An Infinitely Expandable Cloning Strategy plus Repeat-Proof PCR for Working with Multiple shRNA

Vector construction with restriction enzymes (REs) typically involves the ligation of a digested donor fragment (insert) to a reciprocally digested recipient fragment (vector backbone). Creating a suitable cloning plan becomes increasingly difficult for complex strategies requiring repeated insertions such as constructing multiple short hairpin RNA (shRNA) expression vectors for RNA interference (RNAi) studies. The problem lies in the reduced availability of suitable RE recognition sites with an increasing number of cloning events and or vector size. This report details a technically simple, directional cloning solution using REs with compatible cohesive ends that are repeatedly destroyed and simultaneously re-introduced with each round of cloning. Donor fragments can be made by PCR or sub-cloned from pre-existing vectors and inserted ad infinitum in any combination. The design incorporates several cloning cores in order to be compatible with as many donor sequences as possible. We show that joining sub-combinations made in parallel is more time-efficient than sequential construction (of one cassette at a time) for any combination of 4 or more insertions. Screening for the successful construction of combinations using Taq polymerase based PCR became increasingly difficult with increasing number of repeated sequence elements. A Pfu polymerase based PCR was developed and successfully used to amplify combinations of up to eleven consecutive hairpin expression cassettes. The identified PCR conditions can be beneficial to others working with multiple shRNA or other repeated sequences, and the infinitely expandable cloning strategy serves as a general solution applicable to many cloning scenarios.     

Depression and bone mineral density: is there a relationship in elderly Asian men? Results from Mr. Os (Hong Kong)

Previous epidemiological study has suggested that depression might be associated with low bone mass in Caucasian women. This has not been studied in Asian men. Mr. Os (Hong Kong) is the first, large, cohort study on osteoporosis in Asian men, and the current analysis deals with the association between depression and bone mass in this group. Data from the baseline examination of Mr. Os (Hong Kong) were used. Two thousand Hong Kong men aged 65 to 92 years were recruited from the community. Depression was diagnosed by face-to-face interview, using a validated Chinese version of the Geriatric Depression Scale (GDS), with depression being defined as a cut-off score of 8 or more. Bone mineral density (BMD) of the lumbar spine, total hip and total body was measured by dual X-ray densitometry (DEXA) using the Hologic QDR-4500 W densitometer. Multiple regression was used to compare BMD in depressed and non-depressed subjects, controlling for confounding variables. In the study sample 8.5% of men were found to be depressed, and the BMD at the total hip in these subjects was 2.1% lower than in non-depressed subjects (95% CI –0.13 to –4.1), after adjustment for age, body weight, medical history, alcohol consumption, cigarette smoking, calcium intake, physical activity and antidepressant use. Depression was associated with a 1.4-fold (95% CI 1.00 to 2.08) relative risk (RR) of being diagnosed with a T-score equal to or less than –1.0 (low bone mass). We conclude that depression is associated with lower BMD; however, to determine whether depression causes lower BMD or vice versa, we will need to await findings from future prospective studies.Abstract presented at the 25th Annual Meeting of the American Society for Bone and Mineral Research on 21 September 2003, Minnesota, Minneapolis, USA     

Branched-chain amino acid in chronic renal failure patients: respiratory and sleep effects.

Sleep disorders, including a high incidence of sleep apnea, have been recognized as a significant problem in chronic renal failure (CRF) patients. In a preliminary study, we examined CRF patients on maintenance hemodialysis for three nights; one control night, and thereafter randomized to infusion of saline (placebo) for one night and 4% branch-chain amino acid (BCAA) solution for one night. Polysomnographic and respiratory data [respiratory rate, oxygen saturation and end-tidal CO2 (ETCO2)] was recorded continuously throughout the nights and data from each hour compared with baseline (awake) values. The patients studied were characterized by reduced sleep quality and decreased amount of rapid eye movement (REM) sleep. The BCAA infusion was associated with a return of REM sleep to normal and a significant decrease in ETCO2 during both REM and non-REM sleep (P less than 0.05). Our findings demonstrate respiratory stimulation during sleep with infusion of BCAA; this stimulatory effect on respiration (in contrast to many respiratory stimulants) is associated with an increased amount of REM sleep.     

Workshop: Internet Delivery of an Anticoagulation Therapy Management Certificate Program

Journal of Thrombosis and Thrombolysis -     

Proxies and Other External Raters: Methodological Considerations

Objective. The purpose of this paper is to introduce researchers to the measurement and subsequent analysis considerations involved when using externally rated data. We will define and describe two categories of externally rated data, recommend methodological approaches for analyzing and interpreting data in these two categories, and explore factors affecting agreement between self-rated and externally rated reports. We conclude with a discussion of needs for future research.
Data Sources/Study Setting. Data sources for this paper are previous published studies and reviews comparing self-rated with externally rated data.
Study Design/Data Collection/Extraction Methods. This is a psychometric conceptual paper.
Principal Findings. We define two types of externally rated data: proxy data and other-rated data. Proxy data refer to those collected from someone who speaks for a patient who cannot, will not, or is unavailable to speak for him or herself, whereas we use the term other-rater data to refer to situations in which the researcher collects ratings from a person other than the patient to gain multiple perspectives on the assessed construct. These two types of data differ in the way the measurement model is defined, the definition of the gold standard against which the measurements are validated, the analysis strategies appropriately used, and how the analyses are interpreted. There are many factors affecting the discrepancies between self- and external ratings, including characteristics of the patient, the proxy, and of the rated construct. Several psychological theories can be helpful in predicting such discrepancies.
Conclusions. Externally rated data have an important place in health services research, but use of such data requires careful consideration of the nature of the data and how it will be analyzed and interpreted.     

A comparison of social standards and perceived ability in anxious and nonanxious men

This study examined the discrepancy between self-established standards and self-efficacy in social situations. Socially anxious and nonanxious men rated a series of standards for judging the adequacy of their performance in an upcoming social interaction; subjects also rated their social self-efficacy, or perceived ability to handle the interaction. Nonanxious subjects expected their ability to equal or exceed all standards of evaluation. Anxious subjects expected their ability to equal that of the average subject and also believed their performance would match their personal standard. However, anxious subjects believed their social behavior would fall short of what they believed the experimenter expected of them. No support was found for the idea that socially anxious men establish perfectionistic standards. Rather, they believed that others held standards for them that they could not achieve. The results are congruent with cognitive theories of social anxiety.This article is based on a master's thesis by the first author under the supervision of the second author. Preparation of this article was supported by a grant to the second author from the Social Sciences and Humanities Research Council of Canada. The first author was supported by fellowships from the Social Sciences and Humanities Research Council of Canada.