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Background VEGF-targeted therapy has become the mainstay of treatment for majority of mRCC patients. For most patients, benefit is short-lived and therefore treatment remains palliative in intent. HD IL2 is an effective immunotherapy treatment capable of durable remission in some patients but its unselected use has been difficult due to its modest response rate and considerable adverse effects. Using set pathology criteria as a selection tool in clinical practice, we have been able to show improved outcomes in our previous report. Here, we present an updated and extended report of this treatment and seek to explore any pathological, clinical and treatment variables likely to predict better outcomes.Methods This is an extension of a previously reported clinical audit, which includes mRCC cases treated with HD IL2 between 2003 and 2013. Since 2006, tumour specimens of potential candidates were routinely reviewed prospectively and stratified into Favourable or Other categories based on constitution of histological growth pattern, namely alveolar or solid versus papillary and/or sarcomatoid architecture; clear cell versus granular cell cytoplasmic morphology. HD IL2 was preferentially offered to patients with Favourable pathology. Outcome evaluation includes response rates, survival, and treatment tolerance. Multivariate analysis was performed to explore potential prognostic and predictive factors.Results Among prospectively selected patients with Favourable pathology (n = 106), overall response rate was 48.1 % (51/106) with CR rate of 21.6 % (23/106). Median OS was 58.1 months. Factors associated with significantly better response and/or survival includes favourable pathology pattern, higher cycle 1 tolerance and lower number of metastatic organ sites (

Manual loading of samples into horizontal gels, such as the agarose gels commonly used for DNA fragment sizing and quantification, is laborious and prone to errors. Manual-loading times for highthroughput gels can reach 10 min/gel, and human error can result in incorrect identification of samples because of reverse loading or other errors in the loading process. To reduce gel-loading times and to improve reliability, a novel comb has been developed that uses glass capillaries and hydrostatic pressure to simplify sample loading from microplates. Accurate sample metering is ensured by the uniform length and volume of the capillaries. The loaded comb is placed in the gel boat over a pre-cast agarose gel, and buffer is added to a reservoir at the top of the comb. Once the buffer rises over the ends of the capillaries, the samples are pushed into the wells by hydrostatic pressure. This technique was successfully demonstrated for a 24-lane comb. This capillary comb loader reduces loading time, maintains well-to-well uniformity, and retains the same geometry and appearance of manually loaded bands, making this loading method compatible with existing downstream processes and software for subsequent analysis of the gel image.

Background: MicroRNAs are a class of small (~22nt) endogenous RNAs that regulate target transcript expression post-transcriptionally. Previous studies characterized age-related changes in diurnal transcript expression but it is not understood how these changes are regulated, and whether they may be attributed in part to changes in microRNA expression or activity with age. Diurnal small RNA expression changes with age were not previously studied. Methods: To interrogate changes in small RNA expression with age, we collected young (5 day) and old (55 day) Drosophila melanogaster around-the-clock and performed deep sequencing on size-selected RNA from whole heads. Results: We found several microRNAs with changes in rhythmicity after aging, and we investigated microRNAs which are differentially expressed with age. We found that predicted targets of differentially expressed microRNAs have RNA-binding and transcription factor activity. We used a previously published method to identify mRNA transcripts which show evidence of microRNA targeting that is altered after aging, and found several that are involved in muscle development and maintenance. Finally, we identified novel microRNAs using the random-forest-based method miRWoods, which surprisingly also discovered transfer RNA-derived fragments. Conclusions: We showed a decrease in global microRNA expression and a corresponding increase in piRNA expression during aging. We also found an increase in rhythmicity of Drosophila small RNAs during aging, including microRNAs, piRNA clusters, and novel transfer RNA-derived fragments. To our knowledge this is the first study examining diurnal small RNA expression around the clock in young and old Drosophila, and as such it paves the way for future research on changes in small RNA regulatory molecules in the context of aging.