Single fluorescent protein-based Ca2+ sensors with increased dynamic range

<p>Abstract</p> <p>Background</p> <p>Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca²⁺sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).</p> <p>Results</p> <p>Here we report significant progress on the development of the latter type of Ca²⁺sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca²⁺concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca²⁺-free and Ca²⁺-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca²⁺response to a prolonged glutamate treatment in cortical neurons.</p> <p>Conclusion</p> <p>We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.</p

An evolutionary perspective on psychiatry

Recent progress in the evolutionary understanding of behavior may greatly assist psychiatry. Although explanations of psychopathology have traditionally emphasized proximate causes of individual differences, consideration of the evolutionary functions of human behavior is essential for psychiatry, just as biology, ethology, and medicine routinely consider both proximate and evolutionary explanations for a variety of phenomena. The methods and data for testing evolutionary hypotheses are reviewed, and the ways in which evolutionary principles can help to explain maladaptive behaviors are considered. Some psychiatric symptoms that seem maladaptive may, in fact, serve specific survival functions. Hypotheses are proposed about the possible evolutionary significance of overeating, anorexia nervosa, panic attacks, and sexual disorders, and tests of these hypotheses are considered. By incorporating an evolutionary perspective on psychopathology, psychiatry may share the foundation that evolution provides for the rest of natural science.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24655/1/0000068.pd

The Structure of Ca2+ Sensor Case16 Reveals the Mechanism of Reaction to Low Ca2+ Concentrations

Here we report the first crystal structure of a high-contrast genetically encoded circularly permuted green fluorescent protein (cpGFP)-based Ca2+ sensor, Case16, in the presence of a low Ca2+ concentration. The structure reveals the positioning of the chromophore within Case16 at the first stage of the Ca2+-dependent response when only two out of four Ca2+-binding pockets of calmodulin (CaM) are occupied with Ca2+ ions. In such a “half Ca2+-bound state”, Case16 is characterized by an incomplete interaction between its CaM-/M13-domains. We also report the crystal structure of the related Ca2+ sensor Case12 at saturating Ca2+ concentration. Based on this structure, we postulate that cpGFP-based Ca2+ sensors can form non-functional homodimers where the CaM-domain of one sensor molecule binds symmetrically to the M13-peptide of the partner sensor molecule. Case12 and Case16 behavior upon addition of high concentrations of free CaM or M13-peptide reveals that the latter effectively blocks the fluorescent response of the sensor. We speculate that the demonstrated intermolecular interaction with endogenous substrates and homodimerization can impede proper functioning of this type of Ca2+ sensors in living cells

Assessment of rotatory laxity in anterior cruciate ligament-deficient knees using magnetic resonance imaging with Porto-knee testing device

Purpose Objective evaluation of both antero-posterior translation and rotatory laxity of the knee remains a target to be accomplished. This is true for both preoperative planning and postoperative assessment of different ACL reconstruction emerging techniques. The ideal measurement tool should be simple, accurate and reproducible, while enabling to assess both ‘‘anatomy’’ and ‘‘function’’ during the same examination. The purpose of this study is to evaluate the clinical effectiveness of a new in-housedeveloped testing device, the so-called Porto-knee testing device (PKTD). The PKTD is aimed to be used on the evaluation of both antero-posterior and rotatory laxity of the knee during MRI exams. Methods Between 2008 and 2010, 33 patients with ACLdeficient knees were enrolled for the purpose of this study. All patients were evaluated in the office and under anesthesia with Lachman test, lateral pivot-shift test and anterior drawer test. All cases were studied preoperatively with KT-1000 and MRI with PKTD, and examinations performed by independent observers blinded for clinical evaluation. During MRI, we have used a PKTD that applies antero-posterior translation and permits free tibial rotation through a standardized pressure (46.7 kPa) in the proximal posterior region of the leg. Measurements were taken for both knees and comparing side-to-side. Five patients with partial ruptures were excluded from the group of 33. Results For the 28 remaining patients, 3 women and 25 men, with mean age of 33.4 ± 9.4 years, 13 left and 15 right knees were tested. No significant correlation was noticed for Lachman test and PKTD results (n.s.). Pivot-shift had a strong positive correlation with the difference in anterior translation registered in lateral and medial tibia plateaus of injured knees (cor. coefficient = 0.80; p\0.05), and with the difference in this parameter as compared to side-to-side (cor. coefficient = 0.83; p\0.05). Considering the KT-1000 difference between injured and healthy knees, a very strong positive correlation was found for side-to-side difference in medial (cor. coeffi- cient = 0.73; p\0.05) and lateral (cor. coefficient = 0.5; p\0.05) tibial plateau displacement using PKTD. Conclusion The PKTD proved to be a reliable tool in assessment of antero-posterior translation (comparing with KT-1000) and rotatory laxity (compared with lateral pivotshift under anesthesia) of the ACL-deficient knee during MRI examinatio

Understanding biological responses to degraded hydromorphology and multiple stresses. Deliverable 3.2 of REFORM (REstoring rivers FOR effective catchment Management), a Collaborative project (large-scale integrating project) funded by the European Commission within the 7th Framework Programme under Grant Agreement 282656

The aim of this deliverable is to conceptually model and empirically test the response of biota to the effects of both hydromorphological pressures acting in concert with one another or with other types of pressures. Best use is made of existing large national monitoring datasets (Denmark, UK, Finland, France, Germany, Austria & WISER datasets), case studies and modeling to provide evidence of multiple stressors interacting to alter river biota (Biological Quality Elements: BQE)

Culture or Biology? If this sounds interesting, you might be confused

Culture or Biology? The question can seem deep and important. Yet, I argue in this chapter, if you are enthralled by questions about our biological differences, then you are probably confused. My goal is to diagnose the confusion. In debates about the role of biology in the social world it is easy to ask the wrong questions, and it is easy to misinterpret the scientific research. We are intuitively attracted to what is called psychological essentialism, and therefore interpret what is biological as what can be traced to “essences”. On this interpretation, it would be deep and important to know what about, say, the differences between the genders is biological: it would correspond to what is essential to being a man or being a woman, and be opposed to what is a mere accidental feature that some women or some men have. Yet, the psychological essentialist understanding of ‘biological differences’ is deeply mistaken about biology. It has the wrong conception of biological kinds, of biological heritability, and of how genes and hormones work. Those who argue for an important role of ‘biology’ in the explanation of human differences often see ‘the science’ on their side. But this is false – on the interpretation of ‘biological differences’ that is most intuitive and that makes the question appear to be most interesting. Defenders of ‘biology’ often have the science against them. What is often called ‘biology’ is a myth: a myth created by an intuitive tendency that grotesquely distorts real biological research

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Label-free technologies for drug discovery

Over the past two decades the benefits of label-free biosensor analysis have begun to make an impact in the market, and systems are beginning to be used as mainstream research tools in many drug discovery laboratories. Label-Free Technologies For Drug Discovery summarises the latest and emerging developments in label-free detection systems, their underlying technology principles and end-user case studies that reveal the power and limitations of label-free in all areas of drug discovery. Label-free technologies discussed include SPR, NMR, high-throughput mass spectrometry, resonant waveguide plate-based screening, transmitted-light imaging, isothermal titration calorimetry, optical and impedance cell-based assays and other biophysical methods. The technologies are discussed in relation to their use as screening technologies, high-content technologies, hit finding and hit validation strategies, mode of action and ADME/T, access to difficult target classes, cell-based receptor/ligand interactions particularly orphan receptors, and antibody and small molecule affinity and kinetic analysis. Label-Free Technologies For Drug Discovery is an essential guide to this emerging class of tools for researchers in drug discovery and development, particularly high-throughput screening and compound profiling teams, medicinal chemists, structural biologists, assay developers, ADME/T specialists, and others interested in biomolecular interaction analysis

A Chemoinformatics Analysis of Hit Lists Obtained from High-Throughput Affinity-Selection Screening

The online version of this article can be found at