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RNA sequencing (RNAseq) samples the majority of expressed genes infrequently, owing to the large size, complex splicing and wide dynamic range of eukaryotic transcriptomes. This results in sparse sequencing coverage that can hinder robust isoform assembly and quantification. RNA capture sequencing (CaptureSeq) addresses this challenge by using oligonucleotide probes to capture selected genes or regions of interest for targeted sequencing. Targeted RNAseq provides enhanced coverage for sensitive gene discovery, robust transcript assembly and accurate gene quantification. Here we describe a detailed protocol for all stages of RNA CaptureSeq, from initial probe design considerations and capture of targeted genes to final assembly and quantification of captured transcripts. Initial probe design and final analysis can take less than 1 d, whereas the central experimental capture stage requires ∼7 d.

© 2016 John Wiley & Sons Ltd. The second messenger cyclic-di-adenosine monophosphate (c-di-AMP) plays important roles in growth, virulence, cell wall homeostasis, potassium transport and affects resistance to antibiotics, heat and osmotic stress. Most Firmicutes contain only one c-di-AMP synthesizing diadenylate cyclase (CdaA); however, little is known about signals and effectors controlling CdaA activity and c-di-AMP levels. In this study, a genetic screen was employed to identify components which affect the c-di-AMP level in Lactococcus. We characterized suppressor mutations that restored osmoresistance to spontaneous c-di-AMP phosphodiesterase gdpP mutants, which contain high c-di-AMP levels. Loss-of-function and gain-of-function mutations were identified in the cdaA and gdpP genes, respectively, which led to lower c-di-AMP levels. A mutation was also identified in the phosphoglucosamine mutase gene glmM, which is commonly located within the cdaA operon in bacteria. The glmM I154F mutation resulted in a lowering of the c-di-AMP level and a reduction in the key peptidoglycan precursor UDP-N-acetylglucosamine in L. lactis. C-di-AMP synthesis by CdaA was shown to be inhibited by GlmMI154F more than GlmM and GlmMI154F was found to bind more strongly to CdaA than GlmM. These findings identify GlmM as a c-di-AMP level modulating protein and provide a direct connection between c-di-AMP synthesis and peptidoglycan biosynthesis. c-di-AMP is an essential signalling molecule which affects peptidoglycan homeostasis and resistance against various stressors, however little is known regarding how the c-di-AMP level is regulated in the cell. Here we identify the peptidoglycan biosynthesis enzyme GlmM as a modulator of c-di-AMP synthesis through its regulation of diadenylate cyclase enzyme CdaA activity in Lactococcus lactis.

The activity of creatine kinase (CK) and creatine kinase B(CK–B) was measured in 17 patients with injuries to multiple organ systems, including the chest. The patients were closely observed for clinical signs of disturbed cardiac function by means of serial ECG, continuous monitoring of cardiac rhythm, daily cardiac auscultation, serial chest roentgenography and monitoring of central hemodynamic parameters. No statistically significant difference in CK and CK–B activity was found between the group of patients with normal cardiac function and the group with disturbed cardiac function. The CK–B activity was markedly elevated, but CK–B activity relative to CK activity was normal in both groups during the first 7 days after the trauma. The authors conclude that the significance of these enzymes' serum activity, measured with the immunoinhibition method, is diagnostically doubtful not only as regards cardiac contusions, but also in other cardiopathy preceding or following major trauma.

In 10 patients undergoing aortic bypass grafting with peroperative aortic cross-clamping we measured the levels of myoglobin and creatine phosphokinase. The duration of peroperative lower limb arterial clamping ranged from 50 min to 95 min. No significant increase in either serum myoglobin or in serum creatine phosphokinase was found during lower limb arterial clamping or for the first two hours after release of ischemia. Both parameters reached a maximum value at 24 hours after release of ischemia, with a median serum myoglobin concentration of 565 micrograms/l (range: 132-2688 micrograms/l) and a median serum creatine phosphokinase activity of 457 U/l (range: 190-1602 U/l). The increase in serum myoglobin and creatine phosphokinase was not associated with the duration of lower limb arterial clamping. Renal impairment was not found in these patients, as evaluated by the serum concentration of beta 2-microglobulin.