Pristane promotes anaerobic glycolysis to facilitate proinflammatory activation of macrophages and development of arthritis.

Pristane-induced arthritis (PIA) could be adoptively transferred by splenic T cells in rats, and innate immunity should play critical roles in T cell activation. However, in pre-clinical stage, the activation mechanism of innate cells like macrophages remains unclear. Here we found that PIA was dependent on macrophages since cell depletion alleviated disease severity. Splenic macrophages of PIA rats showed M1 phenotypic shifting. The quantitative proteomics analysis suggested that macrophages initiated metabolic reprogramming with the conversion of aerobic oxidation to glycolysis in response to pristane in vivo. Notably, macrophages treated with pristane showed mitochondrial dysregulation and increased glycolysis flux and enzyme activity. Additionally, TNFα production, strongly associating with the glycolysis enzyme Ldha/Ldhb, could be reduced as glycolysis was inhibited or be enhanced as citrate cycle was blocked. This work provides detailed insights into the molecular mechanisms of pristane-mediated metabolic reprogramming in macrophages and suggests a new therapeutic strategy for arthritic disorders.

Cadmium Selenide Formation Influences the Production and Characteristics of Extracellular Polymeric Substances of Anaerobic Granular Sludge.

Feeding cadmium (II) and selenium (IV) simultaneously to anaerobic granular sludge with the aim of synthesizing cadmium selenide (CdSe) nanoparticles induces compositional changes in the extracellular polymeric substances (EPS) matrix of this sludge. A methanogenic anaerobic granular sludge was repeatedly exposed to Cd(II) (10-50 mg L-1) and selenite (79 mg L-1) for 300 days at pH 7.3 and 30 °C in a fed-batch feeding regime for enrichment of Se-reducing bacteria and synthesis of CdSe nanoparticles. EPS fingerprints of the granular sludge, obtained by size exclusion chromatography coupled to a fluorescence detector, showed a significant increase in the intensity of protein-like substances with > 100 kDa apparent molecular weight (aMW) upon repeated exposure to Cd(II) and Se(VI). This was accompanied by a prominent decrease in protein-like substances of aMW < 10 kDa. The fingerprint of the humic-like substances showed emergence of a new peak with aMW of 13 to 300 kDa in the EPS extracted from the Cd/Se fed granular sludge. Experiments on metal(loid)-EPS interactions showed that the CdSe nanoparticles interact mainly with loosely bound EPS (LB-EPS). This study showed that the formation of Se(0) and CdSe nanoparticles occurs in the LB-EPS fraction of the granular sludge and repeated exposure to Cd and Se induces compositional changes in the EPS matrix.

Untargeted Metabolomics Reveals Anaerobic Glycolysis as a Novel Target of the Hepatotoxic Antidepressant Nefazodone.

Mitochondrial damage is considered a hallmark of drug-induced liver injury (DILI). However, despite the common molecular etiology, the evolution of the injury is usually unpredictable, with some cases that are mild and reversible upon discontinuation of the treatment and others characterized by irreversible acute liver failure. This suggests that additional mechanisms of damage play a role in determining the progression of the initial insult. To uncover novel pathways potentially involved in DILI, we investigated in vitro the metabolic perturbations associated with nefazodone, an antidepressant associated with acute liver failure. Several pathways associated with ATP production, including gluconeogenesis, anaerobic glycolysis, and oxidative phosphorylation, were altered in human hepatocellular carcinoma-derived (Huh7) cells after 2-hour exposure to a 50 µM extracellular concentration of nefazodone. In the presence or absence of glucose, ATP production of Huh7 cells was glycolysis- and oxidative phosphorylation-dependent, respectively. In glucose-containing medium, nefazodone-induced ATP depletion from Huh7 cells was biphasic. Huh7 cells in glucose-free medium were more sensitive to nefazodone than those in glucose-containing medium, losing the biphasic inhibition. Nefazodone-induced ATP depletion in primary cultured mouse hepatocytes, mainly dependent on oxidative phosphorylation, was monophasic. At lower extracellular concentrations, nefazodone inhibited the oxygen consumption of Huh7 cells, whereas at higher extracellular concentrations, it also inhibited the extracellular acidification. ATP content was rescued by increasing the extracellular concentration of glucose. In conclusion, nefazodone has a dual inhibitory effect on mitochondrial-dependent and mitochondrial-independent ATP production. SIGNIFICANCE STATEMENT: Mitochondrial damage is a hallmark of drug-induced liver injury, yet other collateral alterations might contribute to the severity and evolution of the injury. Our in vitro study supports previous results arguing that a deficit in hepatic glucose metabolism, concomitant to the mitochondrial injury, might be cardinal in the prognosis of the initial insult to the liver. From a drug development standpoint, coupling anaerobic glycolysis and mitochondrial function assessment might increase the drug-induced liver injury preclinical screening performance.

Low glucose enhanced metformin's inhibitory effect on pancreatic cancer cells by suppressing glycolysis and inducing energy stress via up-regulation of miR-210-5p.

To explore mechanisms underlying the discrepancy in anti-tumor effects of metformin on pancreatic cancer cells PANC-1 under different glucose conditions. We cultured PANC-1 cells in 25 mM and 5 mM glucose media, then treated with or without metformin. It showed that metformin significantly inhibited proliferation and viability, induced apoptosis of PANC-1 cells, which was more pronounced in low-glucose than in high-glucose group. Metformin up-regulated the expression of miR-210-5p in low glucose, but not in high glucose. miR-210-5p mimic inhibited the viability of PANC-1 cells and further enhanced the inhibitory effect of metformin. miR-210-5p down-regulated the expression of PFKFB2, a predicted target gene of miR-210-5p, reduced the activity of PFK1 and LDH. Metformin significantly inhibited the expression of phosphorylation-PFKFB2(p-PFKFB2) in the low-glucose group and inhibited the LDH activity both in the low and high glucose groups, thus inhibiting anaerobic glycolysis and inducing energy stress. Cells in the high glucose group could make a compensatory adaptation to the energy stress induced by metformin through increasing glucose consumption. However, due to the limited glucose supply and high dependence on anaerobic glycolysis of cells in the low glucose group, they couldn't make effective adaptive compensation. Therefore, cells in the low-glucose group were more vulnerable to the toxicity of metformin. In conclusion, the enhanced inhibitory effect of metformin on PANC-1 cells cultured in low glucose may be due to the up-regulation of the expression of miR-210-5p, then inhibiting anaerobic glycolytic flux and inducing energy stress via repressing the expression of p-PFKFB2 and activity of LDH. ABBREVIATIONS: PC: pancreatic cancer; DM: diabetes mellitus; PFKFB2: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase2; PFK1: phosphofructokinases; LDH: lactate dehydrogenase; F-2,6-BP: fructose 2,6-bisphosphate.

Dexmedetomidine alleviated neuropathic pain in dorsal root ganglion neurons by inhibition of anaerobic glycolysis activity and enhancement of ROS tolerance.

Neuropathic pain is a kind of chronic pain that is triggered or caused primarily by damage to the nervous system and neurological dysfunction. It's known that dexmedetomidine is a new type of highly selective alpha2-adrenoceptor agonist with sedation, anti-anxiety, analgesic and other effects. However, the function and mechanism of dexmedetomidine on neuropathic pain are not clear. Rat DRG neurons were isolated and identified using immunofluorescence assay. Following treatment with H2O2, dexmedetomidine or ROS inhibitor (NAC), the apoptosis and ROS levels were examined by flow cytometery; apoptosis- and anaerobic glycolysis-related proteins were determined by Western blot assay; glucose consumption, pyruvic acid, lactic acid and ATP/ADP ratios were also measured. The results revealed that dexmedetomidine inhibited H2O2-induced apoptosis and reactive oxygen species (ROS) in rat DRG neurons and in addition, dexmedetomidine down-regulated the expression levels of anaerobic glycolysis-related proteins, significantly reduced glucose, pyruvic acid and lactic acid levels. It also increased the ATP/ADP ratio in H2O2-treated rat dorsal root ganglion (DRG) neurons. Moreover, we also demonstrated that ROS inhibitor (NAC) also inhibited H2O2-induced apoptosis and anaerobic glycolysis in rat DRG neurons. In conclusion, dexmedetomidine suppressed H2O2-induced apoptosis and anaerobic glycolysis activity by inhibiting ROS, in rat DRG neurons. Therefore, dexmedetomidine might play a pivotal role in neuropathic pain by the inhibition of ROS.

Unravelling the influence of sulfate loading on enhancing anaerobic co-digestion of corn stover and bio-kerosene production wastewater.

This study was conducted to investigate the effect of sulfate loading on methane production and organic matter degradation during the mesophilic anaerobic co-digestion of corn stover and bio-kerosene production wastewater (BKPW). The highest methane production of 192.04 mL/gVS was obtained at a sulfate concentration of 86 mg/L. This was 46.80% higher than that achieved by a sulfate concentration of 113 mg/L. Additional degradation of organic matter was obtained at a sulfate concentration of 113 mg/L because organic matter in the corn stover and BKPW was oxidized by sulfate-reducing bacteria (SRB). The concentration of sulfate declined by approximately 23% after 29 days of anaerobic co-digestion, and this reduction in sulfate was enhanced when the soluble chemical oxygen demand (SCOD)/sulfate ratio was less than 15. The results of a mass balance analysis showed that 34.87% of C element and 10.04% of S element in substrate, respectively, were converted to biogas during anaerobic co-digestion of corn stover and BKPW at a sulfate concentration of 86 mg/L. The microbial community was analysed using 16S rDNA sequencing technology, and the results showed that the relative abundance of Synergistes (related to methane production with acetic acid) at a sulfate concentration of 86 mg/L had obviously increased and was approximately 287% higher than the abundance achieved at a sulfate concentration of 113 mg/L.

The Effects of Thiamine on Breast Cancer Cells.

(1) Background: Thiamine is an important cofactor for multiple metabolic processes. Its role in cancer has been debated for years. Our aim is to determine if thiamine can convert the cellular metabolic state of breast cancer cells from anaerobic to aerobic, thus reducing their growth. (2) Methods: Breast cancer (MCF7) and non-tumorigenic (MCF10A) cell lines were treated with various doses of thiamine and assessed for changes in cell growth. The mechanism of this relationship was identified through the measurement of enzymatic activity and metabolic changes. (3) Results: A high dose of thiamine reduced cell proliferation in MCF7 (63% decrease, p < 0.0001), but didn't affect apoptosis and the cell-cycle profile. Thiamine had a number of effects in MCF7; it (1) reduced extracellular lactate levels in growth media, (2) increased cellular pyruvate dehydrogenase (PDH) activities and the baseline and maximum cellular oxygen consumption rates, and (3) decreased non-glycolytic acidification, glycolysis, and glycolytic capacity. MCF10A cells preferred mitochondrial respiration instead of glycolysis. In contrast, MCF7 cells were more resistant to mitochondrial respiration, which may explain the inhibitory effect of thiamine on their proliferation. (4) Conclusions: The treatment of MCF7 breast cancer cells with 1 µg/mL and 2 µg/mL of thiamine for 24 h significantly reduced their proliferation. This reduction is associated with a reduction in glycolysis and activation of the PDH complex in breast cancer cells.

Anaerobic biodegradation of partially hydrolyzed polyacrylamide in long-term methanogenic enrichment cultures from production water of oil reservoirs.

The increasing usage of partially hydrolyzed polyacrylamide (HPAM) in oilfields as a flooding agent to enhance oil recovery at so large quantities is an ecological hazard to the subsurface ecosystem due to persistence and inertness. Biodegradation of HPAM is a potentially promising strategy for dealing with this problem among many other methods available. To understand the responsible microorganisms and mechanism of HPAM biodegradation under anaerobic conditions, an enrichment culture from production waters of oil reservoirs were established with HPAM as the sole source of carbon and nitrogen incubated for over 328 days, and analyzed using both molecular microbiology and chemical characterization methods. Gel permeation chromatography, High-pressure liquid chromatography and Fourier-transformed infrared spectroscopy results indicated that, after 328 days of anaerobic incubation, some of the amide groups on HPAM were removed and released as ammonia/ammonium and carboxylic groups, while the carbon backbone of HPAM was converted to smaller polymeric fragments, including oligomers and various fatty acids. Based on these results, the biochemical process of anaerobic biodegradation of HPAM was proposed. The phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichments showed that Proteobacteria and Planctomycetes were the dominant bacteria in the culture with HPAM as the source of carbon and nitrogen, respectively. For archaea, Methanofollis was more abundant in the anaerobic enrichment. These results are helpful for understanding the process of HPAM biodegradation and provide significant insights to the fate of HPAM in subsurface environment and for possible bioremediation.

Vascular parameters continue to decrease post-exposure with simultaneous, but not individual exposure to BPA and hypoxia in zebrafish larvae.

How fish respond to hypoxia, a common stressor, can be altered by simultaneous exposure to pollutants like bisphenol A (BPA), a plasticizer. BPA is cardiotoxic and interferes with the hypoxia inducible factor pathway (HIF-1α), therefore disrupting the hypoxic response. Co-exposure to hypoxia and BPA also causes severe bradycardia and reduced cardiac output in zebrafish larvae. The purpose of this work was to determine how the cardiovascular effects of co-exposure vary with BPA concentration and persist beyond exposure. Zebrafish embryos were exposed to 0, 0.01, 0.1, 1, and 100 µg/L of BPA during normoxia (>6.0 mg/L O2) and hypoxia (2.0 ±â€¯0.5 mg/L O2) between 1 h post fertilization (hpf) and late hatching (72-96 hpf). Heart rate, cardiac output, and red blood cell (RBC) velocity were determined through video microscopy and digital motion analysis at late hatching and 10 days post fertilization (dpf), several days post exposure. In comparison to the hypoxic control, RBC velocity was 25% lower with 0.01 µg/L BPA and hypoxia at late hatching. At 10 dpf, the difference in RBC velocity between these treatments doubled, despite several days of recovery. This coincided with a 24% thinner outer diameter for caudal vein but no effect on cardiac or developmental parameters. Statistical interactions between BPA and oxygen concentration were found for arterial RBC velocity at both ages. Because the co-occurrence of both stressors is extremely common, it would be beneficial to understand how BPA and hypoxia interact to affect cardiovascular function during and after exposure.

Low-dose YC-1 combined with glucose and insulin selectively induces apoptosis in hypoxic gastric carcinoma cells by inhibiting anaerobic glycolysis.

This study aimed to establish a therapeutic strategy targeting hypoxic cancer cells in gastric carcinoma (GC). YC-1 is a HIF-1α inhibitor, and we revealed that low-dose YC-1 (10 µM) suppressed HIF-1α expression, and induced hypoxia-dependent apoptosis in the GC cell line 58As9. This hypoxia-specific apoptosis induction by YC-1 involved excessive reactive oxygen species (ROS) generation. The apoptotic effect of 10 µM YC-1 was enhanced by additional glucose (G) and insulin (I) treatments. RT-PCR demonstrated that 10 µM YC-1 reduced hypoxia-induced expression of HIF-1α targets involved in anaerobic glycolysis. Metabolic analysis showed that YC-1 shifted glucose metabolism in hypoxic cells from anaerobic glycolysis to oxidative phosphorylation (OXPHOS). Additional GI accelerated membranous GLUT1 translocation, elevating glucose uptake, and increased acetyl-CoA levels, leading to more ROS generation in hypoxic YC-1-treated cells. Finally, we evaluated the anti-cancer effect of low-dose YC-1 (1 mg/kg) + G (2 g/kg) and I (1 unit/3 g G) treatment in xenograft models. YC-1 + GI therapy strongly inhibited tumour growth. Immunohistochemical analysis demonstrated that YC-1 + GI reduced HIF-1α expression and pimonidazole accumulation in tumours. Conversely, YC-1 + GI increased intra-tumoral 8-OHdG and levels of apoptosis markers. Low-dose YC-1 + GI is a unique therapy targeting hypoxic GC cells that generates lethal ROS via forced activation of OXPHOS.

Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA.

Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, which is highly prevalent worldwide and has a major impact on reproductive and neonatal health. The superbug status of N. gonorrhoeae necessitates the development of drugs with different mechanisms of action. Here, we focused on targeting the nitrite reductase AniA, which is a pivotal component of N. gonorrhoeae anaerobic respiration and biofilm formation. Our studies showed that gonococci expressing AniA containing the altered catalytic residues D137A and H280A failed to grow under anaerobic conditions, demonstrating that the nitrite reductase function is essential. To facilitate the pharmacological targeting of AniA, new crystal structures of AniA were refined to 1.90-Å and 2.35-Å resolutions, and a phage display approach with libraries expressing randomized linear dodecameric peptides or heptameric peptides flanked by a pair of cysteine residues was utilized. Biopanning experiments led to the identification of 29 unique peptides, with 1 of them, C7-3, being identified multiple times. Evaluation of their ability to interact with AniA using enzyme-linked immunosorbent assay and computational docking studies revealed that C7-3 was the most promising inhibitor, binding near the type 2 copper site of the enzyme, which is responsible for interaction with nitrite. Subsequent enzymatic assays and biolayer interferometry with a synthetic C7-3 and its derivatives, C7-3m1 and C7-3m2, demonstrated potent inhibition of AniA. Finally, the MIC50 value of C7-3 and C7-3m2 against anaerobically grown N. gonorrhoeae was 0.6 mM. We present the first peptide inhibitors of AniA, an enzyme that should be further exploited for antigonococcal drug development.

Acetoacetate is a more efficient energy-yielding substrate for human mesenchymal stem cells than glucose and generates fewer reactive oxygen species.

Stem cells have been assumed to demonstrate a reliance on anaerobic energy generation, suited to their hypoxic in vivo environment. However, we found that human mesenchymal stem cells (hMSCs) have an active oxidative metabolism with a range of substrates. More ATP was consistently produced from substrate oxidation than glycolysis by cultured hMSCs. Strong substrate preferences were shown with the ketone body, acetoacetate, being oxidised at up to 35 times the rate of glucose. ROS-generation was 45-fold lower during acetoacetate oxidation compared with glucose and substrate preference may be an adaptation to reduce oxidative stress. The UCP2 inhibitor, genipin, increased ROS production with either acetoacetate or glucose by 2-fold, indicating a role for UCP2 in suppressing ROS production. Addition of pyruvate stimulated acetoacetate oxidation and this combination increased ATP production 27-fold, compared with glucose alone, which has implications for growth medium composition. Oxygen tension during culture affected metabolism by hMSCs. Between passages 2 and 5, rates of both glycolysis and substrate-oxidation increased at least 2-fold for normoxic (20% O2)- but not hypoxic (5% O2)-cultured hMSCs, despite declining growth rates and no detectable signs of differentiation. Culture of the cells with 3-hydroxybutyrate abolished the increased rates of these pathways. These findings have implications for stem cell therapy, which necessarily involves in vitro culture of cells, since low passage number normoxic cultured stem cells show metabolic adaptations without detectable changes in stem-like status.

Effects of Feedstock Sources on Inoculant Acclimatization: Start-up Strategies and Reactor Performance.

Different inoculum sources and acclimatization methods result in different substrate adaptation and biodegradability. To increase straw degradation rate, shorten the digester start-up time, and enhance the biogas production, we domesticated anaerobic sludge by adding microcrystalline cellulose (MCC). During acclimatization, the start-up strategies and reactor performance were investigated to analyze changes in feedstock adaption, biodegradability, and methanogen activity. The effect of the domesticated inoculum was evaluated by testing batch un-pretreated corn stover with a dewatered sludge (DS)-domesticated inoculum as a control. The results showed that (1) using MCC as a substrate rapidly improved microorganism biodegradability and adaptation. (2) MCC as domesticated substrate has relatively stable system and high mass conversion, but with low buffer capacity. (3) Macro- and micronutrients should be added for improving the activity of methanogenic and system's buffer capacity. (4) Using the domesticated inoculums and batch tests to anaerobically digest untreated corn stover yielded rapid biogas production of 292 mL, with an early peak value on the first day. The results indicated that cultivating directional inoculum can efficiently and quickly start-up digester. These investigated results to promote anaerobic digestion of straw for producing biogas speed up the transformation of achievements of biomass solid waste utilization have a positive promoting significance.

Overcoming the Heat Endurance of Tumor Cells by Interfering with the Anaerobic Glycolysis Metabolism for Improved Photothermal Therapy.

In this study, we developed a general method to decorate plasmonic gold nanorods (GNRs) with a CD44-targeting functional polymer, containing a hyaluronic acid (HA)-targeting moiety and a small molecule Glut1 inhibitor of diclofenac (DC), to obtain GNR/HA-DC. This nanosystem exhibited the superiority of selectively sensitizing tumor cells for photothermal therapy (PTT) by inhibiting anaerobic glycolysis. Upon specifically targeting CD44, sequentially time-dependent DC release could be achieved by the trigger of hyaluronidase (HAase), which abundantly existed in tumor tissues. The released DC depleted the Glut1 level in tumor cells and induced a cascade effect on cellular metabolism by inhibiting glucose uptake, blocking glycolysis, decreasing ATP levels, hampering heat shock protein (HSP) expression, and ultimately leaving malignant cells out from the protection of HSPs to stress (e.g., heat), and then tumor cells were more easy to kill. Owing to the sensitization effect of GNR/HA-DC, CD44 overexpressed tumor cells could be significantly damaged by PTT with an enhanced therapeutic efficiency in vitro and in vivo.

Ethylene-Regulated Glutamate Dehydrogenase Fine-Tunes Metabolism during Anoxia-Reoxygenation.

Ethylene is an essential hormone in plants that is involved in low-oxygen and reoxygenation responses. As a key transcription factor in ethylene signaling, ETHYLENE INSENSITIVE3 (EIN3) activates targets that trigger various responses. However, most of these targets are still poorly characterized. Through analyses of our microarray data and the published Arabidopsis (Arabidopsis thaliana) EIN3 chromatin immunoprecipitation sequencing data set, we inferred the putative targets of EIN3 during anoxia-reoxygenation. Among them, GDH2, which encodes one subunit of glutamate dehydrogenase (GDH), was chosen for further studies for its role in tricarboxylic acid cycle replenishment. We demonstrated that both GDH1 and GDH2 are induced during anoxia and reoxygenation and that this induction is mediated via ethylene signaling. In addition, the results of enzymatic assays showed that the level of GDH during anoxia-reoxygenation decreased in the ethylene-insensitive mutants ein2-5 and ein3eil1 Global metabolite analysis indicated that the deamination activity of GDH might regenerate 2-oxoglutarate, which is a cosubstrate that facilitates the breakdown of alanine by alanine aminotransferase when reoxygenation occurs. Moreover, ineffective tricarboxylic acid cycle replenishment, disturbed carbohydrate metabolism, reduced phytosterol biosynthesis, and delayed energy regeneration were found in gdh1gdh2 and ethylene mutants during reoxygenation. Taken together, these data illustrate the essential role of EIN3-regulated GDH activity in metabolic adjustment during anoxia-reoxygenation.

Icariin Metabolism by Human Intestinal Microflora.

Icariin is a major bioactive compound of Epimedii Herba, a traditional oriental medicine exhibiting anti-cancer, anti-inflammatory and anti-osteoporosis activities. Recently, the estrogenic activities of icariin drew significant attention, but the published scientific data seemed not to be so consistent. To provide fundamental information for the study of the icaritin metabolism, the biotransformation of icariin by the human intestinal bacteria is reported for the first time. Together with human intestinal microflora, the three bacteria Streptococcus sp. MRG-ICA-B, Enterococcus sp. MRG-ICA-E, and Blautia sp. MRG-PMF-1 isolated from human intestine were reacted with icariin under anaerobic conditions. The metabolites including icariside II, icaritin, and desmethylicaritin, but not icariside I, were produced. The MRG-ICA-B and E strains hydrolyzed only the glucose moiety of icariin, and icariside II was the only metabolite. However, the MRG-PMF-1 strain metabolized icariin further to desmethylicaritin via icariside II and icaritin. From the results, along with the icariin metabolism by human microflora, it was evident that most icariin is quickly transformed to icariside II before absorption in the human intestine. We propose the pharmacokinetics of icariin should focus on metabolites such as icariside II, icaritin and desmethylicaritin to explain the discrepancy between the in vitro bioassay and pharmacological effects.

Intermittent hypoxia leads to functional reorganization of mitochondria and affects cellular bioenergetics in marine molluscs.

Fluctuations in oxygen (O2) concentrations represent a major challenge to aerobic organisms and can be extremely damaging to their mitochondria. Marine intertidal molluscs are well-adapted to frequent O2 fluctuations, yet it remains unknown how their mitochondrial functions are regulated to sustain energy metabolism and prevent cellular damage during hypoxia and reoxygenation (H/R). We used metabolic control analysis to investigate the mechanisms of mitochondrial responses to H/R stress (18 h at <0.1% O2 followed by 1 h of reoxygenation) using hypoxia-tolerant intertidal clams Mercenaria mercenaria and hypoxia-sensitive subtidal scallops Argopecten irradians as models. We also assessed H/R-induced changes in cellular energy balance, oxidative damage and unfolded protein response to determine the potential links between mitochondrial dysfunction and cellular injury. Mitochondrial responses to H/R in scallops strongly resembled those in other hypoxia-sensitive organisms. Exposure to hypoxia followed by reoxygenation led to a strong decrease in the substrate oxidation (SOX) and phosphorylation (PHOS) capacities as well as partial depolarization of mitochondria of scallops. Elevated mRNA expression of a reactive oxygen species-sensitive enzyme aconitase and Lon protease (responsible for degradation of oxidized mitochondrial proteins) during H/R stress was consistent with elevated levels of oxidative stress in mitochondria of scallops. In hypoxia-tolerant clams, mitochondrial SOX capacity was enhanced during hypoxia and continued rising during the first hour of reoxygenation. In both species, the mitochondrial PHOS capacity was suppressed during hypoxia, likely to prevent ATP wastage by the reverse action of FO,F1-ATPase. The PHOS capacity recovered after 1 h of reoxygenation in clams but not in scallops. Compared with scallops, clams showed a greater suppression of energy-consuming processes (such as protein turnover and ion transport) during hypoxia, indicated by inactivation of the translation initiation factor EIF-2α, suppression of 26S proteasome activity and a dramatic decrease in the activity of Na(+)/K(+)-ATPase. The steady-state levels of adenylates were preserved during H/R exposure and AMP-dependent protein kinase was not activated in either species, indicating that the H/R exposure did not lead to severe energy deficiency. Taken together, our findings suggest that mitochondrial reorganizations sustaining high oxidative phosphorylation flux during recovery, combined with the ability to suppress ATP-demanding cellular functions during hypoxia, may contribute to high resilience of clams to H/R stress and help maintain energy homeostasis during frequent H/R cycles in the intertidal zone.

Anaerobic toxicity of cationic silver nanoparticles.

The microbial toxicity of silver nanoparticles (AgNPs) stabilized with different capping agents was compared to that of Ag(+) under anaerobic conditions. Three AgNPs were investigated: (1) negatively charged citrate-coated AgNPs (citrate-AgNPs), (2) minimally charged polyvinylpyrrolidone coated AgNPs (PVP-AgNPs) and (3) positively charged branched polyethyleneimine coated AgNPs (BPEI-AgNPs). The AgNPs investigated in this experiment were similar in size (10-15nm), spherical in shape, but varied in surface charge which ranged from highly negative to highly positive. While, at AgNPs concentrations lower than 5mgL(-1), the anaerobic decomposition process was not influenced by the presence of the nanoparticles, there was an observed impact on the diversity of the microbial community. At elevated concentrations (100mgL(-1) as silver), only the cationic BPEI-AgNPs demonstrated toxicity similar in magnitude to that of Ag(+). Both citrate and PVP-AgNPs did not exhibit toxicity at the 100mgL(-1) as measured by biogas evolution. These findings further indicate the varying modes of action for nanoparticle toxicity and represent one of the few studies that evaluate end-of-life management concerns with regards to the increasing use of nanomaterials in our everyday life. These findings also highlight some of the concerns with a one size fits all approach to the evaluation of environmental health and safety concerns associated with the use of nanoparticles.

The effects of chronic cadmium exposure on repeat swimming performance and anaerobic metabolism in brown trout (Salmo trutta) and lake whitefish (Coregonus clupeaformis).

This study investigates the effect of chronic Cd exposure on the ability to perform repeat swim challenges in brown trout (Salmo trutta) and lake whitefish (Coregonus clupeaformis). Fish were exposed to waterborne Cd (18nM) in moderately hard water (120mgL(-1) CaCO3) for 30 days. This level of exposure has been shown to cause sublethal physiological disruption and acclimation responses but no impairment of sustained swimming capacity (Ucrit) in single swim challenges. Swim trials were done over the course of the exposure and each one consisted of an initial swim to 85% of the Ucrit of control fish, a 30min recovery period and finally a second swim challenge to determine Ucrit. Plasma and tissue samples were collected before and after each of the swim periods. As expected from previous studies, Cd exposure resulted in significant accumulation of Cd in gills, liver and kidney but not in white muscle. Exposure also induced a loss of plasma Ca followed by subsequent recovery (in lake whitefish but not brown trout) with few mortalities (100% survival for lake whitefish and 93% for brown trout). Both control and exposed fish swam to 85% of the single swim Ucrit and no differences in performance were seen. The Ucrit of unexposed controls in the second swim challenges were not different from the single swim Ucrit. However, second swim performance was significantly reduced in Cd exposed fish, particularly after a week of exposure where 31% and 38% reductions were observed for brown trout and lake whitefish respectively. Swimming to 85% Ucrit resulted in metabolic expenditure with little recovery after 30min. Few differences were observed between control and Cd exposed fish with the exception of a reduction in resting white muscle ATP stores of Cd exposed fish after 1 week of exposure. The results show that chronic sublethal Cd exposure results in an impairment of swimming ability in repeat swim challenges but this impairment is generally not related to metabolic processes in white muscle.

Metformin improves performance in high-intensity exercise, but not anaerobic capacity in healthy male subjects.

The aim of this study was to determine the ergogenic effects of metformin in high-intensity exercise, as well as its effects on anaerobic capacity, in healthy and physically active men. Ten subjects (mean (± standard deviation) maximal oxygen uptake (V˙O2max ) 38.6 ± 4.5 mL/kg per min) performed the following tests in a cycle ergometer: (i) an incremental test; (ii) six submaximal constant workload tests at 40%-90% (V˙O2max ); and (iii) two supramaximal tests (110% (V˙O2max ). Metformin (500 mg) or placebo was ingested 60 min before the supramaximal test. There were no significant differences between the placebo and metformin groups in terms of maximum accumulated oxygen deficit (2.8 ± 0.6 vs 3.0 ± 0.8 L, respectively; P = 0.08), lactate concentrations (7.8 ± 2.6 vs 7.5 ± 3.0 mmol/L, respectively; P = 0.75) or O2 consumed in either the last 30 s of exercise (40.4 ± 4.4 vs 39.9 ± 4.0 mL/kg per min, respectively; P = 0.35) or the first 110 s of exercise (29.0 ± 2.5 vs 29.5 ± 3.0 mL/kg per min, respectively; P = 0.42). Time to exhaustion was significantly higher after metformin than placebo ingestion (191 ± 33 vs 167 ± 32 s, respectively; P = 0.001). The fast component of V˙O2 recovery was higher in the metformin than placebo group (12.71 vs 12.18 mL/kg per min, respectively; P = 0.025). Metformin improved performance and anaerobic alactic contribution during high-intensity exercise, but had no effect on overall anaerobic capacity in healthy subjects.