Genetic and Non-genetic Predictors of LINE-1 Methylation in Leukocyte DNA.

Background: Altered DNA methylation has been associated with various diseases. Objective: We evaluated the association between levels of methylation in leukocyte DNA at long interspersed nuclear element 1 (LINE-1) and genetic and non-genetic characteristics of 892 control participants from the Spanish Bladder Cancer/EPICURO study. Methods: We determined LINE-1 methylation levels by pyrosequencing. Individual data included demographics, smoking status, nutrient intake, toenail concentrations of 12 trace elements, xenobiotic metabolism gene variants, and 515 polymorphisms among 24 genes in the one-carbon metabolism pathway. To assess the association between LINE-1 methylation levels (percentage of methylated cytosines) and potential determinants, we estimated beta coefficients (βs) by robust linear regression. Results: Women had lower levels of LINE-1 methylation than men (β = –0.7, p = 0.02). Persons who smoked blond tobacco showed lower methylation than nonsmokers (β = –0.7, p = 0.03). Arsenic toenail concentration was inversely associated with LINE-1 methylation (β = –3.6, p = 0.003). By contrast, iron (β = 0.002, p = 0.009) and nickel (β = 0.02, p = 0.004) were positively associated with LINE-1 methylation. Single nucleotide polymorphisms (SNPs) in DNMT3A (rs7581217-per allele, β = 0.3, p = 0.002), TCN2 (rs9606756-GG, β = 1.9, p = 0.008; rs4820887-AA, β = 4.0, p = 4.8 × 10–7; rs9621049-TT, β = 4.2, p = 4.7 × 10–9), AS3MT (rs7085104-GG, β = 0.7, p = 0.001), SLC19A1 (rs914238, TC vs. TT: β = 0.5 and CC vs. TT: β = –0.3, global p = 0.0007) and MTHFS (rs1380642, CT vs. CC: β = 0.3 and TT vs. CC; β = –0.8, global p = 0.05) were associated with LINE-1 methylation. Conclusions: We identified several characteristics, environmental factors, and common genetic variants that predicted DNA methylation among study participants.This work was partially supported by the Association for International Cancer Research (AICR; grant 09-0780, and a doctoral scholarship awarded to S.M.T.); Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, MINECO, Spain (grants 00/0745, PI051436, PI061614, PI09-02102, and G03/174); Red Temática de Investigación Cooperativa en Cáncer (grant RD06/0020-RTICC); the U.S. National Institutes of Health (grant RO1-CA089715); a postdoctoral fellowship awarded to A.F.S.A. from the Fundación Científica de la AECC; Fundació Marató TV3; and The Johns Hopkins University Vredenburg Scholarship awarded to A.L.C

The cloning and characterization of the human transcobalamin II gene

Transcobalamin II (TCII) is a plasma protein that binds vitamin B12 (cobalamin; Cbl) and facilitates the cellular uptake of the vitamin by receptor-mediated endocytosis. In genetic disorders that are characterized by congenital deficiency of TCII, intracellular Cbl deficiency occurs, resulting in an early onset of megaloblastic anemia that is sometimes accompanied by a neurologic disorder. To define the genetic basis for TCII deficiency, we have cloned and characterized the human gene that encodes this protein. The gene spans a minimum of 18 kbp and contains nine exons and eight introns, with a polyadenylation signal sequence located 509 bp downstream from the termination codon and a transcription initiation site beginning 158 bp upstream from the ATG translation start site. The 5′ flanking DNA does not have a TATA or CCAAT regulatory element, but a 34-nucleotide stretch beginning just upstream of the CAP site contains four tandemly organized 5′-CCCC-3′ tetramers. This sequence is a motif for a trans-active transcription factor (ETF) that regulates expression of the epidermal growth factor receptor gene (EGFR), which also lacks TATA and CCAAT regulatory elements. A GC-rich sequence that binds the SP1 protein is located 356 nucleotides upstream from the first of the series of CCCC tetramers. Although this GC sequence is at an unusual location with respect to the CAP site, a 507-bp fragment containing this GC box drives the chloramphenicol acetyltransferase (CAT) reporter gene after transient transfection into NIH 3T3 cells. No CAT activity was observed when a 420-bp fragment lacking this GC box but containing the ETF-binding domains was similarly transfected into this cell line. One consensus and two atypical motifs for the c-myc ligand are located downstream and upstream, respectively, of the GC box, and this could explain the elevated plasma TCII observed in some patients with multiple myeloma, as the c-myc product is overexpressed in some myeloma cells. Restriction endonuclease digestion of genomic DNA from eight normal subjects with Taq I, Hinfl, Msp I, and Bgl I identified three patterns of restriction fragment length polymorphism (RFLP). A number of the exon/intron splice junctions of human TCII, TCI, and IF genes are located in homologous regions of these proteins, providing evidence that these genes have evolved by duplication of an ancestral gene. This characterization of the TCII gene and the RFLP should facilitate the identification of the mutation(s) responsible for the genetic abnormalities of TCII expression.</jats:p

Exploring vibroacoustic therapy in adults experiencing pain: a scoping review

Objective To explore the characteristics and outcomes of vibroacoustic therapy (VAT) in adults experiencing pain. To give directions for future research and clinical applications of VAT in pain management for adults.Design Scoping review.Data sources BMČ, CINAHL Plus, Cochrane Library, EBSCOhost, EBM Reviews, EMBASE, Epistemonikos, ERIC, MEDLINE complete, Scopus, Web of Science, Google Scholar, ProQuest, hand search in unpublished sources.Study selection All quantitative and qualitative research studies and systematic reviews, without any date or language limit.Data extraction Two independent reviewers extracted data on the study design, location and setting, the causes of pain, participants, vibroacoustic intervention, measurement tools, and key findings related to pain.Results From 430 records, 20 were included for narrative synthesis. Fifteen studies researched chronic pain, two studies acute pain, two studies both types of pain and one study experimentally induced pain. The description of VAT applied in studies usually included the description of research experiments, vibroacoustic devices and frequencies of sinusoidal sound. There was high heterogeneity in study protocols, however, 40 Hz was predominantly used, most sessions ranged between 20 and 45 min, and the frequency of treatment was higher for acute pain (daily) compared with chronic pain (daily to once a week). Outcomes related to pain focused mainly on perceived pain; however, other surrogate measures were also considered, for example, an increased number of treatment days or pain medication usage.Conclusions Research in this area is too sparse to identify properties of VAT that are beneficial for pain management. We suggest VAT researchers describe a minimum of four measurements—frequency, amplitude, pulsation and loudness. Randomised controlled trials are needed to establish reliable scientific proof of VAT effectiveness for both acute and chronic pain. Furthermore, clinical practice would benefit from researching patients’ experiences and preferences of vibroacoustic treatment and its psychosocial components

Exploring vibroacoustic therapy in adults experiencing pain: a scoping review

ObjectiveTo explore the characteristics and outcomes of vibroacoustic therapy (VAT) in adults experiencing pain. To give directions for future research and clinical applications of VAT in pain management for adults.DesignScoping review.Data sourcesBMČ, CINAHL Plus, Cochrane Library, EBSCOhost, EBM Reviews, EMBASE, Epistemonikos, ERIC, MEDLINE complete, Scopus, Web of Science, Google Scholar, ProQuest, hand search in unpublished sources.Study selectionAll quantitative and qualitative research studies and systematic reviews, without any date or language limit.Data extractionTwo independent reviewers extracted data on the study design, location and setting, the causes of pain, participants, vibroacoustic intervention, measurement tools, and key findings related to pain.ResultsFrom 430 records, 20 were included for narrative synthesis. Fifteen studies researched chronic pain, two studies acute pain, two studies both types of pain and one study experimentally induced pain. The description of VAT applied in studies usually included the description of research experiments, vibroacoustic devices and frequencies of sinusoidal sound. There was high heterogeneity in study protocols, however, 40 Hz was predominantly used, most sessions ranged between 20 and 45 min, and the frequency of treatment was higher for acute pain (daily) compared with chronic pain (daily to once a week). Outcomes related to pain focused mainly on perceived pain; however, other surrogate measures were also considered, for example, an increased number of treatment days or pain medication usage.ConclusionsResearch in this area is too sparse to identify properties of VAT that are beneficial for pain management. We suggest VAT researchers describe a minimum of four measurements—frequency, amplitude, pulsation and loudness. Randomised controlled trials are needed to establish reliable scientific proof of VAT effectiveness for both acute and chronic pain. Furthermore, clinical practice would benefit from researching patients’ experiences and preferences of vibroacoustic treatment and its psychosocial components.</jats:sec

Potential Early Rabies Infection Detected in Two Raccoon Cases by LN34 pan-lyssavirus real-time RT-PCR Assay in Pennsylvania

AbstractDuring 2017 – 2019, the Pennsylvania Department of Health Bureau of Laboratories (PABOL) tested 6,855 animal samples for rabies using both the gold standard direct fluorescent antibody (DFA) test and LN34 pan-lyssavirus reverse transcriptase quantitative PCR (RT-qPCR). Two samples (0.03 %) were identified as LN34 RT-qPCR positive after failure to detect rabies virus antigen during initial DFA testing: an adult raccoon collected in 2017 and a juvenile raccoon collected in 2019. After the positive PCR result, additional tissues were collected and re-tested by DFA, where very sparse, disperse antigen was observed. Tissues from both animals were submitted to the Centers for Disease Control and Prevention (CDC) for confirmatory testing, and were confirmed positive. At both PABOL and CDC, rabies virus antigen and RNA levels were much lower than for a typical rabies case. In addition, rabies virus antigen and RNA levels were higher in brain stem and rostral spinal cord than cerebellum, hippocampus and cortex. Cross-contamination was ruled out in the case of the 2019 juvenile raccoon by sequencing, as nucleoprotein and glycoprotein gene sequences displayed &gt;1% nucleotide differences to sequences from all positive samples processed at PABOL within two weeks of the juvenile raccoon. Taken together, the low level of rabies virus in the central nervous system combined with presence in more caudal brain structures suggest the possibility of an early infection in both cases. These two cases highlight the increased sensitivity and ease of interpretation of LN34 RT-qPCR in rabies diagnostics for the identification of low positive cases.</jats:p

Detection of Apparent Early Rabies Infection by LN34 Pan-Lyssavirus Real-Time RT-PCR Assay in Pennsylvania

The Pennsylvania Department of Health Bureau of Laboratories (PABOL) tested 6855 animal samples for rabies using both the direct fluorescent antibody test (DFA) and LN34 pan-lyssavirus reverse transcriptase quantitative PCR (RT-qPCR) during 2017–2019. Only two samples (0.03%) were initially DFA negative but positive by LN34 RT-qPCR. Both cases were confirmed positive upon re-testing at PABOL and confirmatory testing at the Centers for Disease Control and Prevention by LN34 RT-qPCR and DFA. Rabies virus sequences from one sample were distinct from all positive samples processed at PABOL within two weeks, ruling out cross-contamination. Levels of rabies virus antigen and RNA were low in all brain structures tested, but were higher in brain stem and rostral spinal cord than in cerebellum, hippocampus or cortex. Taken together, the low level of rabies virus combined with higher abundance in more caudal brain structures suggest early infection. These cases highlight the increased sensitivity and ease of interpretation of LN34 RT-qPCR for low positive cases