Analysis of the miR-34 family functions in breast cancer reveals annotation error of miR-34b.

The microRNAs in the miR-34 family, consisting of miR-34a, miR-34b and miR-34c, are tumour suppressors. The annotated human miR-34b-5p has one additional base at the 5' end of the common miR-34 family seed sequence, compared to miR-34a-5p and miR-34c-5p. This extra base results in a shift of the seed sequence, which would affect the target gene repertoire and have functional consequences. During our studies of miR-34 functions, we investigated the precise sequence of mature miR-34b-5p in human cells by deep sequencing. We found that a miR-34b-5p without the extra base was the predominant form in both non-malignant and malignant cells derived from several human tissues, indicating that the miR-34b annotation is misleading. We evaluated the functional implications of the seed shift, by comparing the effect of mimics representing the alternative miR-34b-5p sequences in MDA-MB-231 cells. In contrast to the annotated miR-34b, the endogenously expressed miR-34b displayed tumour suppressive characteristics in vitro similarly to miR-34c. These data demonstrate the importance of determining the precise sequence of a mature microRNA before exploring miRNA functions.

Technical improvement of human pancreatic islet isolation.

INTRODUCTION: A key factor for successful islet isolation is to place the optimal amount of enzyme into the pancreatic ducts prior to starting digestion of pancreatic glands. To improve this procedure, we introduced novel techniques to identify and repair tissue damage resulting in leakage of collagenase solution. MATERIALS AND METHODS: One hundred twelve standardized consecutive islet isolations were for the effects of dye and glue on islet yield, islet function using a perifusion assay, and the possibility of clinical transplantation. One group of pancreata (n = 26) obtained en bloc together with duodenum were carefully detached with ligation of accessory ducts in an isolation unit (WPD group), whereas the pancreata were dissected from the duodenum in the operating room in the other 86 isolations. In 28 of 86 isolations, whole glands were used (WP group), while only the body and tail area were applied in the remaining 58 isolations (PP group). RESULTS: Both dye and glue effectively prevented leakage of collagenase from the gland. Both islet yield and success rate were higher with these tools without adverse effects on islet function or collagenase activity. The success rate of isolations and islet yield were significantly higher in the WPD group (P = .02 and .001, respectively). CONCLUSIONS: Dye and glue may be useful tools to improve human islet isolation procedures. In addition, the use of the whole pancreas further improves the outcome.

Key factors for human islet isolation and clinical transplantation.

BACKGROUND: To further improve the outcome of clinical islet transplantation analysis of the impact of donor- and process-related factors could be of great importance. MATERIALS AND METHODS: Thirty-eight consecutive clinical islet transplantations were performed with consecutive islet isolations. Univariate analysis for donor- and isolation-related variables were correlated with recipient C-peptide levels at 2 and 4 weeks after transplantation. "Warm ischemia time" was defined as the time from start of University of Wisconsin solution perfusion in the donor until the pancreas was removed to the back table. RESULTS: Short "warm ischemia time" (WIT), low expression of tissue factor (TF) in pancreatic tissue, and high creatinine levels in the donor were variables related to high C-peptide values after islet transplantation. Furthermore, hospitalization length longer than 4 days was associated with low C-peptide levels. The number of islet equivalents (IEQ) did not correlate with the clinical outcome, possibly due to the fact that IEQ number was included in the release criteria for clinical islet transplantation CONCLUSIONS: Successful clinical islet transplantation is strongly correlated with donor and pancreas procurement factors rather than isolation process-related variables. "WIT" may induce TF expression in the pancreatic tissues. TF has been identified as the main trigger of the instant blood-mediated-inflammatory reaction in clinical islet transplantation. Therefore, assay of TF expression in pancreatic tissues could be applied as useful screening tool to identify "good" pancreata for clinical transplantation.