Assessing the effectiveness of marine nature-based solutions with climate risk assessments.

Prospective risks from climate change impacts in ocean and coastal systems are urging the implementation of nature-based solutions (NBS). These are climate-resilient strategies to maintain biodiversity and the delivery of ecosystem services, contributing to the adaptation of social-ecological systems and the mitigation of climate-related impacts. However, the effectiveness of measures like marine restoration or conservation is not exempt from the impacts of climate change, and the degree to which they can sustain biodiversity and ecosystem services remains unknown. Such uncertainty, together with the slow pace of implementation, causes decision-makers and societies to demand a better understanding of NBS effects. To address this gap, in this study, we use the risk mitigation capacity of marine NBS as a proxy for their effectiveness while providing a toolset for the implementation of the method. The method considers environmental data and relies on expert elicitation, allowing us to go beyond current practice to evaluate the effectiveness of NBS in reducing habitat or species risks under different future socio-political and climate-change scenarios. As a result, we present a ready-to-use tool, and supporting materials, for the implementation of the Climate Risk Assessment method and an illustrative example considering the application of the NBS "nature-inclusive harvesting" in two shellfisheries. The method works as a rapid assessment that guarantees comparability across sites and species due to its low data or resource demand, so it can be widely incorporated to adaptation policies across the marine realm.

Assessment of sodium-glucose cotransporter 2 inhibitors (SGLT2i) and other antidiabetic agents in Alzheimer's disease: A population-based study.

The lack of effective treatments for dementia has led to explore the potential of antidiabetic agents as a possible approach. This cross-sectional and population-based study aimed to investigate the relationship between each antidiabetic drug and their defined daily doses (DDDs) and the use of anti-Alzheimer's disease (AD) drugs in order to establish new possible hypotheses about the role of antidiabetic drugs in AD. For that purpose, a database containing information on medications prescribed to 233183 patients aged 50 years or older between 2018 and 2020 was used. DDDs were calculated according to the ATC/DDD index 2023. Statistical analyses, with logistic regression, were carried out to assess antidiabetic and anti-AD drugs consumption. A total of 91836 patients who were prescribed at least one antihypertensive, antidiabetic, or lipid-modifying agent were included in the study; specifically, 29260 patients were prescribed antidiabetic medication. Among the antidiabetic agents, glucagon-like peptide-1 analogs (GLP-1) DDDs were likely to have a positive association with anti-AD drugs in people aged between 70 and 80 years. Additionally, sodium-glucose cotransporter 2 inhibitors (SGLT2i) were prone to have a positive association with anti-AD drug usage across almost every age. However, insulin usage was associated with an increased usage of anti-AD agents. In conclusion, there is evidence suggesting a correlation between certain antidiabetic agents and dementia. Specifically, GLP-1 and SGLT2i might be associated with lower odds of anti-AD drugs usage, while insulins might be linked to higher odds of using anti-AD drugs.

Nomogram based on baseline clinicopathological characteristics for predicting bladder cancer-specific survival to neoadjuvant chemotherapy in muscle-invasive bladder cancer.

PURPOSE: To develop a risk score based on a prognostic model and a nomogram integrating baseline clinicopathological variables to predict bladder cancer-specific survival (BCSS) to neoadjuvant chemotherapy (NAC) in muscle-invasive bladder cancer (MIBC) patients. METHODS: We retrospectively identified a consecutive sample of 247 MIBC patients treated with cisplatin-based NAC-plus-cystectomy in two Spanish hospitals between 2000 and 2019. Age at MIBC diagnosis, sex, histology, lymphovascular invasion, previous non-MIBC, hydronephrosis, and clinical TNM were included in the initial Cox regression model. A risk score was computed based on the final prognostic model and a nomogram was used to estimate BCSS at 2 and 5 years. RESULTS: Median age was 66 years; 89% were males; 83% had pure urothelial carcinoma; 16.2% had previous non-MIBC. Clinical stage was T2N0, T3-4aN0, and Tx-4N + in 24%, 57%, and 19% of patients, respectively. Complete pathological response was seen in 29.4% and downstaging to non-MIBC (ypT1, ypTa, ypTis) in 12.5% of patients. Overall 5-year BCSS was 59%. Four prognostic factors were identified: variant histology, previous non-MIBC, female sex and hydronephrosis. By adding the points attributed to each of these factors, we categorized patients in three groups: low-risk (0 points); intermediate-risk (1-9 points); high-risk (≥ 10 points). Five-year BCSS was 72%, 53%, and 15%, respectively (p < 0.0001). CONCLUSION: We developed a nomogram and risk score based on four baseline clinicopathological characteristics to predict BCSS to NAC-plus-cystectomy in MIBC patients. If validated in prospective studies, this nomogram can be useful for selecting patients likely to benefit from NAC.

Immune Checkpoint Inhibitors: A Promising Treatment Option for Metastatic Castration-Resistant Prostate Cancer?

Since 2010, several treatment options have been available for men with metastatic castration-resistant prostate cancer (mCRPC), including immunotherapeutic agents, although the clinical benefit of these agents remains inconclusive in unselected mCRPC patients. In recent years, however, immunotherapy has re-emerged as a promising therapeutic option to stimulate antitumor immunity, particularly with the use of immune checkpoint inhibitors (ICIs), such as PD-1/PD-L1 and CTLA-4 inhibitors. There is increasing evidence that ICIs may be especially beneficial in specific subgroups of patients with high PD-L1 tumor expression, high tumor mutational burden, or tumors with high microsatellite instability/mismatch repair deficiency. If we are to improve the efficacy of ICIs, it is crucial to have a better understanding of the mechanisms of resistance to ICIs and to identify predictive biomarkers to determine which patients are most likely to benefit. This review focuses on the current status of ICIs for the treatment of mCRPC (either as monotherapy or in combination with other drugs), mechanisms of resistance, potential predictive biomarkers, and future challenges in the management of mCRPC.

Supercritical Antisolvent Fractionation of Antioxidant Compounds from Salvia officinalis.

The increasing interest towards greener antioxidants obtained via natural sources and more sustainable processes encourages the development of new theoretical and experimental methods in the field of those compounds. Two advanced separation methods using supercritical CO2 are applied to obtain valuable antioxidants from Salvia officinalis, and a first approximation to a QSAR model relating molecular structure with antioxidant activity is explored in order to be used, in the future, as a guide for the preselection of compounds of interest in these processes. Separation experiments through antisolvent fractionation with supercritical CO2 were designed using a Response Surface Methodology to study the effect of pressure and CO2 flow rate on both mass yields and capability to obtain fractions enriched in three antioxidant compounds: chlorogenic acid, caffeic acid and rosmarinic acid which were tracked using HPLC PDA. Rosmarinic acid was completely retained in the precipitation vessel while chlorogenic and caffeic acids, though distributed between the two separated fractions, had a major presence in the precipitation vessel too. The conditions predicted for an optimal overall yield and enrichment were 148 bar and 10 g/min. Although a training dataset including much more compounds than those now considered can be recommended, descriptors calculated from the σ-profiles provided by COSMO-RS model seem to be adequate for estimating the antioxidant activity of pure compounds through QSAR.

On the use of n-octyl gallate and salicylhydroxamic acid to study the alternative oxidase role.

The alternative oxidase (AOX) catalyzes the transfer of electrons from ubiquinol to oxygen without the translocation of protons across the inner mitochondrial membrane. This enzyme has been proposed to participate in the regulation of cell growth, sporulation, yeast-mycelium transition, resistance to reactive oxygen species, infection, and production of secondary metabolites. Two approaches have been used to evaluate AOX function: incubation of cells for long periods of time with AOX inhibitors or deletion of AOX gene. However, AOX inhibitors might have different targets. To test non-specific effects of n-octyl gallate (nOg) and salicylhydroxamic acid (SHAM) on fungal physiology we measured the growth and respiratory capacity of two fungal strains lacking (Ustilago maydis-Δaox and Saccharomyces cerevisiae) and three species containing the AOX gene (U. maydis WT, Debaryomyces hansenii, and Aspergillus nidulans). For U. maydis, a strong inhibition of growth and respiratory capacity by SHAM was observed, regardless of the presence of AOX. Similarly, A. nidulans mycelial growth was inhibited by low concentrations of nOg independently of AOX expression. In contrast, these inhibitors had no effect or had a minor effect on S. cerevisiae and D. hansenii growth. These results show that nOg and SHAM have AOX independent effects which vary in different microorganisms, indicating that studies based on long-term incubation of cells with these inhibitors should be considered as inconclusive.

The Wright-Fisher model with efficiency.

In this article, we propose a Wright-Fisher model with two types of individuals: the inefficient individuals, those who need more resources to reproduce and can have a higher growth rate, and the efficient individuals. In this model, the total amount of resource N is fixed, and the population size varies randomly depending on the number of efficient individuals. We show that, as N increases, the frequency process of efficient individuals converges to a diffusion which is a generalization of the Wright-Fisher diffusion with selection. The genealogy of this model is given by a branching-coalescing process that we call the Ancestral Selection/Efficiency Graph, and that is an extension of the Ancestral Selection Graph (Krone and Neuhauser, 1997a,b). The main contribution of this paper is that, in evolving populations, inefficiency can arise as a promoter of selective advantage and not necessarily as a trade-off.

Rapamycin induces morphological and physiological changes without increase in lipid content in Ustilago maydis.

The evolutionarily conserved serine/threonine kinase TOR recruits different subunits to assemble the Target of Rapamycin Complex 1 (TORC1), which is inhibited by rapamycin and regulates ribosome biogenesis, autophagy, and lipid metabolism by regulating the expression of lipogenic genes. In addition, TORC1 participates in the cell cycle, increasing the length of the G2 phase. In the present work, we investigated the effect of rapamycin on cell growth, cell morphology and neutral lipid metabolism in the phytopathogenic fungus Ustilago maydis. Inhibition of TORC1 by rapamycin induced the formation of septa that separate the nuclei that were formed after mitosis. Regarding neutral lipid metabolism, a higher accumulation of triacylglycerols was not detected, but the cells did contain large lipid bodies, which suggests that small lipid bodies became fused into big lipid droplets. Vacuoles showed a similar behavior as the lipid bodies, and double labeling with Blue-CMAC and BODIPY indicates that vacuoles and lipid bodies were independent organelles. The results suggest that TORC1 has a role in cell morphology, lipid metabolism, and vacuolar physiology in U. maydis.

Moving towards Personalized Medicine in Muscle-Invasive Bladder Cancer: Where Are We Now and Where Are We Going?

Neoadjuvant cisplatin-based chemotherapy followed by radical cystectomy is the recommended treatment, with the highest level of evidence, for patients with muscle-invasive bladder cancer (MIBC). However, only a minority of patients receive this treatment, mainly due to patient comorbidities, the relatively small survival benefit, and the lack of predictive biomarkers to select those patients most likely to benefit from this multimodal approach. In addition, adjuvant chemotherapy has been recommended for patients with high-risk MIBC, although randomized trials have not provided conclusive evidence on the impact of this approach. At present, however, this situation is changing, largely due to our improved knowledge of the molecular biology of bladder cancer, which has enabled us to identify new prognostic and predictive biomarkers that can be used to select the most appropriate treatment for each patient. Moreover, new active treatments, especially immunotherapy, have shown promising results in the neoadjuvant setting. In addition, the gene expression profile of bladder tumors can be used to classify them into different subtypes, which correlate with specific clinical-pathological characteristics and with treatment response or resistance. Therefore, the main objective for the near future is to introduce these translational breakthroughs into routine clinical practice in order to personalize treatment for each patient.

New Insights of Ustilago maydis as Yeast Model for Genetic and Biotechnological Research: A Review.

The basidiomycete Ustilago maydis is a biotrophic organism responsible for corn smut disease. In recent years, it has become one of the most promising models for biochemical and biotechnological research due to advantages, such as rapid growth, and easy genetic manipulation. In some aspects, this yeast is more similar to complex eukaryotes, such as humans, compared to standard laboratory yeast models. U. maydis can be employed as a tool to explore physiological processes with more versatility than other fungi. Previously, U. maydis was only considered as a phytopathogenic fungus, but different studies have shown its potential as a research model. Therefore, numerous promising studies have focused on deepening our understanding of the natural interactions, enzyme production, and biotechnological capacity. In this review, we explore general characteristics of U. maydis, both as pathogenic and "innocuous" basidiomycete. Additionally, a comparison with other yeast models focusing on genetic, biochemical, and biotechnological research are analyzed, to emphasize the versatility, dynamism, and novelty that U. maydis has as a research model. In this review, we highlight the applications of the yeast form of the fungus; however, since the filamentous form is also of relevance, it is addressed in the present work, as well.

Membrane Solubilization by Styrene-Maleic Acid Copolymers: Delineating the Role of Polymer Length.

Styrene-maleic acid (SMA) copolymers have attracted interest in membrane research because they allow the solubilization and purification of membrane-spanning proteins from biological membranes in the form of native-like nanodisks. However, our understanding of the underlying SMA-lipid interactions is hampered by the fact that SMA preparations are very polydisperse. Here, we obtained fractions of the two most commonly used SMA preparations: SMA 2:1 and SMA 3:1 (both with specified Mw ∼10 kD), with different number-average molecular weight (Mn) and styrene content. The fractionation is based on the differential solubility of styrene-maleic anhydride (SMAnh) in hexane and acetone mixtures. SMAnh fractions were hydrolyzed to SMA and added to lipid self-assemblies. It was found that SMA fractions inserted in monolayers and solubilized vesicles to a different extent, with the highest efficiency being observed for low-Mn SMA polymers. Electron microscopy and dynamic light scattering size analyses confirmed the presence of nanodisks independent of the Mn of the SMA polymers forming the belt, and it was shown that the nanodisks all have approximately the same size. However, nanodisks bounded by high-Mn SMA polymers were more stable than those bounded by low-Mn polymers, as indicated by a better retention of the native lipid thermotropic properties and by slower exchange rates of lipids between nanodisks. In conclusion, we here present a simple method to separate SMAnh molecules based on their Mn from commercial SMAnh blends, which allowed us to obtain insights into the importance of SMA length for polymer-lipid interactions.

Translational Research Opportunities Regarding Homologous Recombination in Ovarian Cancer.

Homologous recombination (HR) is a DNA repair pathway that is deficient in 50% of high-grade serous ovarian carcinomas (HGSOC). Deficient HR (DHR) constitutes a therapeutic opportunity for these patients, thanks to poly (ADP-ribose) polymerases (PARP) inhibitors (PARPi; olaparib, niraparib, and rucaparib are already commercialized). Although initially, PARPi were developed for patients with BRCA1/2 mutations, robust clinical data have shown their benefit in a broader population without DHR. This breakthrough in daily practice has raised several questions that necessitate further research: How can populations that will most benefit from PARPi be selected? At which stage of ovarian cancer should PARPi be used? Which strategies are reasonable to overcome PARPi resistance? In this paper, we present a summary of the literature and discuss the present clinical research involving PARPi (after reviewing ClinicalTrials.gov) from a translational perspective. Research into the functional biomarkers of DHR and clinical trials testing PARPi benefits as first-line setting or rechallenge are currently ongoing. Additionally, in the clinical setting, only secondary restoring mutations of BRCA1/2 have been identified as events inducing resistance to PARPi. The clinical frequency of this and other mechanisms that have been described in preclinics is unknown. It is of great importance to study mechanisms of resistance to PARPi to guide the clinical development of drug combinations.

Structural and kinetics characterization of the F1F0-ATP synthase dimer. New repercussion of monomer-monomer contact.

Ustilago maydis is an aerobic basidiomycete that fully depends on oxidative phosphorylation for its supply of ATP, pointing to mitochondria as a key player in the energy metabolism of this organism. Mitochondrial F1F0-ATP synthase occurs in supramolecular structures. In this work, we isolated the monomer (640kDa) and the dimer (1280kDa) and characterized their subunit composition and kinetics of ATP hydrolysis. Mass spectrometry revealed that dimerizing subunits e and g were present in the dimer but not in the monomer. Analysis of the ATPase activity showed that both oligomers had Michaelis-Menten kinetics, but the dimer was 7 times more active than the monomer, while affinities were similar. The dimer was more sensitive to oligomycin inhibition, with a Ki of 24nM, while the monomer had a Ki of 169nM. The results suggest that the interphase between the monomers in the dimer state affects the catalytic efficiency of the enzyme and its sensitivity to inhibitors.

Lipid droplets accumulation and other biochemical changes induced in the fungal pathogen Ustilago maydis under nitrogen-starvation.

In many organisms, the growth under nitrogen-deprivation or a poor nitrogen source impacts on the carbon flow distribution and causes accumulation of neutral lipids, which are stored as lipid droplets (LDs). Efforts are in progress to find the mechanism of LDs synthesis and degradation, and new organisms capable of accumulating large amounts of lipids for biotechnological applications. In this context, when Ustilago maydis was cultured in the absence of a nitrogen source, there was a large accumulation of lipid bodies containing mainly triacylglycerols. The most abundant fatty acids in lipid bodies at the stationary phase were palmitic, linoleic, and oleic acids, and they were synthesized de novo by the fatty-acid synthase. In regard to the production of NADPH for the synthesis of fatty acids, the cytosolic NADP+-dependent isocitrate dehydrogenase and the glucose-6-phosphate and 6-phosphogluconate dehydrogenases couple showed the highest specific activities, with a lower activity of the malic enzyme. The ATP-citrate lyase activity was not detected in any of the culture conditions, which points to a different mechanism for the transfer of acetyl-CoA into the cytosol. Protein and RNA contents decreased when U. maydis was grown without a nitrogen source. Due to the significant accumulation of triacylglycerols and the particular composition of fatty acids, U. maydis can be considered an alternative model for biotechnological applications.

Solubilization of lipids and lipid phases by the styrene-maleic acid copolymer.

A promising tool in membrane research is the use of the styrene-maleic acid (SMA) copolymer to solubilize membranes in the form of nanodiscs. Since membranes are heterogeneous in composition, it is important to know whether SMA thereby has a preference for solubilization of either specific types of lipids or specific bilayer phases. Here, we investigated this by performing partial solubilization of model membranes and analyzing the lipid composition of the solubilized fraction. We found that SMA displays no significant lipid preference in homogeneous binary lipid mixtures in the fluid phase, even when using lipids that by themselves show very different solubilization kinetics. By contrast, in heterogeneous phase-separated bilayers, SMA was found to have a strong preference for solubilization of lipids in the fluid phase as compared to those in either a gel phase or a liquid-ordered phase. Together the results suggest that (1) SMA is a reliable tool to characterize native interactions between membrane constituents, (2) any solubilization preference of SMA is not due to properties of individual lipids but rather due to properties of the membrane or membrane domains in which these lipids reside and (3) exploiting SMA resistance rather than detergent resistance may be an attractive approach for the isolation of ordered domains from biological membranes.

Crowdsourcing reproducible seizure forecasting in human and canine epilepsy.

SEE MORMANN AND ANDRZEJAK DOI101093/BRAIN/AWW091 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE : Accurate forecasting of epileptic seizures has the potential to transform clinical epilepsy care. However, progress toward reliable seizure forecasting has been hampered by lack of open access to long duration recordings with an adequate number of seizures for investigators to rigorously compare algorithms and results. A seizure forecasting competition was conducted on kaggle.com using open access chronic ambulatory intracranial electroencephalography from five canines with naturally occurring epilepsy and two humans undergoing prolonged wide bandwidth intracranial electroencephalographic monitoring. Data were provided to participants as 10-min interictal and preictal clips, with approximately half of the 60 GB data bundle labelled (interictal/preictal) for algorithm training and half unlabelled for evaluation. The contestants developed custom algorithms and uploaded their classifications (interictal/preictal) for the unknown testing data, and a randomly selected 40% of data segments were scored and results broadcasted on a public leader board. The contest ran from August to November 2014, and 654 participants submitted 17 856 classifications of the unlabelled test data. The top performing entry scored 0.84 area under the classification curve. Following the contest, additional held-out unlabelled data clips were provided to the top 10 participants and they submitted classifications for the new unseen data. The resulting area under the classification curves were well above chance forecasting, but did show a mean 6.54 ± 2.45% (min, max: 0.30, 20.2) decline in performance. The kaggle.com model using open access data and algorithms generated reproducible research that advanced seizure forecasting. The overall performance from multiple contestants on unseen data was better than a random predictor, and demonstrates the feasibility of seizure forecasting in canine and human epilepsy.media-1vid110.1093/brain/aww045_video_abstractaww045_video_abstract.

Response of Ustilago maydis against the Stress Caused by Three Polycationic Chitin Derivatives.

Chitosan is a stressing molecule that affects the cells walls and plasma membrane of fungi. For chitosan derivatives, the action mode is not clear. In this work, we used the yeast Ustilago maydis to study the effects of these molecules on the plasma membrane, focusing on physiologic and stress responses to chitosan (CH), oligochitosan (OCH), and glycol-chitosan (GCH). Yeasts were cultured with each of these molecules at 1 mg·mL-1 in minimal medium. To compare plasma membrane damage, cells were cultivated in isosmolar medium. Membrane potential (Δψ) as well as oxidative stress were measured. Changes in the total plasma membrane phospholipid and protein profiles were analyzed using standard methods, and fluorescence-stained mitochondria were observed. High osmolarity did not protect against CH inhibition and neither affected membrane potential. The OCH did produce higher oxidative stress. The effects of these molecules were evidenced by modifications in the plasma membrane protein profile. Also, mitochondrial damage was evident for CH and OCH, while GCH resulted in thicker cells with fewer mitochondria and higher glycogen accumulation.

Mitochondrial proteases act on STARD3 to activate progesterone synthesis in human syncytiotrophoblast.

BACKGROUND: STARD1 transports cholesterol into mitochondria of acutely regulated steroidogenic tissue. It has been suggested that STARD3 transports cholesterol in the human placenta, which does not express STARD1. STARD1 is proteolytically activated into a 30-kDa protein. However, the role of proteases in STARD3 modification in the human placenta has not been studied. METHODS: Progesterone determination and Western blot using anti-STARD3 antibodies showed that mitochondrial proteases cleave STARD3 into a 28-kDa fragment that stimulates progesterone synthesis in isolated syncytiotrophoblast mitochondria. Protease inhibitors decrease STARD3 transformation and steroidogenesis. RESULTS: STARD3 remained tightly bound to isolated syncytiotrophoblast mitochondria. Simultaneous to the increase in progesterone synthesis, STARD3 was proteolytically processed into four proteins, of which a 28-kDa protein was the most abundant. This protein stimulated mitochondrial progesterone production similarly to truncated-STARD3. Maximum levels of protease activity were observed at pH7.5 and were sensitive to 1,10-phenanthroline, which inhibited steroidogenesis and STARD3 proteolytic cleavage. Addition of 22(R)-hydroxycholesterol increased progesterone synthesis, even in the presence of 1,10-phenanthroline, suggesting that proteolytic products might be involved in mitochondrial cholesterol transport. CONCLUSION: Metalloproteases from human placental mitochondria are involved in steroidogenesis through the proteolytic activation of STARD3. 1,10-Phenanthroline inhibits STARD3 proteolytic cleavage. The 28-kDa protein and the amino terminal truncated-STARD3 stimulate steroidogenesis in a comparable rate, suggesting that both proteins share similar properties, probably the START domain that is involved in cholesterol binding. GENERAL SIGNIFICANCE: Mitochondrial proteases are involved in syncytiotrophoblast-cell steroidogenesis regulation. Understanding STARD3 activation and its role in progesterone synthesis is crucial to getting insight into its action mechanism in healthy and diseased syncytiotrophoblast cells.

Membrane potential regulates mitochondrial ATP-diphosphohydrolase activity but is not involved in progesterone biosynthesis in human syncytiotrophoblast cells.

ATP-diphosphohydrolase is associated with human syncytiotrophoblast mitochondria. The activity of this enzyme is implicated in the stimulation of oxygen uptake and progesterone synthesis. We reported previously that: (1) the detergent-solubilized ATP-diphosphohydrolase has low substrate specificity, and (2) purine and pyrimidine nucleosides, tri- or diphosphates, are fully dephosphorylated in the presence of calcium or magnesium (Flores-Herrera 1999, 2002). In this study we show that ATP-diphosphohydrolase hydrolyzes first the nucleoside triphosphate to nucleoside diphosphate, and then to nucleotide monophosphate, in the case of all tested nucleotides. The activation energies (Ea) for ATP, GTP, UTP, and CTP were 6.06, 4.10, 6.25, and 5.26 kcal/mol, respectively; for ADP, GDP, UDP, and CDP, they were 4.67, 5.42, 5.43, and 6.22 kcal/mol, respectively. The corresponding Arrhenius plots indicated a single rate-limiting step for each hydrolyzed nucleoside, either tri- or diphosphate. In intact mitochondria, the ADP produced by ATP-diphosphohydrolase activity depolarized the membrane potential (ΔΨm) and stimulated oxygen uptake. Mitochondrial respiration showed the state-3/state-4 transition when ATP was added, suggesting that ATP-diphosphohydrolase and the F1F0-ATP synthase work in conjunction to avoid a futile cycle. Substrate selectivity of the ATP-diphosphohydrolase was modified by ΔΨm (i.e. ATP was preferred over GTP when the inner mitochondrial membrane was energized). In contrast, dissipation of ΔΨm by CCCP produced a loss of substrate specificity and so the ATP-diphosphohydrolase was able to hydrolyze ATP and GTP at the same rate. In intact mitochondria, ATP hydrolysis increased progesterone synthesis as compared with GTP. Although dissipation of ΔΨm by CCCP decreased progesterone synthesis, NADPH production restores steroidogenesis. Overall, our results suggest a novel physiological role for ΔΨm in steroidogenesis.

Molecular model for the solubilization of membranes into nanodisks by styrene maleic Acid copolymers.

A recent discovery in membrane research is the ability of styrene-maleic acid (SMA) copolymers to solubilize membranes in the form of nanodisks allowing extraction and purification of membrane proteins from their native environment in a single detergent-free step. This has important implications for membrane research because it allows isolation as well as characterization of proteins and lipids in a near-native environment. Here, we aimed to unravel the molecular mode of action of SMA copolymers by performing systematic studies using model membranes of varying compositions and employing complementary biophysical approaches. We found that the SMA copolymer is a highly efficient membrane-solubilizing agent and that lipid bilayer properties such as fluidity, thickness, lateral pressure profile, and charge density all play distinct roles in the kinetics of solubilization. More specifically, relatively thin membranes, decreased lateral chain pressure, low charge density at the membrane surface, and increased salt concentration promote the speed and yield of vesicle solubilization. Experiments using a native membrane lipid extract showed that the SMA copolymer does not discriminate between different lipids and thus retains the native lipid composition in the solubilized particles. A model is proposed for the mode of action of SMA copolymers in which membrane solubilization is mainly driven by the hydrophobic effect and is further favored by physical properties of the polymer such as its relatively small cross-sectional area and rigid pendant groups. These results may be helpful for development of novel applications for this new type of solubilizing agent, and for optimization of the SMA technology for solubilization of the wide variety of cell membranes found in nature.