Construction and validation of the APOCHIP, a spotted oligo-microarray for the study of beta-cell apoptosis

BACKGROUND: Type 1 diabetes mellitus (T1DM) is a autoimmune disease caused by a long-term negative balance between immune-mediated beta-cell damage and beta-cell repair/regeneration. Following immune-mediated damage the beta-cell fate depends on several genes up- or down-regulated in parallel and/or sequentially. Based on the information obtained by the analysis of several microarray experiments of beta-cells exposed to pro-apoptotic conditions (e.g. double stranded RNA (dsRNA) and cytokines), we have developed a spotted rat oligonucleotide microarray, the APOCHIP, containing 60-mer probes for 574 genes selected for the study of beta-cell apoptosis. RESULTS: The APOCHIP was validated by a combination of approaches. First we performed an internal validation of the spotted probes based on a weighted linear regression model using dilution series experiments. Second we profiled expression measurements in ten dissimilar rat RNA samples for 515 genes that were represented on both the spotted oligonucleotide collection and on the in situ-synthesized 25-mer arrays (Affymetrix GeneChips). Internal validation showed that most of the spotted probes displayed a pattern of reaction close to that predicted by the model. By using simple rules for comparison of data between platforms we found strong correlations (r(median)= 0.84) between relative gene expression measurements made with spotted probes and in situ-synthesized 25-mer probe sets. CONCLUSION: In conclusion our data suggest that there is a high reproducibility of the APOCHIP in terms of technical replication and that relative gene expression measurements obtained with the APOCHIP compare well to the Affymetrix GeneChip. The APOCHIP is available to the scientific community and is a useful tool to study the molecular mechanisms regulating beta-cell apoptosis

How can surgical skills in laparoscopic colon surgery be objectively assessed?—a scoping review

BACKGROUND: In laparoscopic colorectal surgery, higher technical skills have been associated with improved patient outcome. With the growing interest in laparoscopic techniques, pressure on surgeons and certifying bodies is mounting to ensure that operative procedures are performed safely and efficiently. The aim of the present review was to comprehensively identify tools for skill assessment in laparoscopic colon surgery and to assess their validity as reported in the literature. METHODS: A systematic search was conducted in EMBASE and PubMed/MEDLINE in May 2021 to identify studies examining technical skills assessment tools in laparoscopic colon surgery. Available information on validity evidence (content, response process, internal structure, relation to other variables, and consequences) was evaluated for all included tools. RESULTS: Fourteen assessment tools were identified, of which most were procedure-specific and video-based. Most tools reported moderate validity evidence. Commonly not reported were rater training, assessment correlation with variables other than training level, and validity reproducibility and reliability in external educational settings. CONCLUSION: The results of this review show that several tools are available for evaluation of laparoscopic colon cancer surgery, but few authors present substantial validity for tool development and use. As we move towards the implementation of new techniques in laparoscopic colon surgery, it is imperative to establish validity before surgical skill assessment tools can be applied to new procedures and settings. Therefore, future studies ought to examine different aspects of tool validity, especially correlation with other variables, such as patient morbidity and pathological reports, which impact patient survival. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00464-021-08914-z

Perceptions about screening for prostate cancer using genetic lifetime risk assessment: a qualitative study

Background Most health authorities do not recommend screening for prostate cancer with PSA tests in asymptomatic patients who are not at increased risk. However, opportunistic screening for prostate cancer is still wanted by many patients and it is widely used in primary care clinics, with potential for overdiagnosis and overtreatment. Better tools for risk assessment have been called for, to better target such opportunistic screening. Our aim was to explore perceptions about prostate cancer risk and subsequent opportunistic screening among patients who were not at increased risk of prostate cancer after a first PSA test plus a genetic lifetime risk assessment. Methods We undertook semi-structured patient interviews with recording and verbatim transcription of interviews. Data were analysed thematically. Results Three themes were identified: uncertainty of the nature of prostate cancer; perceived benefits of testing; and conflicting public health recommendations. Prostate cancer was spoken of as an inescapable risk in older age. The aphorism “you die with it, not from it” was prominent in the interviews but patients focused on the benefits of testing now rather than the future risks associated with treatment relating to potential overdiagnosis. Many expressed frustration with perceived mixed messages about early detection of cancer, in which on one side men feel that they are encouraged to seek medical testing to act responsibly regarding the most common cancer disease in men, and on the other side they are asked to refrain from opportunistic testing for prostate cancer. Taken together, personal risks of prostate cancer were perceived as high in spite of a normal PSA test and a genetic lifetime risk assessment showing no increased risk. Conclusion Patients saw prostate cancer risk as high and increasing with age. They focused on the perceived benefit of early detection using PSA testing. It was also commonly acknowledged that most cases are indolent causing no symptoms and not shortening life expectancy. There was a frustration with mixed messages about the benefit of early detection and risk of overdiagnosis. These men’s genetic lifetime risk assessment showing no increased risk did not appear to influence current intentions to get PSA testing in the future

Functional analysis of the mismatch repair system in bladder cancer

In bladder cancer the observed microsatellite instability indicates that mismatch repair deficiency could be a frequently involved factor in bladder cancer progression. To investigate this hypothesis we analysed extracts of seven bladder cancer cell lines and, as a novel approach, five clinical cancer samples for mismatch repair activity. We found that one cell line (T24) and three of the clinical samples had a reduced repair capacity, measured to ~20% or less. The T24 cell extract was unable to repair a G-G mismatch and showed reduced repair of a 2-base loop, consistent with diminished function of the MSH2-MSH6 heterodimer. The functional assay was combined with measurement for mutation frequency, microsatellite analysis, sequencing, MTT assay, immunohistochemical analysis and RT-PCR analysis of the mismatch repair genes MSH2, MSH3, MSH6, PMS1, PMS2 and MLH1. A >7-fold relative increase in mutation frequency was observed for T24 compared to a bladder cancer cell line with a fully functional mismatch repair system. Neither microsatellite instability, loss of repair nor mismatch repair gene mutations were detected. However, RT-PCR analysis of mRNA levels did detect changes in the ratio of expression of the Mut S and Mut L homologues. The T24 cell line had the lowest MSH6 expression level of the cell lines tested. Identical RT-PCR analysis of seventeen clinical samples (normal urothelium, 7; pTa low stage, 5; and pT1-4 high stage, 5) indicated a significant change in the expression ratio between MSH3/MSH6 (P< 0.004), MSH2/MSH3 (P< 0.012) and PMS2/MLH1 P< 0.005, in high stage bladder tumours compared to normal urothelium and low stage tumours. Collectively, the data suggest that imbalanced expression of mismatch repair genes could lead to partial loss of mismatch repair activity that is associated with invasive bladder cancer. © 2001 Cancer Research Campaign http://www.bjcancer.co