RAPID THERMOCYCLER SYSTEM FOR RAPID AMPLIFICATION OF NUCLEIC ACIDS AND RELATED METHODS

A thermo cycling device and method of operating a thermocycler instrument, the instrument including a sample holder, at least one thermal cycling element, and at least one first and second temperature sensors, for causing the sample holder containing the at least one sample to undergo polymerase chain reaction amplification by repeated cycling between at least a denaturation heating stage and an aunealing cooling stage. The first temperature corresponding with the temperature of the sample holder is monitored using the at least one first temperature sensor, and a second temperature corresponding with the temperature external of the sample holder is monitored using the at least one second temperature sensor. Based upon the first temperature and the second temperature, the power that is delivered to the at least one thermal cycling element of the instrument is dynamically controlled

Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler

Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653 bp) and pUC19 β-lactamase (929 bp) in under 20 min. However, rapid integrated PCA–PCR was found to be problematic when attempted with the TM-1 gene (1509 bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers

Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler

Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653 bp) and pUC19 β-lactamase (929 bp) in under 20 min. However, rapid integrated PCA–PCR was found to be problematic when attempted with the TM-1 gene (1509 bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers

Perceived Injustice and Chronic Pain Acceptance: Are We Measuring Two Separate Constructs or Opposite Poles of the Same Dimension?

Perceived injustice has recently emerged as an important construct in the chronic pain literature. Perceived injustice has been shown to be a risk factor for various problematic pain outcomes, such as increased pain severity, depressive symptoms, and pain-related disability. At present, research on perceived injustice in chronic pain is lacking a theoretical model to facilitate understanding of its influence on chronic pain outcomes. It has been suggested that it might be useful to conceptualize perceived injustice within the psychological flexibility model of chronic pain.Indeed, there is preliminary evidencethatperceived injustice is negatively related to chronic pain acceptance, which is an important process within this model; however, the nature of this association is uncertain at present. In particular, it is unclear whether current measures of perceived injustice and chronic acceptance simplyreflect different poles of the same dimension, or theoretically separate, but related, constructs. This study aims to further examine the relation between perceived injustice and pain acceptance. The sample consisted of 847adults who suffer from chronic pain. Several competing measurement models were tested by means of confirmatory factor analysis. Results indicate that these two constructs appear to be two closely related constructs rather than two opposite poles of the same dimension. Implications of these findings for future research will be discussed

Experimental Validation of a Fundamental Model for PCR Efficiency

Recently a theoretical analysis of PCR efficiency has been published by Booth et al., (2010). The PCR yield is the product of three efficiencies: (i) the annealing efficiency is the fraction of templates that form binary complexes with primers during annealing, (ii)the polymerase binding efficiency is the fraction of binary complexes that bind to polymerase to form ternary complexes and (iii)the elongation efficiency is the fraction of ternary complexes that extend fully. Yield is controlled by the smallest of the three efficiencies and control could shift from one type of efficiency to another over the course of a PCR experiment. Experiments have been designed that are specifically controlled by each one of the efficiencies and the results are consistent with the mathematical model. The experimental data has also been used to quantify six key parameters of the theoretical model. An important application of the fully characterized model is to calculate initial template concentration from real-time PCR data. Given the PCR protocol, the midpoint cycle number (where the template concentration is half that of the final concentration) can be theoretically determined and graphed for a variety of initial DNA concentrations. Real-time results can be used to calculate the midpoint cycle number and consequently the initial DNA concentration, using this graph. The application becomes particularly simple if a conservative PCR protocol is followed where only the annealing efficiency is controlling

Efficiency of the Polymerase Chain Reaction

The polymerase chain reaction (PCR) has found wide application in biochemistry and molecular biology such as gene expression studies, mutation detection, forensic analysis and pathogen detection. Increasingly quantitative real time PCR is used to assess copy numbers from overall yield. In this study the yield is analyzed as a function of several processes: (1) thermal damage of the template and polymerase occurs during the denaturing step, (2) competition exists between primers and templates to either anneal or form dsDNA, (3) polymerase binding to annealed products (primer/ssDNA) to form ternary complexes and (4) extension of ternary complexes. Explicit expressions are provided for the efficiency of each process, therefore reaction conditions can be directly linked to the overall yield. Examples are provided where different processes play the yield-limiting role. The analysis will give researchers a unique understanding of the factors that control the reaction and will aid in the interpretation of experimental results

The enigmatic core L1451-mm: a first hydrostatic core? or a hidden VeLLO?

We present the detection of a dust continuum source at 3-mm (CARMA) and 1.3-mm (SMA), and 12CO(2-1) emission (SMA) towards the L1451-mm dense core. These detections suggest a compact object and an outflow where no point source at mid-infrared wavelengths is detected using Spitzer. An upper limit for the dense core bolometric luminosity of 0.05 Lsun is obtained. By modeling the broadband SED and the continuum interferometric visibilities simultaneously, we confirm that a central source of heating is needed to explain the observations. This modeling also shows that the data can be well fitted by a dense core with a YSO and disk, or by a dense core with a central First Hydrostatic Core (FHSC). Unfortunately, we are not able to decide between these two models, which produce similar fits. We also detect 12CO(2-1) emission with red- and blue-shifted emission suggesting the presence of a slow and poorly collimated outflow, in opposition to what is usually found towards young stellar objects but in agreement with prediction from simulations of a FHSC. This presents the best candidate, so far, for a FHSC, an object that has been identified in simulations of collapsing dense cores. Whatever the true nature of the central object in L1451-mm, this core presents an excellent laboratory to study the earliest phases of low-mass star formation.Comment: 15 pages, 9 figures, emulateapj. Accepted by Ap

Subjective Cognitive Decline Higher Among Sexual and Gender Minorities in the United States, 2015–2018

Introduction: Subjective cognitive decline (SCD) represents self-reported problems with memory, a possible early sign of dementia. Little is known about SCD among sexual and gender minority (SGM) adults who identify as lesbian, gay, bisexual, and/or transgender or gender non-binary. Methods: Data were weighted to represent population estimates from 25 states’ 2015–2018 Behavioral Risk Factor Surveillance System to describe SCD in adults ≥ 45 years by SGM status. Logistic regression tested associations between demographic and health conditions. Results: SCD prevalence was higher in SGM (15.7%; 95% confidence interval [CI]:13.1–18.2) than in non-SGM adults (10.5%; 95% CI:10.1–10.9; P \u3c .0001). SGM adults with SCD were also more likely to report functional limitations due to SCD than non-SGM adults with SCD, 60.8% versus 47.8%, P =.0048. Differences in SCD by SGM status were attenuated after accounting for depression. Discussion: Higher prevalence of SCD in SGM adults highlights the importance of ensuring inclusive screenings, interventions, care services, and resources for SGM adults

ESD Terms and Definitions (Version 12)

Basic terms related to engineering system

Vestibular Rehabilitation for Peripheral Vestibular Hypofunction: An Evidence-Based Clinical Practice Guideline: From the American Physical Therapy Association Neurology Section

Background: Uncompensated vestibular hypofunction results in postural instability, visual blurring with head movement, and subjective complaints of dizziness and/or imbalance. We sought to answer the question, \ Is vestibular exercise effective at enhancing recovery of function in people with peripheral (unilateral or bilateral) vestibular hypofunction?\ Methods: A systematic review of the literature was performed in 5 databases published after 1985 and 5 additional sources for relevant publications were searched. Article types included meta-analyses, systematic reviews, randomized controlled trials, cohort studies, case control series, and case series for human subjects, published in English. One hundred thirty-five articles were identified as relevant to this clinical practice guideline. Results/Discussion: Based on strong evidence and a preponderance of benefit over harm, clinicians should offer vestibular rehabilitation to persons with unilateral and bilateral vestibular hypofunction with impairments and functional limitations related to the vestibular deficit. Based on strong evidence and a preponderance of harm over benefit, clinicians should not include voluntary saccadic or smooth-pursuit eye movements in isolation (ie, without head movement) as specific exercises for gaze stability. Based on moderate evidence, clinicians may offer specific exercise techniques to target identified impairments or functional limitations. Based on moderate evidence and in consideration of patient preference, clinicians may provide supervised vestibular rehabilitation. Based on expert opinion extrapolated from the evidence, clinicians may prescribe a minimum of 3 times per day for the performance of gaze stability exercises as 1 component of a home exercise program. Based on expert opinion extrapolated from the evidence (range of supervised visits: 2-38 weeks, mean = 10 weeks), clinicians may consider providing adequate supervised vestibular rehabilitation sessions for the patient to understand the goals of the program and how to manage and progress themselves independently. As a general guide, persons without significant comorbidities that affect mobility and with acute or subacute unilateral vestibular hypofunction may need once a week supervised sessions for 2 to 3 weeks; persons with chronic unilateral vestibular hypofunction may need once a week sessions for 4 to 6 weeks; and persons with bilateral vestibular hypofunction may need once a week sessions for 8 to 12 weeks. In addition to supervised sessions, patients are provided a daily home exercise program. Disclaimer: These recommendations are intended as a guide for physical therapists and clinicians to optimize rehabilitation outcomes for persons with peripheral vestibular hypofunction undergoing vestibular rehabilitation