Development of a real-time quantitative PCR assay for detection of a stable genomic region of BK virus

<p>Abstract</p> <p>Background</p> <p>BK virus infections can have clinically significant consequences in immunocompromised individuals. Detection and monitoring of active BK virus infections in certain situations is recommended and therefore PCR assays for detection of BK virus have been developed. The performance of current BK PCR detection assays is limited by the existence of viral polymorphisms, unknown at the time of assay development, resulting in inconsistent detection of BK virus. The objective of this study was to identify a stable region of the BK viral genome for detection by PCR that would be minimally affected by polymorphisms as more sequence data for BK virus becomes available.</p> <p>Results</p> <p>Employing a combination of techniques, including amino acid and DNA sequence alignment and interspecies analysis, a conserved, stable PCR target region of the BK viral genomic region was identified within the VP2 gene. A real-time quantitative PCR assay was then developed that is specific for BK virus, has an analytical sensitivity of 15 copies/reaction (450 copies/ml) and is highly reproducible (CV ≤ 5.0%).</p> <p>Conclusion</p> <p>Identifying stable PCR target regions when limited DNA sequence data is available may be possible by combining multiple analysis techniques to elucidate potential functional constraints on genomic regions. Applying this approach to the development of a real-time quantitative PCR assay for BK virus resulted in an accurate method with potential clinical applications and advantages over existing BK assays.</p

Mechanisms underlying the frequency dependence of contraction and [Ca2+]i transients in mouse ventricular myocytes

In most mammalian species force of contraction of cardiac muscle increases with increasing rate of stimulation, i.e. a positive force-frequency relationship. In single mouse ventricular cells, both positive and negative relationships have been described and little is known about the underlying mechanisms. We studied enzymatically isolated single ventricular mouse myocytes, at 30 °C. During field stimulation, amplitude of unloaded cell shortening increased with increasing frequency of stimulation (0.04 ± 0.01 ΔL/L0 at 1 Hz to 0.07 ± 0.01 ΔL/L0 at 4 Hz, n = 12, P < 0.05). During whole cell voltage clamp with 50 μM [K5-fluo-3]pip, both peak and baseline [Ca2+]i increased at higher stimulation frequencies, but the net Δ[Ca2+]i increased only modestly from 1.59 ± 0.08 ΔF/F0 at 1 Hz, to 1.71 ± 0.11 ΔF/F0 at 4 Hz (n = 17, P < 0.05). When a 1 s pause was interposed during stimulation at 2 and 4 Hz, [Ca2+]i transients were significantly larger (at 4 Hz, peak F/F0 increased by 78 ± 2 %, n = 5). SR Ca2+ content assessed during caffeine application, significantly increased from 91 ± 24 μmol l−1 at 1 Hz to 173 ± 20 μmol l−1 at 4 Hz (n = 5, P < 0.05). Peak ICa,L decreased at higher frequencies (by 28 ± 6 % at 2 Hz, and 45 ± 8 % at 4 Hz), due to slow recovery from inactivation. This loss of ICa,L resulted in reduced fractional release. Thus, in mouse ventricular myocytes the [Ca2+]i-frequency response depends on a balance between the increase in SR content and the loss of trigger ICa,L. Small changes in this balance may contribute to variability in frequency-dependent behaviour. In addition, there may be a regulation of the contractile response downstream of [Ca2+]i

Evaluation of Therapeutics for Severely Debilitating or Life‐Threatening Diseases or Conditions: Defining Scope to Enable Global Guidance Development

A significant regulatory gap exists to facilitate global development of therapeutics for nononcology severely debilitating or life-threatening diseases or conditions (SDLTs). In a 2017 publication, a streamlined approach to the development of treatments for SDLTs was proposed to facilitate earlier and continued patient access to new, potentially beneficial therapeutics.1 However, a major hindrance to broad adoption of this streamlined approach has been the lack of universally accepted, objective criteria to define SDLTs. This article serves to extend the 2017 publication by further addressing the challenge of defining SDLT scope in order to stimulate broader discussion and facilitate development of regional and ultimately international guidelines on the development of therapeutics for SDLTs. Using case examples, we describe key attributes of SDLTs and provide criteria for consideration of an SDLT scope definition

Evaluation of Therapeutics for Severely Debilitating or Life-Threatening Diseases or Conditions: Defining Scope to Enable Global Guidance Development

A significant regulatory gap exists to facilitate global development of therapeutics for nononcology severely debilitating or life-threatening diseases or conditions (SDLTs). In a 2017 publication, a streamlined approach to the development of treatments for SDLTs was proposed to facilitate earlier and continued patient access to new, potentially beneficial therapeutics.1 However, a major hindrance to broad adoption of this streamlined approach has been the lack of universally accepted, objective criteria to define SDLTs. This article serves to extend the 2017 publication by further addressing the challenge of defining SDLT scope in order to stimulate broader discussion and facilitate development of regional and ultimately international guidelines on the development of therapeutics for SDLTs. Using case examples, we describe key attributes of SDLTs and provide criteria for consideration of an SDLT scope definition

Changing Attitudes Toward Influenza Vaccination in U.S. Kidney Transplant Programs Over the Past Decade

Background and objectives: Influenza infection in transplant recipients is often associated with significant morbidity. Surveys were conducted in 1999 and 2009 to find out if the influenza vaccination practices in the U.S. transplant programs had changed over the past 10 years