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Autophagy is a central process for degradation of intracellular components that do not operate correctly. Molecular mechanisms underlying this process are extremely difficult to study, since they involve a large number of participants. The main task of autophagy is redistribution of cellular resources in response to environmental changes, such as starvation. Recent studies show that autophagy regulation could be the key to achieve healthy longevity, as well as to create therapeutic agents for treatment of neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases. Thus, development of autophagy activators with established detailed mechanism of action is a really important area of research. Several commercial companies are at various stages of development of such molecules, and some of them have already begun to introduce autophagy activators to the market.

The development of new energy vehicles, particularly electric vehicles (EVs) and hydrogen fuel cell vehicles (HFCVs), represents a strategic initiative to address climate change and foster sustainable development. Integrating PV with hydrogen production into hybrid electricity-hydrogen energy stations enhances land and energy efficiency but introduces scheduling challenges due to uncertainties. A multi-time scale scheduling framework, which includes day-ahead and intraday optimization, is established using fuzzy chance-constrained programming to minimize costs while considering the uncertainties of PV generation and charging/refueling demand. Correspondingly, trapezoidal membership function and triangular membership function are used for the fuzzy quantification of day-ahead and intraday predictions of photovoltaic power generation and load demands. The system achieves 29.37% lower carbon emissions and 17.73% reduced annualized costs compared to day-ahead-only scheduling. This is enabled by real-time tracking of PV/load fluctuations and optimized electrolyzer/fuel cell operations, maximizing renewable energy utilization. The proposed multi-time scale framework dynamically addresses short-term fluctuations in PV generation and load demand induced by weather variability and temporal dynamics. By characterizing PV/load uncertainties through fuzzy methods, it enables formulation of chance-constrained programming models for operational risk quantification. The confidence level – reflecting decision-makers’ reliability expectations – progressively increases with refined temporal resolution, balancing economic efficiency and operational reliability.

Background Respirable crystalline silica causes lung carcinomas and many thousand future cancer cases are expected in e.g. Europe. Critical questions are how silica causes genotoxicity in the respiratory epithelium and if new cases can be avoided by lowered permissible exposure levels. In this study we investigate early DNA damaging effects of low doses of silica particles in respiratory epithelial cells in vitro and in vivo in an effort to understand low-dose carcinogenic effects of silica particles. Results We find DNA damage accumulation already after 5–10 min exposure to low doses (5 μg/cm 2 ) of silica particles (Min-U-Sil 5) in vitro. DNA damage was documented as increased levels of γH2AX, pCHK2, by Comet assay, AIM2 induction, and by increased DNA repair (non-homologous end joining) signaling. The DNA damage response (DDR) was not related to increased ROS levels, but to a NLRP3-dependent mitochondrial depolarization. Particles in contact with the plasma membrane elicited a Ser198 phosphorylation of NLRP3, co-localization of NLRP3 to mitochondria and depolarization. FCCP, a mitochondrial uncoupler, as well as overexpressed NLRP3 mimicked the silica-induced depolarization and the DNA damage response. A single inhalation of 25 μg silica particles gave a similar rapid DDR in mouse lung. Biomarkers (CC10 and GPRC5A) indicated an involvement of respiratory epithelial cells. Conclusions Our findings demonstrate a novel mode of action (MOA) for silica-induced DNA damage and mutagenic double strand breaks in airway epithelial cells. This MOA seems independent of particle uptake and of an involvement of macrophages. Our study might help defining models for estimating exposure levels without DNA damaging effects.

Intracerebral hemorrhage (ICH) is a devastating disease, in which neuroinflammation substantially contributes to brain injury. Uncoupling protein 2 (UCP2) is a member of the mitochondrial anion carrier family, which uncouples oxidative phosphorylation from ATP synthesis by facilitating proton leak across the mitochondrial inner membrane. UCP2 has been reported to modulate inflammation. In this study we investigated whether and how UCP2 modulated neuroinflammation through microglia/macrophages following ICH in vitro and in vivo. We used an in vitro neuroinflammation model in murine BV2 microglia to mimic microglial activation following ICH. ICH in vivo model was established in mice through collagenase infusion into the left striatum. ICH mice were treated with anetholetrithione (ADT, 50 mg· kg −1  ·d −1 , ip) or the classical protonophoric uncoupler FCCP (injected into hemorrhagic striatum). We showed that the expression and mitochondrial location of microglial UCP2 were not changed in both in vitro and in vivo ICH models. Knockdown of UCP2 exacerbated neuroinflammation in BV2 microglia and mouse ICH models, suggesting that endogenous UCP2 inhibited neuroinflammation and therefore played a protective role following ICH. ADT enhanced mitochondrial ROS production thus inducing mitochondrial uncoupling and activating UCP2 in microglia. ADT robustly suppressed neuroinflammation, attenuated brain edema and improved neurological deficits following ICH, and these effects were countered by striatal knockdown of UCP2. ADT enhanced AMP-activated protein kinase (AMPK) activation in the hemorrhagic brain, which was abrogated by striatal knockdown of UCP2. Moreover, striatal knockdown of AMPK abolished the suppression of neuroinflammation by ADT following ICH. On the other hand, FCCP-induced mitochondrial uncoupling was independent of UCP2 in microglia; and striatal knockdown of UCP2 did not abrogate the suppression of neuroinflammation by FCCP in ICH mice. In conclusion, the uncoupling activity is essential for suppression of neuroinflammation by UCP2. We prove for the first time the concept that activators of endogenous UCP2 such as anetholetrithione are a new class of uncouplers with translational significance.

There has been renewed interest in using mitochondrial uncoupler compounds such as niclosamide and carbonyl cyanide p‐(trifluoromethoxy)phenylhydrazone (FCCP) for the treatment of obesity, hepatosteatosis and diseases where oxidative stress plays a role. However, both FCCP and niclosamide have undesirable effects that are not due to mitochondrial uncoupling, such as inhibition of mitochondrial oxygen consumption by FCCP and induction of DNA damage by niclosamide. Through structure–activity analysis, we identified FCCP analogues that do not inhibit mitochondrial oxygen consumption but still provided good, although less potent, uncoupling activity. We also characterized the functional role of the niclosamide 4′‐nitro group, the phenolic hydroxy group and the anilide amino group in mediating uncoupling activity. Our structural investigations provide important information that will aid further drug development. Uncoupler compounds such as niclosamide and FCCP increase fuel oxidation and are a potential treatment for obesity and hepatosteatosis. However, FCCP and niclosamide exhibit undesirable effects by inhibiting mitochondrial oxygen consumption and inducing DNA damage. Through structural investigations, we characterized the role of specific functional groups in mediating uncoupling and side effects, providing important information to aid further drug development.

Hydrogen sulfide (H(2)S) was historically recognized as a toxic gas generated by natural resources. However, its enzymatic production from L-cysteine has recently been demonstrated in mammals. Cystathionine beta-synthase and cystathionine gamma-lyase, both of which can produce H(2)S, were expressed in mouse pancreatic islet cells and the beta-cell line, MIN6. L-cysteine and the H(2)S donor NaHS inhibited glucose-induced insulin release from islets and MIN6 cells. These inhibitory effects were reproduced when insulin release was stimulated by alpha-ketoisocaproate, tolbutamide, or high K+. L-cysteine and NaHS inhibited glucose-potentiated insulin release in the copresence of diazoxide and high K+. Real-time imaging of intracellular Ca2+ concentration ([Ca2+](i)) demonstrated that both L-cysteine and NaHS reversibly suppressed glucose-induced [Ca2+](i) oscillation in a single beta-cell without obvious changes in the mean value. These substances inhibited Ca2+ - or guanosine 5'-0-3-thiotriphosphate-induced insulin release from islets permeabilized with streptolysin-O. L-cysteine and NaHS reduced ATP production and attenuated glucose-induced hyperpolarization of the mitochondrial membrane potential. Finally, L-cysteine increased H(2)S content in MIN6 cells. We suggest here that L-cysteine inhibits insulin release via multiple actions on the insulin secretory process through H(2)S production. Because the activities of H(2)S-producing enzymes and the tissue H(2)S contents are known to increase under diabetic conditions, the inhibition may participate in the deterioration of insulin release in this disease.

Electric cell-substrate impedance sensing (ECIS) is an emerging technique for sensitively monitoring morphological changes of adherent cells in tissue culture. In this study, human mesenchymal stem cells (hMSCs) were exposed to different concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) for 20 h and their subsequent concentration-dependent responses in micromotion and wound healing migration were measured by ECIS. FCCP disrupts ATP synthesis and results in a decrease in cell migration rates. To detect the change of cell micromotion in response to FCCP challenge, time-series resistances of cell-covered electrodes were monitored and the values of variance were calculated to verify the difference. While Seahorse XF-24 extracellular flux analyzer can detect the effect of FCCP at 3 μM concentration, the variance calculation of the time-series resistances measured at 4 kHz can detect the effect of FCCP at concentrations as low as 1 μM. For wound healing migration, the recovery resistance curves were fitted by sigmoid curve and the hill slope showed a concentration-dependent decline from 0.3 μM to 3 μM, indicating a decrease in cell migration rate. Moreover, dose dependent incline of the inflection points from 0.3 μM to 3 μM FCCP implied the increase of the half time for wound recovery migration. Together, our results demonstrate that partial uncoupling of mitochondrial oxidative phosphorylation reduces micromotion and wound healing migration of hMSCs. The ECIS method used in this study offers a simple and sensitive approach to investigate stem cell migration and its regulation by mitochondrial dynamics.

FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) is known to inhibit oxidative phosphorylation as a protonophore, dissipating the proton gradient across the inner mitochondrial membrane. To understand the toxicity of FCCP, 3-day, 2- and 4-week repeated oral dose studies were performed in male rats. In the 3-day and 2-week repeated dose toxicity studies, observations included salivation, increased body temperature, and dead and moribund animals. Increased liver weight was observed in conjunction with hydropic degeneration and centrilobular necrosis of hepatocytes. In addition, pathological changes were observed in the pancreas, testis, epididymal duct, stomach and parotid gland. Electron microscopic examination revealed mitochondrial pleomorphism in the hepatocytes. Swelling of mitochondria was observed in the alpha cells and beta cells of the pancreas. Dilatation of rough endoplasmic reticulum, Golgi bodies and loss of secretory granules were also noted in the beta cells of the pancreas. FCCP was also compared with three other mUncouplers (DNP, OPC-163493 and tolcapone) with regard to in vitro mitochondrial uncoupling (mUncoupling) activities. FCCP produced the peak ΔOCR (oxygen consumption rate) at the lowest concentration (0.4 μM), followed by OPC-163493, tolcapone, and DNP, based on peak values in ascending order of concentration (2.5, 10, and 50 μM, respectively). Considering the relationship between the mUncoupling activity and toxicity profile of the four mUncouplers, there is no parallel relationship between the in vitro mUncoupling activity and the degree of in vivo toxicity. These findings may contribute to the efficient development of new mitochondrial uncoupler candidates.

Flavonoids such as quercetin and kaempferol mediate several health protective effects, e.g., anticancer effects. They are inhibitors of organic anion transporting polypeptides (OATP) and organic cation transporters (e.g., OCT2). However, little is known whether such transporters contribute to the cellular uptake of flavonoids. Therefore, we investigated the cellular uptake of kaempferol and quercetin using HEK293 cell lines stably expressing different human OATPs or OCT1. Kaempferol was not a substrate of any of the investigated transporters (OATP1A2, OATP1B1, OATP1B3, OATP2A1, OATP2B1, OATP3A1, OATP4A1, OATP5A1, and OCT1). Quercetin showed a significantly higher uptake into the HEK293-OATP1A2, HEK293-OATP2A1, HEK293-OATP2B1, and HEK293-OCT1 cells compared to control cells. The OATP1A2-, OATP2B1-, and OCT1-mediated quercetin uptake was inhibited by known inhibitors such as naringin, cyclosporin A, and quinidine, respectively. The cellular accumulation of quercetin into HEK293-OATP2A1 cells was not inhibited by prostaglandin E 2 and diclofenac. The ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) reduced the net uptake of quercetin by increasing the uptake in the HEK293-control cells and causing no significant change in the HEK293-OATP2B1 cells indicating that quercetin follows the FCCP-driven proton flux through the plasma membrane. In addition to passive diffusion, the SLC transporters OATP1A2, OATP2B1, and OCT1 contribute to cellular accumulation of quercetin.

JC was funded by the National Agency for Research and Development (ANID), Ministry of Education, Chile (Scheme: Becas Chile Doctorado en el Extranjero, PFCHA; Grant: 2017/72180128). The authors also acknowledge the support from the Ministry of Science and Innovation, Spain (Grants: RYC-2014-15581 and AGL2017-88329-R), and the Regional Government of Catalonia, Spain (2017-SGR-1229).