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The author reflects on studies regarding the role of a murine knockout of the TREK-1 channel in the regulation of cerebral vasomotor tone. The study conducted by Namiranian and colleagues involves an evaluation of K+ currents in vascular smooth muscle cells that are isolated from cerebral arteries of mice. Furthermore, the study conducted by Blondeau and colleagues shows a minimal role of TREK-1 in the regulation of carotid artery diameter.

Phenanthraquinone (PQ), a compound affixed to diesel exhaust particles, compromises vascular function. In certain populations, particle exposure may exacerbate the impairment. The principal nutrient artery (PNA) is the primary resistance artery that regulates blood flow (BF) to the cortex and marrow in long bones of adult humans and rats. Endothelium-dependent vasodilation of the PNA declines with aging and corresponds with reduced femoral bone BF. Therefore, exposure of the PNA to PQ could further attenuate skeletal perfusion. The purpose of this study was to 1) Determine whether PQ impairs vasodilation in the PNA and 2) determine whether age, gender, or estrogen alters the presumed effects of PQ. Femoral PNAs from intact and ovariectomized (OVX) female rats (6, 14, and 24 mo) and male rats (6 and 24 mo) were isolated and cannulated. Vasodilation to ACh (10-9-10-4M) was assessed before and during incubation with PQ (5 µM). Vasodilation was diminished in 24 mo female and male rats. At 6 mo, PQ attenuated vasodilation by ∼65% in male rats, but had no effect in female rats. At 14 mo (female rats) and 24 mo (female and male), PQ attenuated and abolished vasodilation, respectively. In all OVX rats, PQ abolished vasodilation. Aging impairs endothelium-dependent vasodilation. Following the loss of estrogen, particularly at 6 mo where a cardioprotective effect was observed, PQ obliterated vasodilation. Therefore, the ability of the PNA to regulate bone marrow BF could be compromised after diesel particle exposure in males, the elderly, and post-menopausal women. [ABSTRACT FROM AUTHOR]

Aging is associated with inspiratory muscle dysfunction; however, the impact of aging on diaphragm blood flow (BF) regulation, and whether sex differences exist, is unknown. We tested the hypotheses in young animals that diaphragm BF and vascular conductance (VC) would be greater in females and that aging would decrease the diaphragm's ability to increase BF with contractions. Young (4-6 mo) and old (22-24 mo) Fischer 344 rats were divided into four groups: young female (YF, n = 7), young male (YM, n = 8), old female (OF, n = 9), and old male (OM, n = 9). Diaphragm BF (mL/min/100 g) and VC (mL/mmHg/min/100 g) were determined, via fluorescent microspheres, at rest and during 1 Hz contractions. In YF versus OF, aging blunted the increase in medial costal diaphragm BF (44 ± 5% vs. 16 ± 12%; P < 0.05) and VC (43 ± 7% vs. 21 ± 12%; P < 0.05). Similarly, in YM versus OM, aging blunted the increase in medial costal diaphragm BF (43 ± 6% vs. 24 ± 12%; P < 0.05) and VC (50 ± 6% vs. 34 ± 10%; P < 0.05). In female rats, age increased dorsal costal diaphragm BF, whereas in male rats, age increased crural diaphragm BF ( P < 0.05). Compared with age-matched females, dorsal costal diaphragm BF was lower in YM and OM ( P < 0.05). In conclusion, aging results in an inability to augment medial costal diaphragm BF and alters regional diaphragm BF distribution in response to muscular contractions. Furthermore, sex differences in regional diaphragm BF are present in young and old animals. NEW & NOTEWORTHY This is the first study, to our knowledge, to demonstrate that old age impairs the hyperemic response and alters blood flow distribution in the diaphragm of both female and male rats. In addition, this investigation provides novel evidence of sex differences in regional diaphragm blood flow distribution with contractions. The data presented herein suggest that aging compromises diaphragm vascular function and provides a potential mechanism for the diaphragm contractile dysfunction associated with old age.

Motivation: High-throughput omics methods increasingly result in large datasets including metabolomics data, which are often difficult to analyse., Results: To help researchers to handle and analyse those datasets by mapping and investigating metabolomics data of multiple sampling conditions (e.g. different time points or treatments) in the context of pathways, PathwayNexus has been developed, which presents the mapping results in a matrix format, allowing users to easily observe the relations between the compounds and the pathways. It also offers functionalities like ranking, sorting, clustering, pathway views, and further analytical tools. Its primary objective is to condense large sets of pathways into smaller, more relevant subsets that align with the specific interests of the user., Availability and Implementation: The methodology presented here is implemented in PathwayNexus, an open-source add-on for Vanted available at www.cls.uni-konstanz.de/software/pathway-nexus., Contact: falk.schreiber@unikonstanz.de., Supplementary Information: Website: www.cls.uni-konstanz.de/software/pathway-nexus., (© The Author(s) 2024. Published by Oxford University Press.)

The magnitude of bone formation and remodeling is linked to both the magnitude of strain placed on the bone and the perfusion of bone. It was previously reported that an increase in bone perfusion and bone density occurs in the femur of old rats with moderate aerobic exercise training. This study determined the acute and chronic effects of static muscle stretching on bone blood flow and remodeling. Old male Fischer 344 rats were randomized to either a naive or stretch-trained group. Static stretching of ankle flexor muscles was achieved by placement of a dorsiflexion splint on the left ankle for 30 min/d, 5d/wk for 4wk. The opposite hindlimb served as a contralateral control (nonstretched) limb. Bone blood flow was assessed during and after acute stretching in naive rats, and at rest and during exercise in stretch-trained rats. Vascular reactivity of the nutrient artery of the proximal tibia was also assessed in stretch-trained rats. MicroCT analysis was used to assess bone volume and micro-architecture of the trabecular bone of both tibias near that growth plate. In naive rats, static stretching increased blood flow to the proximal tibial metaphasis. Blood flow to the proximal tibial metaphysis during treadmill exercise was higher in the stretched limb after 4 wk of daily stretching. Daily stretching also increased tibial bone weight and increased total volume in both the proximal and distal tibial metaphyses. In the trabecular bone immediately below the proximal tibial growth plate, total volume and bone volume increased, but bone volume/total volume was unchanged and trabecular connectivity decreased. In contrast, intravascular volume increased in this region of the bone. These data suggest that blood flow to the tibia increases during bouts of static stretching of the hindlimb muscles, and that 4 wk of daily muscle stretching leads to bone remodeling and an increase in intravascular volume of the tibial bone., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

To characterize the hits from a phenotypic neurotoxicity screen, we obtained transcriptomics data for valinomycin, diethylstilbestrol, colchicine, rotenone, 1-methyl-4-phenylpyridinium (MPP), carbaryl and berberine (Ber). For all compounds, the concentration triggering neurite degeneration correlated with the onset of gene expression changes. The mechanistically diverse toxicants caused similar patterns of gene regulation: the responses were dominated by cell de-differentiation and a triggering of canonical stress response pathways driven by ATF4 and NRF2. To obtain more detailed and specific information on the modes-of-action, the effects on energy metabolism (respiration and glycolysis) were measured. Ber, rotenone and MPP inhibited the mitochondrial respiratory chain and they shared complex I as the target. This group of toxicants was further evaluated by metabolomics under experimental conditions that did not deplete ATP. Ber (204 changed metabolites) showed similar effects as MPP and rotenone. The overall metabolic situation was characterized by oxidative stress, an over-abundance of NADH (>1000% increase) and a re-routing of metabolism in order to dispose of the nitrogen resulting from increased amino acid turnover. This unique overall pattern led to the accumulation of metabolites known as biomarkers of neurodegeneration (saccharopine, aminoadipate and branched-chain ketoacids). These findings suggest that neurotoxicity of mitochondrial inhibitors may result from an ensemble of metabolic changes rather than from a simple ATP depletion. The combi-omics approach used here provided richer and more specific MoA data than the more common transcriptomics analysis alone. As Ber, a human drug and food supplement, mimicked closely the mode-of-action of known neurotoxicants, its potential hazard requires further investigation.

The hormone adiponectin has many beneficial effects in atherosclerosis, as gene deficiency in adiponectin or its receptor has shown detrimental effects on plaque burden in mice. Our objective was to understand the potential roles adiponectin deficiency has on aortic plaque content, inflammation, and markers of cardiovascular disease according to sex and age. To study the influence of adiponectin status on sex and atherosclerosis, we used young male and female adipoq -/- apoe -/- , adipoq +/ - apoe -/- , and apoe -/- mice, which were given a high-fat diet (HFD). Even a 50% reduction in the expression of adiponectin led to a plaque reduction in males and an increase in females compared with apoe -/- controls. Changes in plaque were not attributed to changes in cholesterol or cardiovascular disease markers but correlated with inflammatory markers. Plaque reduction in males was associated with reduced monocyte chemoattractant protein 1 (MCP1) and increased colony stimulating factor 3 (CSF3), while the increase in plaque in females correlated with the opposite effect in these markers. In old mice, both adiponectin-deficient genotypes and sexes accumulated more plaque than their respective apoe -/- controls. The increase in plaque with adiponectin deficiency according to age was not explained by a worsening lipid profile but correlated with increased levels of C-C motif chemokine ligand 5 (CCL5). Overall, our study uncovered genotype-specific effects that differed by sex and age of adiponectin deficiency in atherosclerosis.

Analysis of the transcriptomic alterations upon chemical challenge, provides in depth mechanistic information on the compound's toxic mode of action, by revealing specific pathway activation and other transcriptional modulations. Mapping changes in cellular behaviour to chemical insult, facilitates the characterisation of chemical hazard. In this study, we assessed the transcriptional landscape of mitochondrial impairment through the inhibition of the electron transport chain (ETC) in a human renal proximal tubular cell line (RPTEC/TERT1). We identified the unfolded protein response pathway (UPR), particularly the PERK/ATF4 branch as a common cellular response across ETC I, II and III inhibitions. This finding and the specific genes elaborated may aid the identification of mitochondrial liabilities of chemicals in both legacy data and prospective transcriptomic studies., (© 2023. The Author(s).)

Adiponectin (adipoq), the most abundant hormone in circulation, has many beneficial effects on the cardiovascular system, in part by preserving the contractile phenotype of vascular smooth muscle cells (VSMCs). However, the lack of adiponectin or its receptor and treatment with recombinant adiponectin have shown contradictory effects on plaque in mice. RNA sequence of Adipoq +/+ and adipoq -/- VSMCs from male aortas identified a critical role for adiponectin in AKT signaling, the extracellular matrix (ECM), and TGF-β signaling. Upregulation of AKT activity mediated proliferation and migration of adipoq -/- cells. Activation of AMPK with metformin or AdipoRon reduced AKT-dependent proliferation and migration of adipoq -/- cells but did not improve the expression of contractile genes. Adiponectin deficiency impaired oxidative phosphorylation (OXPHOS), increased expression of glycolytic enzymes, and elevated mitochondrial reactive oxygen species (ROS) (superoxide, and hydrogen peroxide). Anti-atherogenic mechanisms targeted the ECM in adipoq -/- cells, downregulating MMP2 and 9 and upregulating decorin (DCN) and elastin (ELN). In vivo, the main sex differences in protein expression in aortas involved a more robust upregulation of MMP3 in females than males. Females also showed a reduction in DCN, which was not affected in males. Our study uncovered the AKT/MAPK/TGF-β network as a central regulator of VSMC phenotype.

Acute aerobic exercise stress is associated with decreased endothelial function that may increase the likelihood of an acute cardiovascular event. Passive stretch (PS) elicits improvements in vascular function, but whether PS can be performed before exercise to prevent declines in vascular function remains unknown. This strategy could be directly applicable in populations that may not be able to perform dynamic exercise. We hypothesized that preexercise PS would provide better vascular resilience after treadmill exercise. Sixteen healthy college-aged males and females participated in a single laboratory visit and underwent testing to assess micro- and macrovascular function. Participants were randomized into either PS group or sham control group. Intermittent calf PS was performed by having the foot in a splinting device for a 5-min stretch and 5-min relaxation, repeated four times. Then, a staged V̇o 2 peak test was performed and 65% V̇o 2 peak calculated for subjects to run at for 30 min. Near-infrared spectroscopy-derived microvascular responsiveness was preserved with the PS group [(pre: 0.53 ± 0.009%/s) (post: 0.56 ± 0.012%/s; P = 0.55)]. However, there was a significant reduction in the sham control group [(pre: 0.67 ± 0.010%/s) (post: 0.51 ± 0.007%/s; P = 0.05)] after treadmill exercise. Flow-mediated vasodilation (FMD) of the popliteal artery showed similar responses. In the PS group, FMD [(pre: 7.23 ± 0.74%) (post: 5.86 ± 1.01%; P = 0.27)] did not significantly decline after exercise. In the sham control group, FMD [(pre: 8.69 ± 0.72%) (post: 5.24 ± 1.24%; P < 0.001)] was significantly reduced after treadmill exercise. Vascular function may be more resilient if intermittent PS is performed before moderate-intensity exercise and, importantly, can be performed by most individuals. NEW & NOTEWORTHY We demonstrate for the first time that popliteal artery and gastrocnemius microvascular responsiveness after acute aerobic exercise are reduced. The decline in vascular function was mitigated in those who performed intermittent passive stretching before the exercise bouts. Collectively, these findings suggest that intermittent passive stretching is a novel method to increase vascular resiliency before aerobic activity.

Proteasome inhibition is associated with parkinsonian pathology in vivo and degeneration of dopaminergic neurons in vitro. We explored here the metabolome (386 metabolites) and transcriptome (3257 transcripts) regulations of human LUHMES neurons, following exposure to MG-132 [100 nM]. This proteasome inhibitor killed cells within 24 h but did not reduce viability for 12 h. Overall, 206 metabolites were changed in live neurons. The early (3 h) metabolome changes suggested a compromised energy metabolism. For instance, AMP, NADH and lactate were up-regulated, while glycolytic and citric acid cycle intermediates were down-regulated. At later time points, glutathione-related metabolites were up-regulated, most likely by an early oxidative stress response and activation of NRF2/ATF4 target genes. The transcriptome pattern confirmed proteostatic stress (fast up-regulation of proteasome subunits) and also suggested the progressive activation of additional stress response pathways. The early ones (e.g., HIF-1, NF-kB, HSF-1) can be considered a cytoprotective cellular counter-regulation, which maintained cell viability. For instance, a very strong up-regulation of AIFM2 (=FSP1) may have prevented fast ferroptotic death. For most of the initial period, a definite life-death decision was not taken, as neurons could be rescued for at least 10 h after the start of proteasome inhibition. Late responses involved p53 activation and catabolic processes such as a loss of pyrimidine synthesis intermediates. We interpret this as a phase of co-occurrence of protective and maladaptive cellular changes. Altogether, this combined metabolomics-transcriptomics analysis informs on responses triggered in neurons by proteasome dysfunction that may be targeted by novel therapeutic intervention in Parkinson's disease.

Microcirculation in skeletal muscle is disturbed with advancing aging, causing limited capillary blood flow and exercise incapacity. Muscle stretch has been widely performed in physical therapy, sports medicine, and health promotion. However, the effect of stretch on microvascular reactivity and muscle blood flow remains unknown. This review focuses on stretch-induced microvascular adaptations based on evidence from cultured cells, small animals, and human studies. Vascular endothelium senses and responds to mechanical stimuli including stretch. This endothelial mechanotransduction potentially plays a vital role in the stretch-induced microvascular adaptation alongside hypoxia. Aging impairs microvascular endothelial function, but muscle stretch has the potential to restore it. Muscle stretch may be an alternative to improve vascular function and enhance exercising blood flow, especially for those who have difficulties in participating in exercise due to medical, functional, or psychological reasons., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hotta and Muller-Delp.)

Adverse Outcome Pathways (AOPs) are increasingly used to support the integration of in vitro data in hazard assessment for chemicals. Quantitative AOPs (qAOPs) use mathematical models to describe the relationship between key events (KEs). In this paper, data obtained in three cell lines, LHUMES, HepG2 and RPTEC/TERT1, using similar experimental protocols, was used to calibrate a qAOP of mitochondrial toxicity for two chemicals, rotenone and deguelin. The objectives were to determine whether the same qAOP could be used for the three cell types, and to test chemical-independence by cross-validation with a dataset obtained on eight other chemicals in LHUMES cells. Repeating the calibration approach for both chemicals in three cell lines highlighted various practical difficulties. Even when the same readouts of KEs are measured, the mathematical functions used to describe the key event relationships may not be the same. Cross-validation in LHUMES cells was attempted by estimating chemical-specific potency at the molecular initiating events and using the rest of the calibrated qAOP to predict downstream KEs: toxicity of azoxystrobin, carboxine, mepronil and thifluzamide was underestimated. Selection of most relevant readouts and accurate characterization of the molecular initiating event for cross-validation are critical when designing in vitro experiments targeted at calibrating qAOPs., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes., (© 2021. The Author(s).)

Introduction: Prolonged mechanical ventilation (MV; ≥6 h) results in large, time-dependent reductions in diaphragmatic blood flow and shear stress. We tested the hypothesis that MV would impair the structural and material properties (ie, increased stress/stretch relation and/or circumferential stretch) of first-order arterioles (1A) from the medial costal diaphragm., Methods: Shear stress was estimated from isolated arterioles and prior blood flow data from the diaphragm during spontaneous breathing (SB) and prolonged MV (6 h MV). Thereafter, female Sprague-Dawley rats (~5 months) were randomly divided into two groups, SB (n = 6) and 6 h MV (n = 6). Following SB and 6 h MV, 1A medial costal diaphragm arterioles were isolated, cannulated, and subjected to stepwise (0-140 cmH 2 O) increases in intraluminal pressure in calcium-free Ringer's solution. Inner diameter and wall thickness were measured at each pressure step and used to calculate wall:lumen ratio, Cauchy-stress, and circumferential stretch., Results: Compared to SB, there was a ~90% reduction in arteriolar shear stress with prolonged MV (9 ± 2 vs 78 ± 20 dynes/cm 2 ; p ≤ .05). In the unloaded condition (0 cmH 2 O), the arteriolar intraluminal diameter was reduced (37 ± 8 vs 79 ± 13 μm) and wall:lumen ratio was increased (120 ± 18 vs 46 ± 10%) compared to SB (p ≤ .05). There were no differences in the passive diameter responses or the circumferential stress/stretch relationship between groups (p > .05), but at each pressure step, circumferential stretch was increased with 6 h MV vs SB (p ≤ .05)., Conclusion: During prolonged MV, medial costal diaphragm arteriolar shear stress is severely diminished. Despite no change in the material behavior (stress/stretch), prolonged MV resulted in altered structural and mechanical properties (ie, elevated circumferential stretch) of medial costal diaphragm arterioles. This provides important novel mechanistic insights into the impaired diaphragm blood flow capacity and vascular dysfunction following prolonged MV., (© 2021 John Wiley & Sons Ltd.)

Until recently, epicardial coronary stenosis has been considered the primary outcome of coronary heart disease, and clinical interventions have been dedicated primarily to the identification and removal of flow-limiting stenoses. However, a growing body of literature indicates that both epicardial stenosis and microvascular dysfunction contribute to damaging myocardial ischemia. In this review, we discuss the coexistence of macro- and microvascular disease, and how the structure and function of the distal microcirculation is impacted by the hemodynamic consequences of an epicardial, flow-limiting stenosis. Mechanisms of endothelial dysfunction as well as alterations of smooth muscle function in the coronary microcirculation distal to stenosis are discussed. Risk factors including diabetes, metabolic syndrome, and aging exacerbate microvascular dysfunction in the myocardium distal to a stenosis, and our current understanding of the role of these factors in limiting collateralization and angiogenesis of the ischemic myocardium is presented. Importantly, exercise training has been shown to promote collateral growth and improve microvascular function distal to stenosis; thus, the current literature reporting the mechanisms that underlie the beneficial effects of exercise training in the microcirculation distal to epicardial stenosis is reviewed. We also discuss recent studies of therapeutic interventions designed to improve microvascular function and stimulate angiogenesis in clinically relevant animal models of epicardial stenosis and microvascular disease. Finally, microvascular adaptation to removal of epicardial stenosis is considered.

Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10-260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.

In the continuing search for novel antibiotics, antimicrobial peptides are promising molecules, due to different mechanisms of action compared to classic antibiotics and to their selectivity for interaction with microorganism cells rather than with mammalian cells. Previously, our research group has isolated the antimicrobial peptide LyeTx I from the venom of the spider Lycosa erythrognatha . Here, we proposed to synthesize three novel shortened derivatives from LyeTx I (LyeTx I mn; LyeTx I mnΔK; LyeTx I mnΔKAc) and to evaluate their toxicity and biological activity as potential antimicrobial agents. Peptides were synthetized by Fmoc strategy and circular dichroism analysis was performed, showing that the three novel shortened derivatives may present membranolytic activity, like the original LyeTx I, once they folded as an alpha helix in 2.2.2-trifluorethanol and sodium dodecyl sulfate. In vitro assays revealed that the shortened derivative LyeTx I mnΔK presents the best score between antimicrobial (↓ MIC) and hemolytic (↑ EC 50 ) activities among the synthetized shortened derivatives, and LUHMES cell-based NeuriTox test showed that it is less neurotoxic than the original LyeTx I (EC 50 [LyeTx I mnΔK] ⋙ EC 50 [LyeTx I]). In vivo data, obtained in a mouse model of septic arthritis induced by Staphylococcus aureus , showed that LyeTx I mnΔK is able to reduce infection, as demonstrated by bacterial recovery assay (∼10-fold reduction) and scintigraphic imaging (less technetium-99m labeled-Ceftizoxime uptake by infectious site). Infection reduction led to inflammatory process and pain decreases, as shown by immune cells recruitment reduction and threshold nociception increment, when compared to positive control group. Therefore, among the three shortened peptide derivatives, LyeTx I mnΔK is the best candidate as antimicrobial agent, due to its smaller amino acid sequence and toxicity, and its greater biological activity.

Read-across approaches are considered key in moving away from in vivo animal testing towards addressing data-gaps using new approach methods (NAMs). Ample successful examples are still required to substantiate this strategy. Here we present and discuss the learnings from two OECD IATA endorsed read-across case studies. They involve two classes of pesticides – rotenoids and strobilurins – each having a defined mode-of-action that is assessed for its neurological hazard by means of an AOP-based testing strategy coupled to toxicokinetic simulations of human tissue concentrations. The endpoint in question is potential mitochondrial respiratory chain mediated neurotoxicity, specifically through inhibition of complex I or III. An AOP linking inhibition of mitochondrial respiratory chain complex I to the degeneration of dopaminergic neurons formed the basis for both cases but was deployed in two different regulatory contexts. The two cases also exemplify several different read-across concepts: analogue versus category approach, consolidated versus putative AOP, positive versus negative prediction (i.e., neurotoxicity versus low potential for neurotoxicity), and structural versus biological similarity. We applied a range of NAMs to explore the toxicodynamic properties of the compounds, e.g., in silico docking as well as in vitro assays and readouts – including transcriptomics – in various cell systems, all anchored to the relevant AOPs. Interestingly, although some of the data addressing certain elements of the read-across were associated with high uncertainty, their impact on the overall read-across conclusion remained limited. Coupled to the elaborate regulatory review that the two cases underwent, we propose some generic learnings of AOP-based testing strategies supporting read-across.

Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.

In the original publication of the article.

Liver receptor homolog-1 (LRH-1, Nr5a2) is an orphan nuclear receptor mainly expressed in tissues of endodermal origin, where its physiological role has been extensively studied. LRH-1 has been implicated in liver cell differentiation and proliferation, as well as glucose, lipid, and bile acid metabolism. In addition, increasing evidence highlights its role in immunoregulatory processes via glucocorticoid synthesis in the intestinal epithelium. Although the direct function of LRH-1 in immune cells is fairly elucidated, a role of LRH-1 in the regulation of macrophage differentiation has been recently reported. In this study, we aimed to investigate the role of LRH-1 in the regulation of pro-inflammatory cytokine production in macrophages. Our data demonstrate that pharmacological inhibition, along with LRH-1 knockdown, significantly reduced the lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines in the macrophage line RAW 264.7 cells, as well as in primary murine macrophages. This inhibitory effect was found to be independent of defects of LRH-1-regulated cell proliferation or toxic effects of the LRH-1 inhibitors. In contrast, LRH-1 inhibition reduced the mitochondrial ATP production and metabolism of macrophages through downregulation of the LRH-1 targets glucokinase and glutminase-2, and thus impairing the LPS-induced macrophage activation. Interestingly, in vivo pharmacological inhibition of LRH-1 also resulted in reduced tumor necrosis factor (TNF) production and associated decreased liver damage in a macrophage- and TNF-dependent mouse model of hepatitis. Noteworthy, despite hepatocytes expressing high levels of LRH-1, pharmacological inhibition of LRH-1 per se did not cause any obvious liver damage. Therefore, this study proposes LRH-1 as an emerging therapeutic target in the treatment of inflammatory disorders, especially where macrophages and cytokines critically decide the extent of inflammation.

Tyrosine nitration is a post-translational protein modification relevant to various pathophysiological processes. Chemical nitration procedures have been used to generate and study nitrated proteins, but these methods regularly lead to modifications at other amino acid residues. A novel strategy employs a genetic code modification that allows incorporation of 3-nitrotyrosine (3-NT) during ribosomal protein synthesis to generate a recombinant protein with defined 3-NT-sites, in the absence of other post-translational modifications. This approach was applied to study the generation and stability of the 3-NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (ASYN) was selected as exemplary protein, relevant in Parkinson's disease (PD). A procedure was established to obtain pure tyrosine-modified ASYN in mg amounts. However, a rapid (t 1/2  = 0.4 h) reduction of 3-NT to 3-aminotyrosine (3-AT) was observed. When screening for potential mechanisms, we found that 3-NT can be reduced enzymatically to 3-AT, whilst biologically relevant low molecular weight reductants, such as NADPH or GSH, did not affect 3-NT. A genetic screen for E.coli proteins, involved in the observed 3-NT reduction, revealed the contribution of several, possibly redundant pathways. Green fluorescent protein was studied as an alternative model protein. These data confirm 3-NT reduction as a broadly-relevant pathway in E.coli. In conclusion, incorporation of 3-NT as a genetically-encoded non-natural amino acid allows for generation of recombinant proteins with specific nitration sites. The potential reduction of the 3-NT moiety by E.coli, however, requires attention to the design of the purification strategy for obtaining pure nitrated protein., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Patients with peripheral arterial disease (PAD) often have walking impairment due to insufficient oxygen supply to skeletal muscle. In aged rats, we have shown that daily stretching of calf muscles improves endothelium-dependent dilation of arterioles from the soleus muscle and increases capillarity and muscle blood flow during exercise. Therefore, we hypothesized that daily muscle stretching of calf muscles would improve endothelium-dependent vasodilation of the popliteal artery and walking function in PAD patients., Methods: We performed a randomized, non-blinded, crossover study whereby 13 patients with stable symptomatic PAD were randomized to undergo either 4 weeks of passive calf muscle stretching (ankle dorsiflexion applied 30 min/d, 5 days/wk) followed by 4 weeks of no muscle stretching and vice versa. Endothelium-dependent flow-mediated dilation (FMD) and endothelium-independent nitroglycerin-induced dilation of the popliteal artery and 6 minute walk test (6MWT) were evaluated at baseline and after each 4 week interval., Results: After 4 weeks of muscle stretching, FMD and 6MWT improved significantly in the muscle stretching group vs. the control (FMD: 5.1 ± 0.5% vs. 3.7 ± 0.3%, P = 0.005; 6MWT continuous walking distance: 304 ± 43 m vs. 182 ± 34 m; P = 0.0006). No difference in nitroglycerin-induced dilation was found between groups (10.9 ± 1.2 vs. 9.9 ± 0.4%, P = 0.48). Post-stretching, 6MWT total walking distance was positively correlated with normalized FMD (R = 0.645, P = 0.02)., Conclusions: Passive calf muscle stretching enhanced vascular endothelial function and improved walking function in elderly patients with stable symptomatic PAD. These findings merit further investigation in a prospective randomized trial., (Copyright © 2019 Elsevier Inc. All rights reserved.)