Application of the SMALP technology to the isolation of GPCRs from low-yielding cell lines

The ability of styrene–maleic acid (SMAc) co-polymers to spontaneously insert into biological membranes can be exploited to extract G protein-coupled receptors (GPCRs) embedded in styrene–maleic acid lipid particles (SMALPs), preserving the native environment around the protein and thus enhancing the feasibility of functional studies. So far, the SMALP technology has been primarily employed on non-mammalian cells and protocols are not optimized for adherent human cell lines, which cannot be harvested in large amounts. In this work, a fine investigation of key parameters affecting the formation of SMALPs was undertaken with the purpose of maximizing the yield of extraction of a recombinant form of human β2-adrenergic receptor (rhβ2AR) from HEK293T cells. The study highlighted an important influence of ionic strength on the membrane solubilization efficiency and GPCR purification yield of SMAc co-polymers: by lowering the salt concentration of all buffers used in previously published SMALP protocols, the water solubility and extraction efficiency of the selected SMAc co-polymer (commercially supplied as a potassium salt) were enhanced. In-line combination of size-exclusion chromatography (SEC) with immobilized metal affinity chromatography (IMAC) allowed further improvement of the final rhβ2AR yield by reducing the loss of SMALP-embedded GPCRs during the fractionation and purification of SMALPs. The overall findings of this study show that the available SMALP protocols can be significantly optimized in several aspects in order to increase the efficiency of GPCR solubilization and isolation from low-yielding expression systems

Tachycardia-induced metabolic rewiring as a driver of contractile dysfunction

Prolonged tachycardia—a risk factor for cardiovascular morbidity and mortality—can induce cardiomyopathy in the absence of structural disease in the heart. Here, by leveraging human patient data, a canine model of tachycardia and engineered heart tissue generated from human induced pluripotent stem cells, we show that metabolic rewiring during tachycardia drives contractile dysfunction by promoting tissue hypoxia, elevated glucose utilization and the suppression of oxidative phosphorylation. Mechanistically, a metabolic shift towards anaerobic glycolysis disrupts the redox balance of nicotinamide adenine dinucleotide (NAD), resulting in increased global protein acetylation (and in particular the acetylation of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), a molecular signature of heart failure. Restoration of NAD redox by NAD+ supplementation reduced sarcoplasmic/endoplasmic reticulum Ca2+-ATPase acetylation and accelerated the functional recovery of the engineered heart tissue after tachycardia. Understanding how metabolic rewiring drives tachycardia-induced cardiomyopathy opens up opportunities for therapeutic intervention

Resveratrol Supplementation Confers Neuroprotection in Cortical Brain Tissue of Nonhuman Primates Fed a High-Fat/Sucrose Diet

Previous studies have shown positive effects of long-term resveratrol (RSV) supplementation in preventing pancreatic beta cell dysfunction, arterial stiffening and metabolic decline induced by high-fat/high-sugar (HFS) diet in nonhuman primates. Here, the analysis was extended to examine whether RSV may reduce dietary stress toxicity in the cerebral cortex of the same cohort of treated animals. Middle-aged male rhesus monkeys were fed for 2 years with HFS alone or combined with RSV, after which whole-genome microarray analysis of cerebral cortex tissue was carried out along with ELISA, immunofluorescence, and biochemical analyses to examine markers of vascular health and inflammation in the cerebral cortices. A number of genes and pathways that were differentially modulated in these dietary interventions indicated an exacerbation of neuroinflammation (e.g., oxidative stress markers, apoptosis, NF-κB activation) in HFS-fed animals and protection by RSV treatment. The decreased expression of mitochondrial aldehyde dehydrogenase 2, dysregulation in endothelial nitric oxide synthase, and reduced capillary density induced by HFS stress were rescued by RSV supplementation. Our results suggest that long-term RSV treatment confers neuroprotection against cerebral vascular dysfunction during nutrient stress

Influence of Environmental Factors on the Production of MIB and Geosmin Metabolites by Bacteria in a Eutrophic Reservoir

Occurrences of odorous bacterial metabolites, 2‐methylisoborneol (MIB) and geosmin (GSM), in drinking water supply reservoirs are considered as a nuisance by the water industry and a source of complaints from customers. In Eagle Creek Reservoir, routine monitoring programs of MIB and GSM highlight intense odorous outbreaks during the spring season when high inflow discharges occur. Cyanobacteria have always been assumed to be source of these metabolites even if no known producers are present in raw water. A copper‐based algaecide is often used to terminate the metabolite production and the algal growth in the reservoir. The current study was designed to investigate and identify other biological sources involved in the biosynthesis of MIB and GSM metabolites as well as environmental factors that could be important triggers for the growth of bacterial producers. The community structure of the bacterioplankton was determined using a 16S rRNA gene sequencing technique, which showed that not only Cyanobacteria but Actinobacteria also were involved in the reservoir internal production. Planktothrix species was identified as the main source of GSM (p < 0.001) while Streptomyces (Actinobacteria) was very likely responsible of MIB (p < 0.01). Application of an algaecide disrupted GSM and the growth of Planktothrix but was less effective against MIB and Streptomyces . Statistical analyses revealed that MIB‐ and GSM‐causing bacteria were found abundant when the water was enriched with nitrogen, temperature cooler, and the water column mixed

Genes Associated with 2-Methylisoborneol Biosynthesis in Cyanobacteria: Isolation, Characterization, and Expression in Response to Light

The volatile microbial metabolite 2-methylisoborneol (2-MIB) is a root cause of taste and odor issues in freshwater. Although current evidence suggests that 2-MIB is not toxic, this compound degrades water quality and presents problems for water treatment. To address these issues, cyanobacteria and actinomycetes, the major producers of 2-MIB, have been investigated extensively. In this study, two 2-MIB producing strains, coded as Pseudanabaena sp. and Planktothricoids raciborskii, were used in order to elucidate the genetic background, light regulation, and biochemical mechanisms of 2-MIB biosynthesis in cyanobacteria. Genome walking and PCR methods revealed that two adjacent genes, SAM-dependent methyltransferanse gene and monoterpene cyclase gene, are responsible for GPP methylation and subsequent cyclization to 2-MIB in cyanobacteria. These two genes are located in between two homologous cyclic nucleotide-binding protein genes that may be members of the Crp-Fnr regulator family. Together, this sequence of genes forms a putative operon. The synthesis of 2-MIB is similar in cyanobacteria and actinomycetes. Comparison of the gene arrangement and functional sites between cyanobacteria and other organisms revealed that gene recombination and gene transfer probably occurred during the evolution of 2-MIB-associated genes. All the microorganisms examined have a common origin of 2-MIB biosynthesis capacity, but cyanobacteria represent a unique evolutionary lineage. Gene expression analysis suggested that light is a crucial, but not the only, active regulatory factor for the transcription of 2-MIB synthesis genes. This light-regulated process is immediate and transient. This study is the first to identify the genetic background and evolution of 2-MIB biosynthesis in cyanobacteria, thus enhancing current knowledge on 2-MIB contamination of freshwater

Ship information systems using smartglasses technology

New technology in the maritime sector often forces shipowners to assemble many new devices on their ship, which can increase the safety of sea travel. With each additional device on the navigation bridge, there are additional sets of data that need to be observed. The following article describes one of the possibilities of using augmented reality technology to support navigational decisions. The research used “smartglasses” technology and AR glasses from Meta Glasses

The concept of determining the ship’s route based on the capability plots

Every year, new vessels equipped with dynamic positioning (DP) systems are built in shipyards around the world. Due to the increasing number of offshore vessels, a client hiring a vessel should analyse the vessel's positioning capability charts to determine which water areas the vessel is designed for. These charts are represented as polar diagrams. In the centre of the chart is a shape symbolising the ship's body, and the values on the chart represent the maximum wind speed that can affect the ship at a given angle, at which the vessel will maintain its position. Vessel capability charts can also be used by the crew during thrusters failures to determine at what angle to the wind direction the vessel should stand to minimise the impact of wind forces. Analyses that determine a vessel's ability to keep position can be performed by classification societies or other companies with approval from classification societies. The article presents the concept of a pathfinding algorithm to determines the route of the ship’s passage with minimal energy consumption. The algorithm uses the information about environmental forces affecting the ship and information about thrust allocation obtained from Capability Plots