A method for detergent-free isolation of membrane proteins in their local lipid environment.

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Harnessing the potential of ligninolytic enzymes for lignocellulosic biomass pretreatment

Abundant lignocellulosic biomass from various industries provides a great potential feedstock for the production of value-added products such as biofuel, animal feed, and paper pulping. However, low yield of sugar obtained from lignocellulosic hydrolysate is usually due to the presence of lignin that acts as a protective barrier for cellulose and thus restricts the accessibility of the enzyme to work on the cellulosic component. This review focuses on the significance of biological pretreatment specifically using ligninolytic enzymes as an alternative method apart from the conventional physical and chemical pretreatment. Different modes of biological pretreatment are discussed in this paper which is based on (i) fungal pretreatment where fungi mycelia colonise and directly attack the substrate by releasing ligninolytic enzymes and (ii) enzymatic pretreatment using ligninolytic enzymes to counter the drawbacks of fungal pretreatment. This review also discusses the important factors of biological pretreatment using ligninolytic enzymes such as nature of the lignocellulosic biomass, pH, temperature, presence of mediator, oxygen, and surfactant during the biodelignification process

Heavy metals health risk assessment for population via consumption of vegetables grown in old mining area; a case study: Banat County, Romania

<p>Abstract</p> <p>Background</p> <p>The aim of this study is to measure the levels of heavy metals (Fe, Mn, Zn, Cu, Ni, Cd and Pb) found in common vegetables (parsley, carrot, onion, lettuce, cucumber and green beans) grown in contaminated mining areas compared with those grown in reference clear area and to determine their potential detrimental effects via calculation of the daily metal intake (DImetal) and Target Hazard Quotients (THQ) for normal daily consumption of these vegetables, for male and female gender.</p> <p>Results</p> <p>Compared with the reference in contaminated areas, soil and plant contents of all analyzed metals are higher, usually over normally content for Mn, Zn, Cu, Cd and Pb. Particularly, in soil, higher values than intervention threshold values (ITV) were found for Cu and Pb and higher than maximum allowable limits (MAL) for Zn, Cu, Cd and Pb for parsley roots and leaves, carrot roots, cabbage, lettuce and cucumber. DImetal and THQ values for male and female were calculated for each vegetable and metal and for which oral reference doses exist. The combined THQ values calculated are concerning in that they are usually below the safe level of THQ<1 for all vegetables grown in reference area. In contaminated Moldova Noua (M) area the combined THQ exceeded the safe level only for parsley roots, while in more contaminated Ruschita (R) area combined THQ exceeded the safe level for parsley and carrot roots, lettuce and cabbage. Cd and Pb, most toxic metals to humans, have an increasing prevalence in the combined THQ for leafy (cabbage and lettuce) and fruit vegetables (cucumber). In the root vegetables only Pb has an increasing prevalence in combined THQ values. In all areas female THQ is higher than male THQ.</p> <p>Conclusion</p> <p>The results of this study regarding metal contents in soils, vegetables, DImetal and THQ suggest that the consumption of some vegetables (especially parsley, carrot and cabbage and less for lettuce, cucumber and green beans) is not free of risks in these areas. The complex THQ parameter use in health risk assessment of heavy metals provides a better image than using only a simple parameter (contents of metals in soils and vegetables).</p

A Membrane Fusion Protein αSNAP Is a Novel Regulator of Epithelial Apical Junctions

Tight junctions (TJs) and adherens junctions (AJs) are key determinants of the structure and permeability of epithelial barriers. Although exocytic delivery to the cell surface is crucial for junctional assembly, little is known about the mechanisms controlling TJ and AJ exocytosis. This study was aimed at investigating whether a key mediator of exocytosis, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP), regulates epithelial junctions. αSNAP was enriched at apical junctions in SK-CO15 and T84 colonic epithelial cells and in normal human intestinal mucosa. siRNA-mediated knockdown of αSNAP inhibited AJ/TJ assembly and establishment of the paracellular barrier in SK-CO15 cells, which was accompanied by a significant down-regulation of p120-catenin and E-cadherin expression. A selective depletion of p120 catenin effectively disrupted AJ and TJ structure and compromised the epithelial barrier. However, overexpression of p120 catenin did not rescue the defects of junctional structure and permeability caused by αSNAP knockdown thereby suggesting the involvement of additional mechanisms. Such mechanisms did not depend on NSF functions or induction of cell death, but were associated with disruption of the Golgi complex and down-regulation of a Golgi-associated guanidine nucleotide exchange factor, GBF1. These findings suggest novel roles for αSNAP in promoting the formation of epithelial AJs and TJs by controlling Golgi-dependent expression and trafficking of junctional proteins

Sustainable theory of a logistic model - Fisher Information approach

Information theory provides a useful tool to understand the evolution of complex nonlinear systems and their sustainability. In particular, Fisher information has been evoked as a useful measure of sustainability and the variability of dynamical systems including self-organising systems. By utilising Fisher information, we investigate the sustainability of the logistic model for different perturbations in the positive and/or negative feedback. Specifically, we consider different oscillatory modulations in the parameters for positive and negative feedback and investigate their effect on the evolution of the system and Probability Density Functions (PDFs). Depending on the relative time scale of the perturbation to the response time of the system (the linear growth rate), we demonstrate the maintenance of the initial condition for a long time, manifested by a broad bimodal PDF. We present the analysis of Fisher information in different cases and elucidate its implications for the sustainability of population dynamics. We also show that a purely oscillatory growth rate can lead to a finite amplitude solution while self-organisation of these systems can break down with an exponentially growing solution due to the periodic fluctuations in negative feedback

“Tourism, water, and gender”—An international review of an unexplored nexus

This international literature review of the tourism–water nexus identifies a gender gap. Tourism development can affect water supply both quantitatively and qualitatively. Many regions will face considerable problems of water availability and quality, affecting their tourism sector and increasing competition with local residents, and other industries especially agriculture. This international review of literature explores the tourism–water nexus, comparing and contrasting literature published in English, Chinese, and Spanish. Securing access to safe water for continued tourism development is a common theme and the vast majority of work has focused on hotels including water pricing, water-saving practices and innovative management methods. In all continents, struggles are apparent, and the unsustainability of tourism is having impacts on water quantity and quality. This article identifies significant gaps in the literature including climate change, the energy-water nexus, and the links with the Sustainable Development Goals. Furthermore, studies from a gendered perspective are minimal and the potential for areas of further gendered studies within the tourism–water nexus are highlighted including intersectionality, water insecurity and sanitation, tourism and gender based violence, and additional unpaid care work

Immunodominant Antigens of Leishmania chagasi Associated with Protection against Human Visceral Leishmaniasis

One of the most striking features of infection by Leishmania chagasi is that infection leads to a spectrum of clinical outcomes ranging from asymptomatic infection to active disease. The existence of asymptomatic infected people has served as an incentive to believe that an effective vaccine is possible, but unfortunately no successful immunological characterization of such cases was obtained. Patients recovered from visceral leishmaniasis show a similar immunological profile to asymptomatic infected individuals and both exhibit a strong cell-mediated immune response against Leishmania antigens and are resistant to disease. Since the past decade several approaches were undertaken to try to shed light on the immunological profile associated with such “resistance” to infections, notwithstanding antigenic recognition profile associated to resistance to infection was not successfully explored. In the present manuscript we describe a specific IgG recognizing pattern associated with resistant individuals (asymptomatic infected people and recovery patients to visceral leishmaniasis). These data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals

Contribution of Cell Elongation to the Biofilm Formation of Pseudomonas aeruginosa during Anaerobic Respiration

Pseudomonas aeruginosa, a gram-negative bacterium of clinical importance, forms more robust biofilm during anaerobic respiration, a mode of growth presumed to occur in abnormally thickened mucus layer lining the cystic fibrosis (CF) patient airway. However, molecular basis behind this anaerobiosis-triggered robust biofilm formation is not clearly defined yet. Here, we identified a morphological change naturally accompanied by anaerobic respiration in P. aeruginosa and investigated its effect on the biofilm formation in vitro. A standard laboratory strain, PAO1 was highly elongated during anaerobic respiration compared with bacteria grown aerobically. Microscopic analysis demonstrated that cell elongation likely occurred as a consequence of defective cell division. Cell elongation was dependent on the presence of nitrite reductase (NIR) that reduces nitrite (NO2−) to nitric oxide (NO) and was repressed in PAO1 in the presence of carboxy-PTIO, a NO antagonist, demonstrating that cell elongation involves a process to respond to NO, a spontaneous byproduct of the anaerobic respiration. Importantly, the non-elongated NIR-deficient mutant failed to form biofilm, while a mutant of nitrate reductase (NAR) and wild type PAO1, both of which were highly elongated, formed robust biofilm. Taken together, our data reveal a role of previously undescribed cell biological event in P. aeruginosa biofilm formation and suggest NIR as a key player involved in such process

Nitric Oxide Synthase Inhibitors into the Clinic at Last

The 1998 nobel prize in medicine and physiology for the discovery of nitric oxide, a nitrogen containing reactive oxygen species (also termed reactive nitrogen or reactive nitrogen/oxygen species) stirred great hopes. Clinical applications, however, have so far pertained exclusively to the downstream signaling of cgmp enhancing drugs such as phosphodiesterase inhibitors and soluble guanylate cyclase stimulators. All clinical attempts, so far, to inhibit nos have failed even though preclinical models were strikingly positive and clinical biomarkers correlated perfectly. This rather casts doubt on our current way of target identification in drug discovery in general and our way of patient stratification based on correlating but not causal biomarkers or symptoms. The opposite, no donors, nitrite and enhancing no synthesis by enos/nos3 recoupling in situations of no deficiency, are rapidly declining in clinical relevance or hold promise but need yet to enter formal therapeutic guidelines, respectively. Nevertheless, nos inhibition in situations of no overproduction often jointly with enhanced superoxide (or hydrogen peroxide production) still holds promise, but most likely only in acute conditions such as neurotrauma (stover et al., j neurotrauma 31(19):1599–1606, 2014) and stroke (kleinschnitz et al., j cereb blood flow metab 1508–1512, 2016; casas et al., proc natl acad sci u s a 116(14):7129–7136, 2019). Conversely, in chronic conditions, long-term inhibition of nos might be too risky because of off-target effects on enos/nos3 in particular for patients with cardiovascular risks or metabolic and renal diseases.graphical abstractnitric oxide synthases (nos) and their role in health (green) and disease (red). Only neuronal/type 1 nos (nos1) has a high degree of clinical validation and is in late stage development for traumatic brain injury, followed by a phase ii safety/efficacy trial in ischemic stroke. The pathophysiology of nos1 (kleinschnitz et al., j cereb blood flow metab 1508–1512, 2016) is likely to be related to parallel superoxide or hydrogen peroxide formation (kleinschnitz et al., j cereb blood flow metab 1508–1512, 2016; casas et al., proc natl acad sci u s a 114(46):12315–12320, 2017; casas et al., proc natl acad sci u s a 116(14):7129–7136, 2019) leading to peroxynitrite and protein nitration, etc. Endothelial/type 3 nos (nos3) is considered protective only and its inhibition should be avoided. The preclinical evidence for a role of high-output inducible/type 2 nos (nos2) isoform in sepsis, asthma, rheumatic arthritis, etc. Was high, but all clinical development trials in these indications were neutral despite target engagement being validated. This casts doubt on the role of nos2 in humans in health and disease (hence the neutral, black coloring).keywordsnitric oxidenitric oxide synthasenosnos inhibitor nos isoforms