Electronic Lab Notebooks and Experimental Design Assistants.

Documentation of experiments is essential for best research practice and ensures scientific transparency and data integrity. Traditionally, the paper lab notebook (pLN) has been employed for documentation of experimental procedures, but over the course of the last decades, the introduction of electronic tools has changed the research landscape and the way that work is performed. Nowadays, almost all data acquisition, analysis, presentation and archiving are done with electronic tools. The use of electronic tools provides many new possibilities, as well as challenges, particularly with respect to documentation and data quality. One of the biggest hurdles is the management of data on different devices with a substantial amount of metadata. Transparency and integrity have to be ensured and must be reflected in documentation within LNs. With this in mind, electronic LNs (eLN) were introduced to make documentation of experiments more straightforward, with the development of enhanced functionality leading gradually to their more widespread use. This chapter gives a general overview of eLNs in the scientific environment with a focus on the advantages of supporting quality and transparency of the research. It provides guidance on adopting an eLN and gives an example on how to set up unique Study-IDs in labs in order to maintain and enhance best practices. Overall, the chapter highlights the central role of eLNs in supporting the documentation and reproducibility of experiments.

Surgical intensive care - current and future challenges?

Bjorn Ibsen, an anesthetist who pioneered positive pressure ventilation as a treatment option during the Copenhagen polio epidemic of 1952, set up the first Intensive Care Unit (ICU) in Europe in 1953. He managed polio patients on positive pressure ventilation together with physicians and physiologists in a dedicated ward, where one nurse was assigned to each patient. In that sense Ibsen is more or less the father of intensive care medicine as a specialty and also an advocate of the one-to-one nursing ratio for critically ill patients. Nowadays, the Surgical Intensive Care Unit (SICU) offers critical care treatment to unstable, severely, or potentially severely ill patients in the perioperative setting, who have life-threatening conditions and require comprehensive care, constant monitoring, and possible emergency interventions. Hence there is one very specific challenge in the surgical setting: the intensivist has to manage the patient flow starting from admission to the hospital through to the operating theater, in the SICU, and postoperatively for the discharge to the ward. In other words, the planning of the resources (most frequently availability of beds) has to be optimized to prevent cancellations of elective surgical procedures but also to facilitate other emergency admissions. SICU intensivists take the role of arbitrators between surgical demand and patient's interests. This means they supervise the safety, efficacy, and workability of the process with respect to all stakeholders. This notion was reported in 2007 when Stawicki and co-workers performed a small prospective study concluding that it appears safe if the dedicated intensivist takes over the role of the last arbitrator supported by a multidisciplinary team.1 However, demographic changes in many countries during the last few decades have given rise to populations which are more elderly and sicker than before. This impacts on the healthcare system in general but on the intensivist and the ICU team too. In addition, in a society with an increased life expectancy, the balance between treatable disease, outcome, and utilization of resources must be maintained. This fact gains even more importance as patients and their families claim "high end" treatment. Such a demand is reflected looking at the developments that have taken place over the last 25 years. Mainly, the focus of intensive care medicine was on technical support or even replacement of failing organ systems such as the lungs, the heart, or the kidneys by veno-venous extracorporeal membrane oxygenation (VV-ECMO), veno-arterial ECMO (VA-ECMO), and continuous veno-venous hemofiltration (CVVH) respectively. This means "technical care" became a core capability and expectation of critical care medicine. In parallel, medical treatment became more standardized. For example, lung protective ventilation strategies, early enteral feeding, and daily sedation vacation are part of modern protocols. As a consequence, ventilator time has been reduced and patients therefore develop delirium less frequently. These measures, beside others, are implemented in care bundles to improve the quality of care of patients by the whole ICU team. The importance of specialty trained teams was already pointed out 35 years ago when Li et al.,2 demonstrated in a study performed in a community hospital that the mortality was decreased if an ICU was managed 24/7 by an on-site physician. The association of improved outcomes and presence of a critical care trained physician (intensivist) has been shown in several studies since that time.3,4,5,6 A modern multidisciplinary critical care team consists at least of an intensivist, ICU nurse, pharmacist, respiratory therapist, physiotherapist, and the primary team physician. Based on clinical needs, the team can be supplemented by oncologists, cardiologists, or other specialties. Again, this approach is supported by research: a recent retrospective cohort study from the California Hospital Assessment and Reporting Taskforce (CHART) on 60,330 patients confirmed the association between improved patient outcome and such a multidisciplinary team.7 If such an intensive care team makes a difference, why do not all patients at risk receive advanced ICU-care? It was already demonstrated by Esteban et al., in a prospective study that patients with severe sepsis had a mortality rate of 26% when not admitted to an ICU in comparison to 11% when they were admitted to an ICU.8 Meanwhile, we know that early referral is particularly important, because for ischemic diseases the timing appears to make a difference in terms of full recovery. So, the following questions arise: Should intensive care be rolled out to each ward and physical admission to an ICU or be restricted to special cases only? For this purpose, the so-called "Rapid Response Teams" (RRT) or "Medical Emergency Team" (MET), which essentially are a form of an ICU outreach team, were implemented. The name, composition, or exact role of such team varies from institution to institution and country to country. Alternatively, should all ward staff be educated to recognize sick patients earlier for a timely transfer to a dedicated area? This would mean that ICU-care would be introduced in the ward. A first attempt to answer this question, whether to deploy critical care resources to deteriorating patients outside the ICU 24/7, was given by Churpek et al.9 The success of the rapid response teams could be related to decreased rates of cardiac arrest outside the ICU setting and in-hospital mortality. Interestingly, an analysis of the registry database of the RRT calls in this study showed that the lowest frequency of calls occurred between 1:00 AM to 6:59 AM time period. In contrast, the mortality was highest around 7 AM and lowest during noon hour. This indicates that not simply the availability of such a team makes a difference but also the alertness of the ward-teams is of high importance to identify deteriorating patients in a timely manner. Essentially, this would necessitate ward staff being trained to provide a higher level of care enabling them to better recognize when patients become sicker to avoid a delayed call to the ICU. Alternatively, a system in which the intensivist plays a major role in daily ward rounds could be beneficial. So, the ward doctor should become an intensivist. However, the latter means the ICU is rolled out across the whole hospital which would consume a huge amount of resources. Another option would be 24/7 remote monitoring of patients at risk that notifies the intensivist or RRT in case of need. The infrastructure, technology, and manpower to put this in place also has associated costs. As the demand for ICU care will rise further in the future, intensivists will play an even more important role in the healthcare system that itself is under enormous economic pressure to ensure the best quality of care for critically ill patients. Besides excellent knowledge and hard skills, intensivists need to be team players, communicators, facilitators, and arbitrators to achieve the best results in collaboration with all involved in patient treatment.

TRPC5 is a Ca2+-activated channel functionally coupled to Ca2+-selective ion channels.

TRPC5 forms non-selective cation channels. Here we studied the role of internal Ca(2+) in the activation of murine TRPC5 heterologously expressed in human embryonic kidney cells. Cell dialysis with various Ca(2+) concentrations (Ca(2+)(i)) revealed a dose-dependent activation of TRPC5 channels by internal Ca(2+) with EC(50) of 635.1 and 358.2 nm at negative and positive membrane potentials, respectively. Stepwise increases of Ca(2+)(i) induced by photolysis of caged Ca(2+) showed that the Ca(2+) activation of TRPC5 channels follows a rapid exponential time course with a time constant of 8.6 +/- 0.2 ms at Ca(2+)(i) below 10 microM, suggesting that the action of internal Ca(2+) is a primary mechanism in the activation of TRPC5 channels. A second slow activation phase with a time to peak of 1.4 +/- 0.1 s was also observed at Ca(2+)(i) above 10 microM. In support of a Ca(2+)-activation mechanism, the thapsigargin-induced release of Ca(2+) from internal stores activated TRPC5 channels transiently, and the subsequent Ca(2+) entry produced a sustained TRPC5 activation, which in turn supported a long-lasting membrane depolarization. By co-expressing STIM1 plus ORAI1 or the alpha(1)C and beta(2) subunits of L-type Ca(2+) channels, we found that Ca(2+) entry through either calcium-release-activated-calcium or voltage-dependent Ca(2+) channels is sufficient for TRPC5 channel activation. The Ca(2+) entry activated TRPC5 channels under buffering of internal Ca(2+) with EGTA but not with BAPTA. Our data support the hypothesis that TRPC5 forms Ca(2+)-activated cation channels that are functionally coupled to Ca(2+)-selective ion channels through local Ca(2+) increases beneath the plasma membrane.

Primary structure, chromosomal localization and expression in immune cells of the murine ORAI and STIM genes.

Recently ORAI and STIM proteins were identified as components of the CRAC channel and the endoplasmic reticulum Ca(2+) sensor, respectively. The ORAI proteins share a predicted structure that includes four transmembrane domains with intracellular N- and C-termini. They share structural similarity with proteins of the tetraspanin superfamily which includes the gamma subunits of voltage-activated Ca(2+) channels (CaVgamma), the transmembrane AMPA regulatory proteins (TARPs), the claudins and the tumor-associated membrane proteins (TMPs). The mouse genome contains four genes which encode the ORAI1, ORAI2 and ORAI3 proteins and two genes which encode the type I single-pass transmembrane STIM1 and STIM2 proteins. ORAI2 transcripts are present in primary cortical neurons and ORAI1, 2, 3 and STIM1, 2 expression is readily detectable in CD3+/CD4+-, CD3+/CD8+-, and CD19+-lymphocytes as well as in mast cells from mouse.

Murine ORAI2 splice variants form functional Ca2+ release-activated Ca2+ (CRAC) channels.

The stimulation of membrane receptors coupled to the phopholipase C pathway leads to activation of the Ca(2+) release-activated Ca(2+) (CRAC) channels. Recent evidence indicates that ORAI1 is an essential pore subunit of CRAC channels. STIM1 is additionally required for CRAC channel activation. The present study focuses on the genomic organization, tissue expression pattern, and functional properties of the murine ORAI2. Additionally, we report the cloning of the murine ORAI1, ORAI3, and STIM1. Two chromosomal loci were identified for the murine orai2 gene, one containing an intronless gene and a second locus that gives rise to the splice variants ORAI2 long (ORAI2L) and ORAI2 short (ORAI2S). Northern blots revealed a prominent expression of the ORAI2 variants in the brain, lung, spleen, and intestine, while ORAI1, ORAI3, and STIM1 appeared to be near ubiquitously expressed in mice tissues. Specific antibodies detected ORAI2 in RBL 2H3 but not in HEK 293 cells, whereas both cell lines appeared to express ORAI1 and STIM1 proteins. Co-expression experiments with STIM1 and either ORAI1 or ORAI2 variants showed that ORAI2L and ORAI2S enhanced substantially CRAC current densities in HEK 293 but were ineffective in RBL 2H3 cells, whereas ORAI1 strongly amplified CRAC currents in both cell lines. Thus, the capability of ORAI2 variants to form CRAC channels depends strongly on the cell background. Additionally, CRAC channels formed by ORAI2S were strongly sensitive to inactivation by internal Ca(2+). When co-expressed with STIM1 and ORAI1, ORAI2S apparently plays a negative dominant role in the formation of CRAC channels.