Analysis of endo-siRNAs in Drosophila.

The small RNA silencing system is an important player in the control of gene expression. In particular analysis of the highly complex sequence repertoire of piRNAs and endogenous siRNAs directed against repetitive elements and transposons has been facilitated by the advent of next-generation sequencing tools. We are providing a detailed protocol for the creation of deep sequencing libraries in combination with a chemical modification step (periodate oxidation and ß-elimination) that enriches for 3'-terminal modified small RNAs. This step can thus facilitate the detection and-since incorporation of the small RNA into the effector complex precedes the terminal methylation-determine the Argonaute-loading state of certain small RNA species. Both, the oxidation as well as the deep sequencing library preparation are adaptations of popular, previously published protocols. The sequencing libraries described here can be sequenced on all flow cell types (i.e., single-end and paired-end) of the Illumina sequencing systems and are compatible with Illumina index reads. It is therefore also possible to analyze the libraries on the MiSeq system, for which currently only paired-end flow cells are available, and combine several libraries in multiplexed experiments. The chapter concludes with considerations concerning quality control and data analysis.

Hsp70, a messenger from hyperthermia for the immune system.

Heat shock proteins (Hsps) hold a dual role depending on their location. Inside cells, they fulfill essential survival functions as molecular chaperones forming complexes with intracellular polypeptides (self or foreign) to help in protein folding, the resolution of protein aggregates and intracellular protein transport. Released from the cell, they act as messengers communicating the cells' interior protein composition to the immune system for initiation of immune responses against intracellular proteins. Here we describe the mechanisms by which Hsp70, the heat-inducible Hsp70 family member, crosstalks with the immune system. Further, we discuss that clinical hyperthermia could be a way to initiate the immunologic activity of Hsp70 by upregulating its expression and facilitating release through local necrosis.