Confocal Microscopy using an InGaN violet laser diode at 406nm

We report on the application of a novel all-solid-state violet laser diode source to confocal microscopy. The source has the potential to replace argon ion lasers in a range of fluorescence based imaging systems. Improvements in system performance and image quality through the use of anamorphic prisms to modify the beam profile have been characterised. These modifications have permitted high quality, optically sectioned images to be obtained from laser diodes operating around 406nm. Living mammalian cells stained with a range of biologically significant fluorophores have been imaged. In addition, it has been shown that at this wavelength it is possible to image dyes that normally require excitation with UV argon laser lines

Modulation of Plasma Lipidomic Profiles in Metastatic Castration-Resistant Prostate Cancer by Simvastatin

Elevated circulating sphingolipids are associated with shorter overall survival and therapeutic resistance in metastatic castration-resistant prostate cancer (mCRPC), suggesting that perturbations in sphingolipid metabolism promotes prostate cancer growth. This study assessed whether addition of simvastatin to standard treatment for mCRPC can modify a poor prognostic circulating lipidomic profile represented by a validated 3-lipid signature (3LS). Men with mCRPC (n = 27) who were not on a lipid-lowering agent, were given simvastatin for 12 weeks (40 mg orally, once daily) with commencement of standard treatment. Lipidomic profiling was performed on their plasma sampled at baseline and after 12 weeks of treatment. Only 11 men had the poor prognostic 3LS at baseline, of whom five (45%) did not retain the 3LS after simvastatin treatment (expected conversion rate with standard treatment = 19%). At baseline, the plasma profiles of men with the 3LS displayed higher levels (p < 0.05) of sphingolipids (ceramides, hexosylceramides and sphingomyelins) than those of men without the 3LS. These plasma sphingolipids were reduced after statin treatment in men who lost the 3LS (mean decrease: 23–52%, p < 0.05), but not in men with persistent 3LS, and were independent of changes to plasma cholesterol, LDL-C or triacylglycerol. In conclusion, simvastatin in addition to standard treatment can modify the poor prognostic circulating lipidomic profile in mCRPC into a more favourable profile at twice the expected conversion rate.Blossom Mak, Hui-Ming Lin, Thy Duong, Kate L. Mahon, Anthony M. Joshua, Martin R. Stockler, Howard Gurney, Francis Parnis, Alison Zhang, Tahlia Scheinberg, Gary Wittert, Lisa M. Butler, David Sullivan, Andrew J. Hoy, Peter J. Meikle, and Lisa G. Horvat

Evaluation of a COVID-19 convalescent plasma program at a U.S. academic medical center

Amidst the therapeutic void at the onset of the COVID-19 pandemic, a critical mass of scientific and clinical interest coalesced around COVID-19 convalescent plasma (CCP). To date, the CCP literature has focused largely on safety and efficacy outcomes, but little on implementation outcomes or experience. Expert opinion suggests that if CCP has a role in COVID-19 treatment, it is early in the disease course, and it must deliver a sufficiently high titer of neutralizing antibodies (nAb). Missing in the literature are comprehensive evaluations of how local CCP programs were implemented as part of pandemic preparedness and response, including considerations of the core components and personnel required to meet demand with adequately qualified CCP in a timely and sustained manner. To address this gap, we conducted an evaluation of a local CCP program at a large U.S. academic medical center, the University of North Carolina Medical Center (UNCMC), and patterned our evaluation around the dimensions of the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework to systematically describe key implementation-relevant metrics. We aligned our evaluation with program goals of reaching the target population with severe or critical COVID-19, integrating into the structure of the hospital-wide pandemic response, adapting to shifting landscapes, and sustaining the program over time during a compassionate use expanded access program (EAP) era and a randomized controlled trial (RCT) era. During the EAP era, the UNCMC CCP program was associated with faster CCP infusion after admission compared with contemporaneous affiliate hospitals without a local program: median 29.6 hours (interquartile range, IQR: 21.2–48.1) for the UNCMC CCP program versus 47.6 hours (IQR 32.6–71.6) for affiliate hospitals; (P<0.0001). Sixty-eight of 87 CCP recipients in the EAP (78.2%) received CCP containing the FDA recommended minimum nAb titer of ≥1:160. CCP delivery to hospitalized patients operated with equal efficiency regardless of receiving treatment via a RCT or a compassionate-use mechanism. It was found that in a highly resourced academic medical center, rapid implementation of a local CCP collection, treatment, and clinical trial program could be achieved through redeployment of highly trained laboratory and clinical personnel. These data provide important pragmatic considerations critical for health systems considering the use of CCP as part of an integrated pandemic response

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Store-operated channels mediate ca2+ influx and contraction in rat pulmonary artery

Cation channels activated by Ca2+ store depletion have been proposed to mediate Ca2+ influx in vascular smooth muscle cells. The aim of this study was to determine if store-operated channels have a functional role in pulmonary artery smooth muscle cells (PASMCs). In intact rat pulmonary artery rings, cyclopiazonic acid (CPA) produced a sustained contraction that was resistant to inhibition by nifedipine, but abolished in Ca2+-free solution and 50% blocked in the presence of 6 µmol/L Cd2+, 10 µmol/L Ni2+, 600 µmol/L La3+, and 7 µmol/L SKF96365. In freshly isolated PASMCs loaded with fura-2, CPA increased the intracellular Ca2+ concentration by stimulating dihydropyridine-resistant Ca2+ influx, which was 50% blocked by 10 µmol/L Ni2+ and 7 µmol/L SKF96365. In perforated-patch recordings, CPA activated a sustained inward current at negative membrane potentials, which persisted in cells dialyzed with BAPTA, showed a near linear dependence on membrane potential when Cs+ was the main intracellular cation, and was blocked by Ni2+, Cd2+, and SKF96365 at concentrations preventing contraction. The current showed a bimodal dependence on extracellular Ca2+, being enhanced 2-fold in the absence of Ca2+ and around 10-fold on reducing Ca from 1.8 to 0.2 mmol/L. RT-PCR revealed the expression of Trp1, Trp3, Trp4, Trp5, and Trp6 mRNA, whereas immunostaining identified Trp1, Trp3, Trp4, and Trp6 channel proteins in isolated PASMCs. At least one of these subunits may contribute to cation channels in PASMCs, which are activated by store depletion to bring about Ca2+ influx and contraction

Regulation of store-operated Ca2+ entry in pulmonary artery smooth muscle cells

Store-operated Ca2+ entry (SOCE) is an important mechanism for Ca2+ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3 μM) potentiated SOCE by 47 ± 2%, while the tyrosine kinase inhibitors genistein (100 μM) and tyrphostin 23 (100 μM) caused a significant reduction in SOCE of 55 ± 9% and 43 ± 7%, respectively. It has been proposed that Ca2+-insensitive phospholipase A2 (iPLA2) is involved in the activation of SOCE in many different cell types. The iPLA2 inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10 μM) and W-7 (10 μM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca2+ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca2+ influx was blocked by Gd3+ (10 μM), but unaffected by 2-APB (75 μM), indicating a pharmacological profile distinct from the classical SOCE pathway

Muscarinic receptors and calcium signalling in rabbit isolated pulmonary artery smooth muscle cells

Acetylcholine (ACh) is an endothelium-dependent vasodilator, but when pulmonary artery endothelium is damaged it causes vasoconstriction through a direct action on smooth muscle muscarinic receptors (Dinh-Xuan et al., 1989). In rabbit main pulmonary artery this action involves M2 and M3 receptors The muscarinic agonist, oxotremorine sesquifumarate (Oxo-S), also contracts this preparation, but the effects appear to be mediated solely through M2 receptors This study characterised responses to ACh and Oxo-S in isolated pulmonary artery smooth muscle cells (PASMC) by monitoring changes in the fluorescence of the Ca2+ indicator, fluo-4. Arteries were obtained from male New Zealand rabbits (2-2.5 kg) killed by lethal injection of sodium pentobarbitone (140 mg kg-' i.v.). PASMC were isolated by enzymatic digestion, loaded with 1 jM fluo-4 at 221C for 45 min, washed and bathed in physiological salt solution (PSS). Fluorescence was excited at 488 nm and measured between 503 and 553 nm using a confocal microscope. Calcium signals were quantified as the change in fluorescence (AF) relative to the background fluorescence (F0). Ca2+-free solution was prepared by replacing CaCl2 in the PSS with equimolar MgC12 and adding 5mM EGTA. To study Gi involvement, PASMC were incubated with pertussis toxin (PTX; 5gig ml-') for 3-4 hr at 22°C and compared with control cells treated in the same way, but without exposure to PTX. Experiments were performed 22°C. Results are expressed as mean ± s.e.m

The effects of paeonol on the electrophysiological properties of cardiac ventricular myocytes

Previous studies have shown that "Mudanpi", a Chinese herbal medicine, has a significant cardioprotective effect against myocardial ischaemia. Based on these findings we hypothesised that paeonol, the main component of Mudanpi, might have an effect on the cellular electrophysiology of cardiac ventricular myocytes. The effects of paeonol on the action potential and ion channels of cardiac ventricular myocytes were studied using the standard whole-cell configuration of the patch-clamp technique. Ventricular myocytes were isolated from the hearts of adult guinea-pig by enzymic dispersion. The myocytes were continuously perfused with various experimental solutions at room temperature and paeonol applied in the perfusate. Action potentials and membrane currents were recorded using both current and voltage clamp modes of the patch-clamp technique. Paeonol, at concentrations 160 mu M and 640 mu M, decreased the action potential upstroke phase, an action associated with the blockade of the voltage-gated, fast sodium channel. The effects of paeonol on both action potential and Na+ current were concentration dependent. Paeonol had a high affinity for inactivated sodium channels. Paeonol also shortened the action potential duration, in a manner not associated with the blockade of the calcium current, or the enhancement of potassium currents. These findings suggest that paeonol, and therefore Mudanpi, may possess antiarrhythmic activity, which may confer its cardioprotective effects. (c) 2006 Elsevier B.V All rights reserved