 ± 5.9 yrs.) via IC through a ParvoMedics metabolic cart. Paired samples t-tests were performed to compare TEE between conditions using HRe and IC. Statistical analysis was performed using IBM Analytics, SPSS v24 with significance set at p\u3c 0.05. A significant difference (p= 0.008) was found between TEE WM and WOM using IC (475.74 ± 98.50 vs. 429.37 ± 121.42), but not between TEE WM and WOM using HRe (p= 2.04; 482.67 ± 151.79 vs. 452.90 ± 164.59). The presence of music and cueing increased TEE when monitored via IC, but not when measured via wearable heart rate technology. Music and cueing does aid in additional caloric expenditure

Voluntary Medical Male Circumcision: A Cross-Sectional Study Comparing Circumcision Self-Report and Physical Examination Findings in Lesotho

BACKGROUND: Overwhelming evidence, including three clinical trials, shows that male circumcision (MC) reduces the risk of HIV infection among men. However, data from recent Lesotho Demographic and Health Surveys do not demonstrate MC to be protective against HIV. These contradictory findings could partially be due to inaccurate self-reported MC status used to estimate MC prevalence. This study describes MC characteristics among men applying for Lesotho Defence Force recruitment and seeks to assess MC self-reported accuracy through comparison with physical-examination-based data. METHODS AND FINDINGS: During Lesotho Defence Force applicant screening in 2009, 241 (77%) of 312 men, aged 18-25 y, consented to a self-administered demographic and MC characteristic survey and physician-performed genital examination. The extent of foreskin removal was graded on a scale of 1 (no evidence of MC) to 4 (complete MC). MC was self-reported by 27% (n = 64/239) of participants. Of the 64 men self-reporting being circumcised, physical exam showed that 23% had no evidence of circumcision, 27% had partial circumcision, and 50% had complete circumcision. Of the MCs reportedly performed by a medical provider, 3% were Grade 1 and 73% were Grade 4. Of the MCs reportedly performed by traditional circumcisers, 41% were Grade 1, while 28% were Grade 4. Among participants self-reporting being circumcised, the odds of MC status misclassification were seven times higher among those reportedly circumcised by initiation school personnel (odds ratio = 7.22; 95% CI = 2.29-22.75). CONCLUSIONS: Approximately 27% of participants self-reported being circumcised. However, only 50% of these men had complete MC as determined by a physical exam. Given this low MC self-report accuracy, countries scaling up voluntary medical MC (VMMC) should obtain physical-exam-based MC data to guide service delivery and cost estimates. HIV prevention messages promoting VMMC should provide comprehensive education regarding the definition of VMMC

Efficacy and safety of emtricitabine/tenofovir alafenamide (FTC/TAF) vs. emtricitabine/tenofovir disoproxil fumarate (FTC/TDF) as a backbone for treatment of HIV-1 infection in virologically suppressed adults: subgroup analysis by third agent of a randomized, double-blind, active-controlled phase 3 trial*

Background: FTC/TAF was shown to be noninferior to FTC/TDF with advantages in markers of renal and bone safety.Objective: To evaluate the efficacy and safety of switching to FTC/TAF from FTC/TDF by third agent (boosted protease inhibitor [PI] vs. unboosted third agent).Methods: We conducted a 48-week subgroup analysis based on third agent from a randomized, double blind study in virologically suppressed adults on a FTC/TDF-containing regimen who switched to FTC/TAF vs. continued FTC/TDF while remaining on the same third agent.Results: We randomized (1:1) 663 participants to either switch to FTC/TAF (N = 333) or continue FTC/TDF (N = 330), each with baseline third agent stratifying by class of third agent in the prior treatment regimen (boosted PI 46%, unboosted third agent 54%). At week 48, significant differences in renal biomarkers and bone mineral density were observed favoring FTC/TAF over FTC/TDF (p < 0.05 for all), with similar improvements in the FTC/TAF arm in those who received boosted PI vs. unb..

Tissue-specific variation in DNA methylation levels along human chromosome 1

<p>Abstract</p> <p>Background</p> <p>DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters.</p> <p>Results</p> <p>Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and <it>vice versa</it>, in most pairs of tissues examined.</p> <p>Conclusion</p> <p>The varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.</p

Chromatin and siRNA pathways cooperate to maintain DNA methylation of small transposable elements in Arabidopsis

BACKGROUND: DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs. However, the relationship between different cytosine methylation pathways and their preferred targets is not known. RESULTS: We used a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci. We found that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that correspond to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants. CONCLUSION: We conclude that the targets of both DNA methylation and histone H3K9 methylation pathways are transposable elements genome-wide, irrespective of element type and position. Our findings also suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism alone. Thus, parallel pathways would be needed to maintain silencing of transposable elements

Loss associated with subtractive health service change: The case of specialist cancer centralization in England

OBJECTIVE: Major system change can be stressful for staff involved and can result in 'subtractive change' - that is, when a part of the work environment is removed or ceases to exist. Little is known about the response to loss of activity resulting from such changes. Our aim was to understand perceptions of loss in response to centralization of cancer services in England, where 12 sites offering specialist surgery were reduced to four, and to understand the impact of leadership and management on enabling or hampering coping strategies associated with that loss. METHODS: We analysed 115 interviews with clinical, nursing and managerial staff from oesophago-gastric, prostate/bladder and renal cancer services in London and West Essex. In addition, we used 134 hours of observational data and analysis from over 100 documents to contextualize and to interpret the interview data. We performed a thematic analysis drawing on stress-coping theory and organizational change. RESULTS: Staff perceived that, during centralization, sites were devalued as the sites lost surgical activity, skills and experienced teams. Staff members believed that there were long-term implications for this loss, such as in retaining high-calibre staff, attracting trainees and maintaining autonomy. Emotional repercussions for staff included perceived loss of status and motivation. To mitigate these losses, leaders in the centralization process put in place some instrumental measures, such as joint contracting, surgical skill development opportunities and trainee rotation. However, these measures were undermined by patchy implementation and negative impacts on some individuals (e.g. increased workload or travel time). Relatively little emotional support was perceived to be offered. Leaders sometimes characterized adverse emotional reactions to the centralization as resistance, to be overcome through persuasion and appeals to the success of the new system. CONCLUSIONS: Large-scale reorganizations are likely to provoke a high degree of emotion and perceptions of loss. Resources to foster coping and resilience should be made available to all organizations within the system as they go through major change

Participation in the Georgia Food for Health program and cardiovascular disease risk factors: A longitudinal observational study

Abstract Objective To assess the relationship between program attendance in a produce prescription program and changes in cardiovascular risk factors. Design The Georgia Food for Health (GF4H) program provided 6 monthly nutrition education sessions, 6 weekly cooking classes, and weekly produce vouchers. Participants became program graduates attending at least 4 of the 6 of both the weekly cooking classes and monthly education sessions. We used a longitudinal, single-arm approach to estimate the association between the number of monthly program visits attended and changes in health indicators. Setting GF4H was implemented in partnership with a large safety-net health system in Atlanta, GA. Participants 331 participants living with or at-risk of chronic disease and food insecurity were recruited from primary care clinics. Over three years, 282 participants graduated from the program. Results After adjusting for program site, year, participant sex, age, race & ethnicity, SNAP participation, and household size, we estimated that each additional program visit attended beyond 4 visits was associated with a 0.06 kg/m2 reduction in BMI (95% CI: -0.12, -0.01; p=0.02), a 0.37 inch reduction in waist circumference (95% CI: -0.48, -0.27; p<0.001), a 1.01 mmHg reduction in systolic blood pressure (95% CI: -1.45, -0.57; p<0.001), and a 0.43 mmHg reduction in diastolic blood pressure (95% CI: -0.69, -0.17; p=0.001). Conclusions Each additional cooking and nutrition education visit attended beyond the graduation threshold was associated with modest but significant improvements in cardiovascular disease risk factors, suggesting that increased engagement in educational components of a produce prescription program improves health outcomes