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Proper regulation of cellular lipid storage and oxidation is indispensable for the maintenance of cellular energy homeostasis and health. Mitochondrial function has been shown to be a main determinant of functional lipid storage and oxidation, which is of particular interest for the adipose tissue, as it is the main site of triacylglyceride storage in lipid droplets (LDs). Recent studies have identified a subpopulation of mitochondria attached to LDs, peridroplet mitochondria (PDM) that can be separated from cytoplasmic mitochondria (CM) by centrifugation. PDM have distinct bioenergetics, proteome, cristae organization and dynamics that support LD build-up, however their role in adipose tissue biology remains largely unexplored. Therefore, understanding the molecular basis of LD homeostasis and their relationship to mitochondrial function and attachment in adipocytes is of major importance.

Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.

After his takeover of Judea, Antiochus III issued a programma that prohibits the introduction of impure animals into Jerusalem. Two Qumran Scrolls contain parallels to this injunction but target a different audience, i.e., Jews, as opposed to the gentile audience of the programma. Consequently, the focus of these texts also differs: pure animals in the scrolls, impure animals in the programma. Nonetheless, the programma, the scrolls, and perhaps also some instructions in the Mishnah reflect a coherent interpretation of the biblical ban on non-sacral slaughter within a certain radius around God’s altar. Furthermore, comparison of these sources reinforces the authenticity of the programma, offers a possible underlying reasoning for a reconstructed ruling in the Temple Scroll, and even alludes to the Vorlage of the biblical text employed for drafting the programma. Further evidence, however, implies that the relevant Jewish halakhah underwent a significant change during the second century BCE.

Within the Herodian temenos in Jerusalem, a warning inscription prohibited non-Jews, under penalty of death, from proceeding any further inward. This was mounted on a low stone balustrade that encircled an area larger than the actual holy ground. As suggested in research, the underlying pentateuchal law for the inscription was הזר הקרב יומת, “the unauthorized encroacher shall be put to death.” The subjection of gentiles to this law, in particular, and its application even when they had not, de facto, trespassed on holy ground remain, however, unexplained. The article suggests that the inscription applied הזר הקרב יומת to a זר, in the sense of “a foreigner,” who merely קרב, “draws near” to sacred ground. A further suggestion is that this reading and implementation of the biblical law reflects a preemptive endeavor to blunt Jewish objection to a major cultic innovation by Herod: granting gentiles access to the Jerusalem temenos.

Ideology plays a central role in conflicts, both on the intergroup and intragroup levels. On the intergroup level, ideology can either contribute to the preservation and escalation of conflicts or serve as the key to resolving them. On the intragroup level, ideology can generate and induce conflicts between opposing ideological groups. However, the interaction between these two levels of conflict and the unique role of ideology in such interactions have not received sufficient systematic scholarly attention so far. We suggest a new theoretical framework emphasizing a possible reciprocal relationship between intractable intergroup conflicts and intragroup ideological polarization: intergroup conflict enhances inner polarization, which in turn, hinders the resolution of the external conflict.

The surface ligands of semiconductor nanocrystals (NCs) are central for determining their properties and for their flexible implementation in diverse applications. Thus far, the thermodynamic characteristics of ligand exchange reactions were attained by indirect methods. Isothermal titration calorimetry is utilized to directly and independently measure both the equilibrium constant and the reaction enthalpy of a model ligand exchange reaction from oleate-capped CdSe NCs to a series of alkylthiols. Increased reaction exothermicity for longer chains, accompanied by a decrease in reaction entropy with an overall enthalpy-entropy compensation behavior is observed, explained by the length-dependent interchain interactions and the organization of the bound ligands on the NCs' surface. An increase in the spontaneity of the reaction with decreasing NC size is also revealed, due to their enhanced surface reactivity. This work provides a fundamental understanding of the physicochemical properties of the NC surface with implications for NC surface ligand design.

Objective: Opioid use and the risk of opioid overdose are growing public health concerns for college-aged adults. Naloxone can temporarily reverse opioid overdoses, but only if easily accessible. O...

Transcriptional inhibition by small interfering RNA (siRNA) delivery using synthetic transfection agents eliminates the subsequent risk of introducing mutations in relevant genes, as opposed to viral vectors. However, synthetic vectors with comparable transfection efficiency to that of viral vectors are yet to be developed. Hence, synthesizing new transfection vehicles with low toxicity is important. In this study, a library of lipid-like molecules (lipidoids) was synthesized by thiolactone chemistry. This library facilitated nonviral delivery of siRNA to mammalian cells, inducing sequence-specific knockdown of a target gene. The liposomal nanoparticles complexed with anti-green fluorescent protein (GFP) siRNA were successfully screened for transfection efficiency using a HeLa-GFP cell line. The five best-performing lipidoids identified in the screening were found to exhibit superior GFP-knockdown efficiency compared with commercially available transfection reagents. The efficiency of siRNA delivery by one of these lipidoids with minimal toxicity was further successfully evaluated in vivo using Kdrl:EGFP zebrafish embryos as a model system. Our study would be important as a facile synthetic route of efficient nonviral nucleic acid delivery to live cells and organisms.

Unpaid or ‘free’ labour is an important element of how precarity has been theorized. It is also an issue that is often seen as endemic to cultural and creative work, rightly attracting a range of criticism. Questions as to the role of unpaid work, for example internships, have become central to understanding the social exclusiveness of many cultural and creative jobs. This paper develops this existing analysis by comparing and contrasting the meaning of 'free' work over the life course of a range of creative occupations, historicising the impact of unpaid labour on the creative sector and showing how it has been stratified by social class, age and career stage. The paper uses two datasets drawn from the Panic! What happened to social mobility in the arts? project, to outline the differing experiences of unpaid labour in cultural and creative occupations. By demonstrating the stratification of unpaid work as a form of precariousness in cultural jobs, along with the social distribution of benign narratives of unpaid work, the paper aims to offer new empirical evidence for those seeking to resist precarious forms of labour.

Mitochondrial turnover is required for proper cellular function. Both mitochondrial biogenesis and mitophagy are impaired in several degenerative and age-related diseases. The search for mitophagy activators recently emerged as a new therapeutical approach; however, there is a lack in suitable tools to follow mitochondrial turnover in a high-throughput manner. We demonstrate that the fluorescent protein, MitoTimer, is a reliable and robust probe to follow mitochondrial turnover. The screening of 15 000 small molecules led us to two chemically-related benzothiophenes that stimulate basal mitophagy in the beta-cell line, INS1. Enhancing basal mitophagy was associated with improved mitochondrial function, higher Complex I activity and Complex II and III expressions in INS1 cells, as well as better insulin secretion performance in mouse islets. The possibility of further enhancing mitophagy in the absence of mitochondrial stressors points to the existence of a ‘basal mitophagy spare capacity'. To this end, we found two small molecules that can be used as models to better understand the physiological regulation of mitophagy.

The biological activity of sulfur and selenium, despite their similarity, shows some remarkable differences that have been recognized in many different scenarios. However, the underlying cause has not been completely clarified yet. The difference in the redox behavior of these two chalcogens has lately been addressed as justification of the presence of selenium in some essential biological systems. In particular, selenium is found in some peroxidases, i.e. glutathione peroxidases (GPx), whose redox activity relies on a fast-reacting selenocysteine and is fundamental in metabolizing harmful peroxides. In this work, a systematic in silico investigation on model systems, i.e. phenylchalcogenides, containing sulfur, selenium and tellurium is presented. Sequential oxidation reactions of these chalcogen-based substrates by hydrogen peroxide are carried out spanning the range of the biologically relevant chalcogen oxidation numbers [Advances in Molecular Toxicology, ed. J. C. Fishbein, Elsevier, 2010, vol. 4, pp. 183–222.] (2−, 0, 2+ and 4+) and analyzed through the calculation of intrinsic reaction coordinate paths and the application of the activation strain model. The results allowed us to highlight the different behaviors of S, Se and Te in highly oxidizing environments.

We estimate the kinetic constants of a series of archetypal S

SummaryTo date, there is no effective oral antiviral against SARS-CoV-2 that is also anti-inflammatory. Herein, we show that the mitochondrial antioxidant mitoquinone/mitoquinol mesylate (Mito-MES), a dietary supplement, has potent antiviral activity against SARS-CoV-2 and its variants of concern in vitro and in vivo. Mito-MES had nanomolar in vitro antiviral potency against the Beta and Delta SARS-CoV-2 variants as well as the murine hepatitis virus (MHV-A59). Mito-MES given in SARS-CoV-2 infected K18-hACE2 mice through oral gavage reduced viral titer by nearly 4 log units relative to the vehicle group. We found in vitro that the antiviral effect of Mito-MES is attributable to its hydrophobic dTPP+ moiety and its combined effects scavenging reactive oxygen species (ROS), activating Nrf2 and increasing the host defense proteins TOM70 and MX1. Mito-MES was efficacious reducing increase in cleaved caspase-3 and inflammation induced by SARS-CoV2 infection both in lung epithelial cells and a transgenic mouse model of COVID-19. Mito-MES reduced production of IL-6 by SARS-CoV-2 infected epithelial cells through its antioxidant properties (Nrf2 agonist, coenzyme Q10 moiety) and the dTPP moiety. Given established safety of Mito-MES in humans, our results suggest that Mito-MES may represent a rapidly applicable therapeutic strategy that can be added in the therapeutic arsenal against COVID-19. Its potential long-term use by humans as diet supplement could help control the SARS-CoV-2 pandemic, especially in the setting of rapidly emerging SARS-CoV-2 variants that may compromise vaccine efficacy.One-Sentence SummaryMitoquinone/mitoquinol mesylate has potent antiviral and anti-inflammatory activity in preclinical models of SARS-CoV-2 infection.

Surface ligands of semiconductor nanocrystals (NCs) play key roles in determining their colloidal stability and physicochemical properties and are thus enablers also for the NCs flexible manipulation toward numerous applications. Attention is usually paid to the ligand binding group, while the impact of the ligand chain backbone structure is less discussed. Using isothermal titration calorimetry (ITC), we studied the effect of structural changes in the ligand chain on the thermodynamics of the exchange reaction for oleate coated CdSe NCs, comparing linear and branched alkylthiols. The investigated alkylthiol ligands differed in their backbone length, branching position, and branching group length. Compared to linear ligands, lower exothermicity and entropy loss were observed for an exchange with branched ligands, due to steric hindrance in ligand packing, thereby justifying their previous classification as "entropic ligands". Mean-field calculations for ligand binding demonstrate the contribution to the overall entropy originating from ligand conformational entropy, which is diminished upon binding mainly by packing of NC-bound ligands. Model calculations and the experimental ITC data both point to an interplay between the branching position and the backbone length in determining the entropic nature of the branched ligand. Our findings suggest that the most entropic ligand should be a short, branched ligand with short branching group located toward the middle of the ligand chain. The insights provided by this work also contribute to a future smarter NC surface design, which is an essential tool for their implementation in diverse applications.

Additional file 3: Supplementary Figure 1. Validation of the ectopic expression of CD44 and/or TrkA in COS-7 cells. The expression of all CD44 isoforms (A), CD44 variant 3 (B), CD44 variant 6 (C) and TrkA (hyaluronan [HA]; D) was evaluated by RT–qPCR (normalized using PUM-1). No expression of CD44, CD44 variants [3 or 6] or TrkA was detected in COS-7 cells (A-D) compared with that in cells transfected with the expression plasmid carrying each protein. Supplementary figure 2. TrkA does not interact with CD44s or CD44v6. Wild-type cos7 cells (A) or CD44S- (B) or CD44v6 (C) cells transfected with TrkA were treated with 100 ng/ml NGF (0, 5 or 30 min) and fixed. Quantification of the PLA results was performed using ImageJ software (30 randomly chosen fields per condition of three different experiments). Statistical analyses were performed using one-way ANOVA followed by Bonferroni’s posttest. The error bars represent the standard error of the mean (S.E.M.); ns, not significant. Supplementary Figure 3. CD44v3 and TrkA are recruited to the plasma membrane through NGF stimulation. CD44v3 and CD44v6 levels at the plasma membrane were assessed by flow cytometry (A-D). The plasma membrane and total TrkA and CD44v3 levels were also quantified by confocal microscopy (E-H). Statistical analyses were performed using one-way ANOVA followed by Bonferroni’s posttest. The error bars represent the standard error of the mean (S.E.M.); * p

Acid Sphingomyelinase (ASM) is a human phosphodiesterase that catalyzes the metabolism of sphingomyelin (SM) to ceramide and phosphocholine. ASM is involved in the plasma membrane cell repair and is associated with the lysosomal inner lipid membrane by nonbonding interactions. The disruption of those interaction would result in ASM release into the lysosomal lumen and consequent degradation of its structure. Furthermore, SARS-CoV-2 infection has been linked with ASM activation and with a ceramide domain formation in the outer leaflet of the plasma membrane that is thought to be crucial for the viral particles recognition by the host cells. In this study, we have explored in silico the behavior of fluoxetine and related drugs as potential inhibitors of ASM. Theoretically, these drugs would be able to overpass lysosomal membrane and reach the interactions that sustain ASM structure, breaking them and inhibiting the ASM. The analyses of docking data indicated that fluoxetine allocated mainly in the N-terminal saposin domain via nonbonding interactions, mostly of hydrophobic nature. Similar results were obtained for venlafaxine, citalopram, atomoxetine, nisoxetine and fluoxetine’s main metabolite norfluoxetine. In conclusion, it was observed that the saposin allocation may be a good indicative of the drugs inhibition mechanism, once this domain is responsible for the binding of ASM to lysosomal membrane and some of those drugs have previously been reported to inhibit the phosphodiesterase by releasing its structure in the lysosomal lumen. Our MD data also provides some insight about natural ligand C18 sphingomyelin conformations on saposin. Communicated by Ramaswamy H. Sarma

The mechanochemical syntheses of allyl and indenyl palladate complexes are reported. All compounds were obtained in quantitative yields and microanalytically pure without the need of any workup. These complexes are stable in chlorinated and polar (DMSO or DMSO/H2O solutions) solvents. In chlorinated solvents, they appear as ionic pairs of which crystals suitable for single X-ray diffraction studies have been obtained. Bonding and solvation properties are rationalized through scalar relativistic DFT calculations. Moreover, most complexes showed excellent cytotoxicity towards ovarian cancer cell lines, with IC50 values comparable or lower than cisplatin. The potent anticancer activity of two IPrCl and IPr*-based palladate complexes was examined in a high-grade serous ovarian cancer (HGSOC) patient-derived tumoroid. Moreover, the inhibition of the antioxidant enzyme thioredoxin reductase (TrxR) was noticed, and structure-activity relationships could be derived, suggesting the ROS detoxifying system is involved in the mode of action.

Supplemental material, sj-docx-1-soc-10.1177_00380385221129953 for Social Mobility and ‘Openness’ in Creative Occupations since the 1970s by Orian Brook, Andrew Miles, Dave O’Brien and Mark Taylor in Sociology

Phenotypic plasticity allows carcinoma cells to transiently acquire the quasi-mesenchymal features necessary to detach from the primary mass and proceed along the invasion-metastasis cascade. A broad spectrum of epigenetic mechanisms is likely to cause the epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) transitions necessary to allow local dissemination and distant metastasis. Here, we report on the role played by alternative splicing (AS) in eliciting phenotypic plasticity in epithelial malignancies with focus on colon cancer. By taking advantage of the coexistence of subpopulations of fully epithelial (EpCAMhi) and quasi-mesenchymal and highly metastatic (EpCAMlo) cells in conventional human cancer cell lines, we here show that the differential expression of ESRP1 and other RNA-binding proteins (RBPs) downstream of the EMT master regulator ZEB1 alters the AS pattern of a broad spectrum of targets including CD44 and NUMB, thus resulting in the generation of specific isoforms functionally associated with increased invasion and metastasis. Additional functional and clinical validation studies indicate that both the newly identified RBPs and the CD44s and NUMB2/4 splicing isoforms promote local invasion and distant metastasis and are associated with poor survival in colon cancer. The systematic elucidation of the spectrum of EMT-related RBPs and AS targets in epithelial cancers, apart from the insights in the mechanisms underlying phenotypic plasticity, will lead to the identification of novel and tumor-specific therapeutic targets.

The human eye can distinguish as many as 10,000 different colours but is far less sensitive to variations in intensity1, meaning that colour is highly desirable when interpreting images. However, most biological samples are essentially transparent, and nearly invisible when viewed using a standard optical microscope2. It is therefore highly desirable to be able to produce coloured images without needing to add any stains or dyes, which can alter the sample properties. Here we demonstrate that colorimetric histology images can be generated using full-sized plasmonically active microscope slides. These slides translate subtle changes in the dielectric constant into striking colour contrast when samples are placed upon them. We demonstrate the biomedical potential of this technique, which we term histoplasmonics, by distinguishing neoplastic cells from normal breast epithelium during the earliest stages of tumorigenesis in the mouse MMTV-PyMT mammary tumour model. We then apply this method to human diagnostic tissue and validate its utility in distinguishing normal epithelium, usual ductal hyperplasia, and early-stage breast cancer (ductal carcinoma in situ). The colorimetric output of the image pixels is compared to conventional histopathology. The results we report here support the hypothesis that histoplasmonics can be used as a novel alternative or adjunct to general staining. The widespread availability of this technique and its incorporation into standard laboratory workflows may prove transformative for applications extending well beyond tissue diagnostics. This work also highlights opportunities for improvements to digital pathology that have yet to be explored.

The discoveries leading to our present understanding of the glutathione peroxidases (GPxs) are recalled. The cytosolic GPx, now GPx1, was first described by Mills in 1957 and claimed to depend on selenium by Rotruck et al., in 1972. With the determination of a stoichiometry of one selenium per subunit, GPx1 was established as the first selenoenzyme of vertebrates. In the meantime, the GPxs have grown up to a huge family of enzymes that prevent free radical formation from hydroperoxides and, thus, are antioxidant enzymes, but they are also involved in regulatory processes or synthetic functions. The kinetic mechanism of the selenium-containing GPxs is unusual in neither showing a defined K

The soft lattice of lead-halide perovskite nanocrystals (NCs) allows tuning their optoelectronic characteristics via anion exchange by introducing halide salts to a solution of perovskite NCs. Similarly, cross-anion exchange can occur upon mixing NCs of different perovskite halides. This process, though, is detrimental for applications requiring perovskite NCs with different halides in close proximity. We study the effects of various stabilizing surface ligands on the kinetics of the cross-anion exchange reaction, comparing zwitterionic and ionic ligands. The kinetic analysis, inspired by the "cage effect" for solution reactions, showcases a mechanism where the surface capping ligands act as anion carriers that diffuse to the NC surface, forming an encounter pair enclosed by the surrounding ligands that initiates the anion exchange process. The zwitterionic ligands considerably slow down the cross-anion exchange process, and while they do not fully inhibit it, they confer improved stability alongside enhanced solubility relevant for various applications., Nano Letters, 22 (11), ISSN:1530-6984, ISSN:1530-6992

Objective The contribution of beta-cell dysfunction to type 2 diabetes (T2D) is not restricted to insulinopenia in the late stages of the disease. Elevated fasting insulinemia in normoglycemic humans is a major factor predicting the onset of insulin resistance and T2D, demonstrating an early alteration of beta-cell function in T2D. Moreover, an early and chronic increase in fasting insulinemia contributes to insulin resistance in high-fat diet (HFD)-fed mice. However, whether there are genetic factors that promote beta-cell-initiated insulin resistance remains undefined. Human variants of the mitochondrial transporter ABCB10, which regulates redox by increasing bilirubin synthesis, have been associated with an elevated risk of T2D. The effects of T2D ABCB10 variants on ABCB10 expression and the actions of ABCB10 in beta-cells are unknown. Methods The expression of beta-cell ABCB10 was analyzed in published transcriptome datasets from human beta-cells carrying the T2D-risk ABCB10 variant. Insulin sensitivity, beta-cell proliferation, and secretory function were measured in beta-cell-specific ABCB10 KO mice (Ins1Cre-Abcb10flox/flox). The short-term role of beta-cell ABCB10 activity on glucose-stimulated insulin secretion (GSIS) was determined in isolated islets. Results Carrying the T2Drisk allele G of ABCB10 rs348330 variant was associated with increased ABCB10 expression in human beta-cells. Constitutive deletion of Abcb10 in beta-cells protected mice from hyperinsulinemia and insulin resistance by limiting HFD-induced beta-cell expansion. An early limitation in GSIS and H2O2-mediated signaling caused by elevated ABCB10 activity can initiate an over-compensatory expansion of beta-cell mass in response to HFD. Accordingly, increasing ABCB10 expression was sufficient to limit GSIS capacity. In health, ABCB10 protein was decreased during islet maturation, with maturation restricting beta-cell proliferation and elevating GSIS. Finally, ex-vivo and short-term deletion of ABCB10 in islets isolated from HFD-fed mice increased H2O2 and GSIS, which was reversed by bilirubin treatments. Conclusions Beta-cell ABCB10 is required for HFD to induce insulin resistance in mice by amplifying beta-cell mass expansion to maladaptive levels that cause fasting hyperinsulinemia., Highlights • ABCB10 deletion in mouse beta-cells protects from HFD-induced insulin resistance. • ABCB10 deletion increases mouse beta-cell function and limits HFD-induced beta cell expansion. • T2D ABCB10 variant rs348330 is associated with increased ABCB0 expression in beta-cells. • ABCB10 gain-of-function decreases glucose stimulated insulin secretion.

Abstract: Low-molecular-mass selenium (LMM-Se) molecules, such as ebselen and diphenyldiselenide, have many biological and potential therapeutic activities; however, little is known about their mechanism of action. It has been stipulated that LMM-Se can modify the physiological chemistry of endogenous thiol (–SH) and selenol (–SeH) groups by different mechanisms. Generically, LMM-Se compounds are poor mimetics of glutathione peroxidase (GPx) enzyme, suggesting that their thiol-modifier effect is more reasonable to justify their biological action. Unfortunately, the LMM-Se interactions with their targets are relatively non-specific. Here, the action of LMM-Se as potential therapeutic agents will be discussed, as well as the bottleneck and myths about their potential use as therapeutic agents.

We present a multiscale, parameter-free approach to the estimation of a series of archetypal SN2 substitutions of halides in halomethane.

It has been previously shown (Phys. Rev. E 66, 066305, 2002) that a non-rotating turbulent convection with nonuniform large-scale flows contributes to the turbulent heat flux. As a result, the turbulent heat flux depends explicitly not only on the gradients of the large-scale temperature, but it also depends on the gradients of the large-scale velocity. This is because the nonuniform large-scale flows produce anisotropic velocity fluctuations which modify the turbulent heat flux. This effect causes an excitation of a convective-wind instability and formation of large-scale semi-organised coherent structures (large-scale convective cells). We perform mean-field numerical simulations of shear-free convection which take into account the modification of the turbulent heat flux by nonuniform large-scale flows. The redistribution of the turbulent heat flux by the nonuniform large-scale motions in turbulent convection plays a crucial role in the formation of the large-scale semi-organised coherent structures. This effect results in a strong reduction of the critical effective Rayleigh number (based on the eddy viscosity and turbulent temperature diffusivity) required for the formation of the large-scale convective cells. The convective-wind instability is excited when the scale separation ratio between the height of the convective layer and the integral turbulence scale is large. The level of the mean kinetic energy at saturation increases with the scale separation ratio. Inside the large-scale convective cells, there are local regions with the positive vertical gradient of the potential temperature which implies that these regions are stably stratified., Comment: 13 pages, 14 figures, revtex4-2

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Mitochondrial depolarization can initiate reversal activity of ATP synthase, depleting ATP by its hydrolysis. We have recently shown that increased ATP hydrolysis contributes to ATP depletion leading to a maladaptation in mitochondrial disorders, where maximal hydrolytic capacity per CV content is increasing. However, despite its importance, ATP hydrolysis is not a commonly studied parameter because of the limitations of the currently available methods. Methods that measure CV hydrolytic activity indirectly require the isolation of mitochondria and involve the introduction of detergents, preventing their utilization in clinical studies or any high-throughput analyses. Here, we describe a novel approach to assess maximal ATP hydrolytic capacity and maximal respiratory capacity in a single assay in cell lysates, PBMCs, and tissue homogenates that were previously frozen. The methodology described here has the potential to be used in clinical samples to determine adaptive and maladaptive adjustments of CV function in diseases, with the added benefit of being able to use frozen samples in a high-throughput manner and to explore ATP hydrolysis as a drug target for disease treatment.

The reactive oxygen species (ROS) scavenging capacities of ginkgolides and bilobalide, which are the peculiar constituents of the extract of Ginkgo biloba, are investigated in silico (level of theory: (SMD)-M06-2X/6-311+G(d,p)//M06-2X/6-31G(d)). Unlike other popular antioxidant natural substances, the carbon backbones of these compounds are entirely aliphatic and exclusively single C–C bonds are present. The selectivity for alkoxyl radicals via hydrogen-atom transfer (HAT) is assessed; importantly, the scavenging of peroxyl radicals is also possible from a peculiar site, here labeled C10 both for ginkgolides and bilobalide. The energetics are described in detail, and the analysis discloses that the studied compounds are powerful scavengers, with thermodynamic and kinetic properties similar to those of Trolox and melatonin, and that, in addition, they display selectivity for peroxyl radicals. These are all chemical-reactivity features contributing to the therapeutic action of the extract of G. biloba.