A new method to prevent carry-over contaminations in two-step PCR NGS library preparations.

Two-step PCR procedures are an efficient and well established way to generate amplicon libraries for NGS sequencing. However, there is a high risk of cross-contamination by carry-over of amplicons from first to second amplification rounds, potentially leading to severe misinterpretation of results. Here we describe a new method able to prevent and/or to identify carry-over contaminations by introducing the K-box, a series of three synergistically acting short sequence elements. Our K-boxes are composed of (i) K1 sequences for suppression of contaminations, (ii) K2 sequences for detection of possible residual contaminations and (iii) S sequences acting as separators to avoid amplification bias. In order to demonstrate the effectiveness of our method we analyzed two-step PCR NGS libraries derived from a multiplex PCR system for detection of T-cell receptor beta gene rearrangements. We used this system since it is of high clinical relevance and may be affected by very low amounts of contaminations. Spike-in contaminations are effectively blocked by the K-box even at high rates as demonstrated by ultra-deep sequencing of the amplicons. Thus, we recommend implementation of the K-box in two-step PCR-based NGS systems for research and diagnostic applications demanding high sensitivity and accuracy.

Treatment with ribociclib shows favourable immunomodulatory effects in patients with hormone receptor-positive breast cancer-findings from the RIBECCA trial.

BACKGROUND: Inhibitors of the cyclin-dependent kinases 4 and 6 (CDK4/6i) have significantly improved clinical outcomes in patients with advanced hormone receptor-positive (HR+) breast cancer and have demonstrated favourable antitumour immune responses in preclinical studies. METHODS: Here, we investigated peripheral immune responses to ribociclib in patients with metastatic HR+ breast cancer as a preplanned exploratory subanalysis of the RIBECCA trial (NCT03096847). Peripheral blood mononuclear cells were subjected to immune cell profiling, gene expression analysis of immune-related signatures, and deep T cell receptor profiling before treatment started and after 12 weeks of treatment with ribociclib. RESULTS: Gene expression analysis revealed an upregulation of signatures associated with an activated adaptive immune system and a decrease in immunosuppressive cytokine signalling during treatment with ribociclib. Profiling of peripheral immune cell subpopulations showed a decrease in Treg cell frequencies, which was associated with treatment response. Furthermore, induction of CD4+ naive T cells could be seen, whereas effector and memory T cell populations remained largely unchanged. Correspondingly, T cell repertoire diversity remained mostly unchanged during treatment, although an increase in clonality could be observed in single patients. CONCLUSIONS: We show that treatment with ribociclib has significant effects on the peripheral innate and adaptive immune response in patients with HR+ breast cancer. Our data suggest that these effects lead to an activation of an already existing immune response rather than a de novo induction and make a strong case for future combination strategies of CDK4/6i with immunotherapies to enhance the adaptive immune response in HR+ breast cancer.

Nuclear import of the histone acetyltransferase complex SAS-I in Saccharomyces cerevisiae.

The protein complex SAS-I links histone acetylation to the assembly of repressed chromatin in Saccharomyces cerevisiae. Sas2p, the histone acetyltransferase subunit of SAS-I, forms a complex with Sas4p and Sas5p, which are both required for maximal complex activity. In this study, we found that Sas4p was the central subunit of the SAS-I complex, bridging Sas2p and Sas5p. We demonstrated that the nuclear import of Sas2p and Sas5p was mediated by two karyopherins/importins, Kap123p and Pse1p, and both were associated in vivo with these importins. By contrast, Sas4p was not a substrate of Kap123p or Pse1p, suggesting that the nuclear import of the SAS-I subunits occurred independently of each other. Several other non-essential karyopherins were not involved in the nuclear import of SAS-I subunits. When the putative nuclear localization signal (NLS) of Sas2p was deleted, nuclear accumulation of Sas2p was significantly decreased. By contrast, deletion of the proposed NLS of Sas4p had no influence on its nuclear localization. An unknown signal region was located in the N-terminal domain of Sas5p and was responsible for the nuclear import by Kap123p and Pse1p. We found a striking similarity between the NLS sequences of Sas2p and those of histones H3 and H4, which were recently reported to be further import substrates of Kap123p and Pse1p. A database search based on the aligned consensus sequence revealed potential new import substrates of the Kap123p and Pse1p nuclear import pathways, which are connected to chromatin function.