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It is generally accepted that alterations in metabolism are critical for the metastatic process; however, the mechanisms by which these metabolic changes are controlled by the major drivers of the metastatic process remain elusive. Here, we found that S100 calcium-binding protein A4 (S100A4), a major metastasis-promoting protein, confers metabolic plasticity to drive tumor invasion and metastasis of non-small cell lung cancer cells. Investigating how S100A4 regulates metabolism, we found that S100A4 depletion decreases oxygen consumption rates, mitochondrial activity, and ATP production and also shifts cell metabolism to higher glycolytic activity. We further identified that the 49-kDa mitochondrial complex I subunit NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NDUFS2) is regulated in an S100A4-dependent manner and that S100A4 and NDUFS2 exhibit co-occurrence at significant levels in various cancer types as determined by database-driven analysis of genomes in clinical samples using cBioPortal for Cancer Genomics. Importantly, we noted that S100A4 or NDUFS2 silencing inhibits mitochondrial complex I activity, reduces cellular ATP level, decreases invasive capacity in three-dimensional growth, and dramatically decreases metastasis rates as well as tumor growth in vivo. Finally, we provide evidence that cells depleted in S100A4 or NDUFS2 shift their metabolism toward glycolysis by up-regulating hexokinase expression and that suppressing S100A4 signaling sensitizes lung cancer cells to glycolysis inhibition. Our findings uncover a novel S100A4 function and highlight its importance in controlling NDUFS2 expression to regulate the plasticity of mitochondrial metabolism and thereby promote the invasive and metastatic capacity in lung cancer.

Ceramide and diacylglycerol (DAG) are bioactive lipids and mediate many cellular signaling pathways. Sphingomyelin synthase (SMS) is the single metabolic link between the two, while SMS2 is the only SMS form located at the plasma membrane. SMS2 functions were investigated in HepG2 cell lines stably expressing SMS2. SMS2 overexpression did not alter sphingomyelin (SM), phosphatidylcholine (PC), or ceramide levels. DAG content increased by approx. 40% and led to downregulation of DAG-dependent protein kinase C (PKC). SMS2 overexpression also induced senescence, characterized by positivity for β-galactosidase activity and heterochromatin foci. HepG2-SMS2 cells exhibited protruded mitochondria and suppressed mitochondrial respiration rates. ATP production and the abundance of Complex V were substantially lower in HepG2-SMS2 cells as compared to controls. SMS2 overexpression was associated with inflammasome activation based on increases in IL-1β and nlpr3 mRNA levels. HepG2-SMS2 cells exhibited lipid droplet accumulation, constitutive activation of AMPK based on elevated 172Thr phosphorylation, increased AMPK abundance, and insensitivity to insulin suppression of AMPK. Thus, our results show that SMS2 regulates DAG homeostasis and signaling in hepatocytes and also provide proof of principle for the concept that offset in bioactive lipids’ production at the plasma membrane can drive the senescence program in association with steatosis and, seemingly, by cell-autonomous mechanisms.

Treatment of advanced hepatocellular carcinoma (HCC) remains a challenge due to the high tumor heterogeneity. In the present study, we aim to evaluate the impact of the β-catenin inhibitor, FH535, alone or in combination with the Ras/Raf/MAPK inhibitor Sorafenib, on the bioenergetics profiles of the HCC cell lines Huh7 and PLC/PRF/5. Single low-dose treatments with FH535 or Sorafenib promoted different effects on mitochondrial respiration and glycolysis in a cell type specific manner. However, the combination of these drugs significantly reduced both mitochondrial respiration and glycolytic rates regardless of the HCC cells. The significant changes in mitochondrial respiration observed in cells treated with the Sorafenib-FH535 combination may correspond to differential targeting of ETC complexes and changes in substrate utilization mediated by each drug. Moreover, the bioenergetics changes and the loss of mitochondrial membrane potential that were evidenced by treatment of HCC cells with the combination of FH535 and Sorafenib, preceded the induction of cell apoptosis. Overall, our results demonstrated that Sorafenib-FH535 drug combination induce the disruption of the bioenergetics of HCC by the simultaneous targeting of mitochondrial respiration and glycolytic flux that leads the synergistic effect on inhibition of cell proliferation. These findings support the therapeutic potential of combinatory FH535-Sorafenib treatment of the HCC heterogeneity by the simultaneous targeting of different molecular pathways.

Juvenile neuronal ceroid lipofuscinosis (JNCL, aka. juvenile Batten disease or CLN3 disease) is a lysosomal storage disease characterized by progressive blindness, seizures, cognitive and motor failures, and premature death. JNCL is caused by mutations in the Ceroid Lipofuscinosis, Neuronal 3 (CLN3) gene, whose function is unclear. Although traditionally considered a neurodegenerative disease, CLN3 disease displays eye-specific effects: Vision loss not only is often one of the earliest symptoms of JNCL, but also has been reported in non-syndromic CLN3 disease. Here we described the roles of CLN3 protein in maintaining healthy retinal pigment epithelium (RPE) and normal vision. Using electroretinogram, fundoscopy and microscopy, we showed impaired visual function, retinal autofluorescent lesions, and RPE disintegration and metaplasia/hyperplasia in a Cln3 ~ 1 kb-deletion mouse model [1] on C57BL/6J background. Utilizing a combination of biochemical analyses, RNA-Seq, Seahorse XF bioenergetic analysis, and Stable Isotope Resolved Metabolomics (SIRM), we further demonstrated that loss of CLN3 increased autophagic flux, suppressed mTORC1 and Akt activities, enhanced AMPK activity, and up-regulated gene expression of the autophagy-lysosomal system in RPE-1 cells, suggesting autophagy induction. This CLN3 deficiency induced autophagy induction coincided with decreased mitochondrial oxygen consumption, glycolysis, the tricarboxylic acid (TCA) cycle, and ATP production. We also reported for the first time that loss of CLN3 led to glycogen accumulation despite of impaired glycogen synthesis. Our comprehensive analyses shed light on how loss of CLN3 affect autophagy and metabolism. This work suggests possible links among metabolic impairment, autophagy induction and lysosomal storage, as well as between RPE atrophy/degeneration and vision loss in JNCL.

To increase the efficacy of radiation, iron oxide nanoparticles can be utilized for their ability to produce reactive oxygen species (ROS). Radiation therapy promotes leakage of electrons from the electron transport chain and leads to an increase in mitochondrial production of the superoxide anion which is converted to hydrogen peroxide by superoxide dismutase. Iron oxide nanoparticles can then catalyze the reaction from hydrogen peroxide to the highly reactive hydroxyl radical. Therefore, the overall aim of this project was to utilize iron oxide nanoparticles conjugated to a cell penetrating peptide, TAT, to escape lysosomal encapsulation after internalization by cancer cells and catalyze hydroxyl radical formation. It was determined that TAT functionalized iron oxide nanoparticles and uncoated iron oxide nanoparticles resulted in permeabilization of the lysosomal membranes. Additionally, mitochondrial integrity was compromised when A549 cells were treated with both TAT-functionalized nanoparticles and radiation. Pre-treatment with TAT-functionalized nanoparticles also significantly increased the ROS generation associated with radiation. A long term viability study showed that TAT-functionalized nanoparticles combined with radiation resulted in a synergistic combination treatment. This is likely due to the TAT-functionalized nanoparticles sensitizing the cells to subsequent radiation therapy, because the nanoparticles alone did not result in significant toxicities.

Experimental and preclinical evidence suggest that adoptive transfer of regulatory T (Treg) cells could be an appropriate therapeutic strategy to induce tolerance and improve graft survival in transplanted patients. The University of Kentucky Transplant Service Line is developing a novel phase I/II clinical trial with ex vivo expanded autologous Treg cells as an adoptive cellular therapy in renal transplant recipients who are using everolimus (EVR)-based immunosuppressive regimen.The aim of this study was to determine the mechanisms of action and efficacy of EVR for the development of functionally competent Treg cell-based adoptive immunotherapy in transplantation to integrate a common EVR-based regimen in vivo (in the patient) and ex vivo (in the expansion of autologous Treg cells). CD25 Treg cells were selected from leukapheresis product with a GMP-compliant cell separation system and placed in 5-day (short) or 21-day (long) culture with EVR or rapamycin (RAPA). Multi-parametric flow cytometry analyses were used to monitor the expansion rates, phenotype, autophagic flux, and suppressor function of the cells. phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway profiles of treated cells were analyzed by Western blot and cell bioenergetic parameters by extracellular flux analysis.EVR-treated cells showed temporary slower growth, lower metabolic rates, and reduced phosphorylation of protein kinase B compared with RAPA-treated cells. In spite of these differences, the expansion rates, phenotype, and suppressor function of long-term Treg cells in culture with EVR were similar to those with RAPA.Our results support the feasibility of EVR to expand functionally competent Treg cells for their clinical use.

Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.

The PI3K/mTOR signaling cascade is fundamental in T-cell activation and fate decisions. We showed the distinct regulation of PI3K/mTOR in regulatory and effector T-cells and proposed the potential therapeutic benefit of targeting this pathway to control the balance between effector and regulatory T-cell activities. Substantial adverse effects in long-term clinical usage of Rapamycin suggest the use of alternative treatments in restraining effector T-cell function in transplant patients. We hypothesize that dual PI3K/mTOR inhibitors may represent an immunosuppressant alternative. Here we show that dual PI3K/mTOR PI-103 and PKI-587 inhibitors interfered IL-2-dependent responses in T-cells. However, in contrast to the inhibitory effects in non-Treg T-cell proliferation and effector functions, dual inhibitors increased the differentiation, preferential expansion and suppressor activity of iTregs. Rapamycin, PI-103 and PKI-587 targeted different signaling events and induced different metabolic patterns in primary T-cells. Similar to Rapamycin, in vivo administration of PI-103 and PKI-587 controlled effectively the immunological response against allogenic skin graft. These results characterize specific regulatory mechanisms of dual PI3K/mTOR inhibitors in T-cells and support their potential as a novel therapeutic option in transplantation. This article is protected by copyright. All rights reserved.

Thermotherapy, as a method of treating cancer, has recently attracted considerable attention from basic and clinical investigators. A number of studies and clinical trials have shown that thermotherapy can be successfully used as a therapeutic approach for various cancers. However, the effects of temperature on cancer bioenergetics have not been studied in detail with a real time, microplate based, label-free detection approach. This study investigates how changes in temperature affect the bioenergetics characteristics (mitochondrial function and glycolysis) of three colorectal cancer (CRC) cell lines utilizing the Seahorse XF96 technology. Experiments were performed at 32°C, 37°C and 42°C using assay medium conditions and equipment settings adjusted to produce equal oxygen and pH levels ubiquitously at the beginning of all experiments. The results suggest that temperature significantly changes multiple components of glycolytic and mitochondrial function of all cell lines tested. Under hypothermia conditions (32°C), the extracellular acidification rates (ECAR) of CRC cells were significantly lower compared to the same basal ECAR levels measured at 37°C. Mitochondrial stress test for SW480 cells at 37°C vs 42°C demonstrated increased proton leak while all other OCR components remained unchanged (similar results were detected also for the patient-derived xenograft cells Pt.93). Interestingly, the FCCP dose response at 37°C vs 42°C show significant shifts in profiles, suggesting that single dose FCCP experiments might not be sufficient to characterize the mitochondrial metabolic potential when comparing groups, conditions or treatments. These findings provide valuable insights for the metabolic and bioenergetic changes of CRC cells under hypo- and hyperthermia conditions that could potentially lead to development of better targeted and personalized strategies for patients undergoing combined thermotherapy with chemotherapy.

Increased glucose metabolism is considered as one of the most important metabolic alterations adapted by cancer cells in order to generate energy as well as high levels of glycolytic intermediates to support rapid proliferation. PH domain leucine-rich repeat protein phosphatase (PHLPP) belongs to a novel family of Ser/Thr protein phosphatases that function as tumor suppressors in various types of human cancer. Here we determined the role of PHLPP in regulating glucose metabolism in colon cancer cells. Knockdown of PHLPP increased the rate of glucose consumption and lactate production, whereas overexpression of PHLPP had the opposite effect. Bioenergetic analysis using Seahorse Extracelluar Flux Analyzer revealed that silencing PHLPP expression induced a glycolytic shift in colon cancer cells. Mechanistically, we found that PHLPP formed a complex with Akt and hexokinase 2 (HK2) in the mitochondrial fraction of colon cancer cells and knockdown of PHLPP enhanced Akt-mediated phosphorylation and mitochondrial localization of HK2. Depletion of HK2 expression or treating cells with Akt and HK2 inhibitors reversed PHLPP loss-induced increase in glycolysis. Furthermore, PHLPP knockdown cells became addicted to glucose as a major energy source in that glucose starvation significantly decreased cancer cell survival. As HK2 is the key enzyme that determines the direction and magnitude of glucose flux, our study identified PHLPP as a novel regulator of glucose metabolism by controlling HK2 activity in colon cancer cells.

Doxorubicin (DOX), one of the most effective anticancer drugs, is known to generate progressive cardiac damage, which is due, in part, to DOX-induced reactive oxygen species (ROS). The elevated ROS often induce oxidative protein modifications that result in alteration of protein functions. This study demonstrates that the level of proteins adducted by 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, is significantly increased in mouse heart mitochondria following DOX treatment. A redox proteomics method involving 2D electrophoresis followed by mass spectrometry and investigation of protein data bases identified several HNE-modified mitochondria proteins, which were verified by HNE-specific immunoprecipitation in cardiac mitochondria from the DOX-treated mice. The majority of the identified proteins are related to mitochondrial energy metabolism. These include proteins in the citric acid cycle (TCA) and electron transport chain (ETC). The enzymatic activities of the HNE-adducted proteins were significantly reduced in DOX-treated mice. Consistent with the decline in the function of the HNE adducted proteins, the respiratory function of cardiac mitochondria as determined by oxygen consumption rate (OCR) was also significantly reduced after DOX treatment. The treatment with Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnP, an SOD mimic, averted the doxorubicin-induced mitochondrial dysfunctions as well as the HNE protein adductions. Together, the results demonstrate that free radical-mediated alteration of energy metabolism is an important mechanism mediating DOX-induced cardiac injury suggesting that metabolic intervention may represent a novel approach to preventing cardiac injury after chemotherapy.

Diastolic dysfunction is a clinically significant problem for patients with diabetes and often reflects increased ventricular stiffness. Attached cross-bridges contribute to myocardial stiffness and produce short-range forces, but it is not yet known whether these forces are altered in diabetes. In this study, we tested the hypothesis that cross-bridge-based short-range forces are increased in the streptozotocin (STZ) induced rat model of Type 1 diabetes. Chemically permeabilized myocardial preparations were obtained from 12 week old rats that had been injected with STZ or vehicle 4 weeks earlier, and activated in solutions with pCa (=−log10[Ca2+]) values ranging from 9.0 to 4.5. The short-range forces elicited by controlled length changes were ~67% greater in the samples from the diabetic rats than in the control preparations. This change was mostly due to an increased elastic limit (the length change at the peak short-range force) as opposed to increased passive muscle stiffness. The STZ-induced increase in short-ranges forces is thus unlikely to reflect changes to titin and/or collagen filaments. Gel electrophoresis showed that STZ increased the relative expression of β myosin heavy chain. This molecular mechanism can explain the increased short-ranges forces observed in the diabetic tissue if β myosin molecules remain bound between the filaments for longer durations than α molecules during imposed movements. These results suggest that interventions that decrease myosin attachment times may be useful treatments for diastolic dysfunction associated with diabetes.

It is generally accepted that alterations in metabolism are critical for the metastatic process; however, the mechanisms by which the metabolic changes are controlled by the major drivers of the metastatic process remain elusive. Here, we find that S100A4, a major metastasis-promoting protein, alters metabolic plasticity to drive tumor invasion and metastasis. Investigating how S100A4 regulates metabolism, we find that depletion of S100A4 decreases oxygen consumption rate, mitochondrial activity, and ATP production, and shifts cell metabolism to be more glycolytically active. We further identify that the 49 KD mitochondrial complex I subunit NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NDUFS2) is regulated in an S100A4-dependent manner and that S100A4 and NDUFS2 exhibit co-occurrence at significant levels in various cancer types (cBioPortal). Importantly, we find that knockdown of NDUFS2 inhibits mitochondrial complex I activity, reduces cellular ATP level, and decreases the invasive capacity in three-dimensional (3D) growth, thus mimicking the biological effect resulting from S100A4 knockdown in A549 cells. Likewise, silencing S100A4 and NDUFS2 dramatically decreases the rate of metastasis as well as tumor growth in vivo. Finally, we provide evidence that cells with S100A4 and NDUFS2 depletion shift metabolism toward glycolysis through upregulating hexokinases expression and that suppressing S100A4 signaling sensitizes lung cancer cells to glycolysis inhibition. Together, our findings uncover a novel function of S100A4 in mitochondrial metabolism and highlight the importance of NDUFS2 in regulating the plasticity of mitochondrial metabolism by S100A4 to promote the invasion and metastasis in lung cancer. Citation Format: Lili Liu, Lei Qi, Teresa Knifley, Dava W. Piecoro, Piotr Rychahou, Jinpeng Liu, Mihail I. Mitov, Jeremiah Martin, Chi Wang, Jianrong Wu, Heidi L. Weiss, D. Allan Butterfield, B. Mark Evers, Kathleen L. O'Connor, Min Chen. S100A4 alters mitochondrial metabolism to promote invasion and metastasis of non-small cell lung cancer cells through upregulation of NDUFS2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1119.

Obesity has been associated with increased incidence and mortality of a wide variety of human cancers including colorectal cancer. However, the molecular mechanism by which adipocytes regulate the metabolism of colon cancer cells remains elusive. In this study, we showed that adipocytes isolated from adipose tissues of colon cancer patients have an important role in modulating cellular metabolism to support tumor growth and survival. Abundant adipocytes were found in close association with invasive tumor cells in colon cancer patients. Co-culture of adipocytes with colon cancer cells led to a transfer of free fatty acids that released from the adipocytes to the cancer cells. Uptake of fatty acids allowed the cancer cells to survive nutrient deprivation conditions by upregulating mitochondrial fatty acid β-oxidation. Mechanistically, co-culture of adipocytes or treating cells with fatty acids induced autophagy in colon cancer cells as a result of AMPK activation. Inhibition of autophagy attenuated the ability of cancer cells to utilize fatty acids and blocked the growth-promoting effect of adipocytes. In addition, we found that adipocytes stimulated the expression of genes associated with cancer stem cells and downregulated genes associated with intestinal epithelial cell differentiation in primary colon cancer cells and mouse tumor organoids. Importantly, the presence of adipocytes promoted the growth of xenograft tumors in vivo. Taken together, our results show that adipocytes in the tumor microenvironment serve as an energy provider and a metabolic regulator to promote the growth and survival of colon cancer cells.

Mitochondria are considered to be the “power plants” of the cell, but can also initiate and execute cell death, stimulated by oxidative stress (OS). OS induced mitochondrial dysfunction is characterized by a loss in oxygen consumption and reduced ATP production. Curcumin, as a potential therapeutic, has been explored as a candidate for mitochondrial OS suppression, but rapid metabolism and aqueous insolubility has prevented it from being effective. Further, efficient delivery of curcumin via the incorporation into nanocarriers has again been limited due to low drug loading capacities and/or significant burst release, resulting in acute cytotoxicity. Hence, to increase the therapeutic potential and reduce the toxic effects of curcumin, curcumin conjugated poly(β-amino ester) nanogels (CNGs) were synthesized using Michael addition chemistry. This approach provided easy control over the nanogel size, with CNGs showing a uniform release of active curcumin over 48 h with no burst release. This controlled release system significantly increased the safety limit for curcumin, with a ten fold increase in the cytotoxic threshold, as compared to free curcumin. Further, real-time mitochondrial response analysis with the Seahorse XF96 showed effective and prolonged suppression of H 2 O 2 induced mitochondrial oxidative stress upon pre-treating endothelial cells with CNGs and this potential of nanogels was studied at different pre-treatment times prior to H 2 O 2 exposure.

Generation of free radicals and oxidative stress play important roles in the physiological response to engineered biomaterials and nanoparticles. Critical to developing new materials and understanding this process is the ability to detect changes in oxidative stress and the red/ox status of cells. In this chapter, a comprehensive overview of most commonly used methods for detection of oxidative stress is presented. Specifically addressed are issues and limitations related to the choice of model (in vitro vs in vivo studies) and approach (direct detection of free radicals vs detection of indexes of oxidative stress). A comprehensive overview of the literature is provided for some of the classical methods, involving the use of highly fluorescent 2’,7’-dichlorofluorescein, thiobarbituric acid reactive species test, etc., and more modern and sophisticated assays, such as the Seahorse XF, Redox proteomics for the detection of changes of cellar function, oxidative stress in contact with biomaterials and nanoparticles..

Recent studies have demonstrated the re-emergence of cell cycle proteins in brain as patients progress from the early stages of mild cognitive impairment (MCI) into Alzheimer's disease (AD). Oxidative stress markers present in AD have also been shown to be present in MCI brain suggesting that these events occur in early stages of the disease. The levels of key cell cycle proteins, such as CDK2, CDK5, cyclin G1, and BRAC1 have all been found to be elevated in MCI brain compared to age-matched control. Further, peptidyl prolyl cis-trans isomerase (Pin1), a protein that plays an important role in regulating the activity of key proteins, such as CDK5, GSK3-β, and PP2A that are involved in both the phosphorylation state of Tau and in the cell cycle, has been found to be oxidatively modified and downregulated in both AD and MCI brain. Hyperphosphorylation of Tau then results in synapse loss and the characteristic Tau aggregation as neurofibrillary tangles, an AD hallmark. In this review, we summarized the role of cell cycle dysregulation in the progression of disease from MCI to AD. Based on the current literature, it is tempting to speculate that a combination of oxidative stress and cell cycle dysfunction conceivably leads to neurodegeneration.

GelBandFitter is a computer program that uses non-linear regression techniques to fit mathematical functions to densitometry profiles of protein gels. This allows for improved quantification of gels with partially overlapping and potentially asymmetric protein bands. The program can also be used to analyze immunoblots with closely spaced bands. GelBandFitter was developed in Matlab and the source code and/or a Windows executable file can be downloaded at no cost to academic users from http://www.gelbandfitter.org.

The mechanical properties of triton-permeabilized ventricular preparations isolated from 4, 18 and 24-month-old F344 rats were analyzed to provide information about the molecular mechanisms that lead to age-related increases in diastolic myocardial stiffness in these animals. Passive stiffness (measured in solutions with minimal free Ca(2+)) did not change with age. This implies that the aging-associated dysfunction is not due to changes in titin or collagen molecules. Ca(2+)-activated preparations exhibited a characteristic short-range force response: force rose rapidly until the muscle reached its elastic limit and less rapidly thereafter. The elastic limit increased from 0.43+/-0.01% l(0) (where l(0) is the initial muscle length) in preparations from 4-month-old animals to 0.49+/-0.01% l(0) in preparations from 24-month-old rats (p

The biogenic elements zinc, manganese and cobalt are essential for metabolic processes in animals. Compounds of nGly.Me2+A. mH2O (Me2+=Zn2+, Mn2+, Co2+; A=Cl−, SO42−, n=1, 2; m=2, 5), as supplements in the diet, were used separately on different experimental groups of male Hisex chickens to correct the mineral deficiency caused byAscaridia galliinfections. An amelioration of body weight gain, reduction of mortality and restoration of trace element levels were estimated in infected chickens. A mathematical model has been proposed forA. gallipopulation kinetics in chickens, taking into account the stimulating effect of these elements on the nematodes. The model parameters are considered as phenomenological constants of the host–parasite system. An agreement with experimental data is observed using, for the parametersψ,α,μandμs, values equal to those calculated in previously investigatedA. galli–chicken systems. For parameterν(immunological constant) the same value was obtained as in a previous experiment with high infection. This model is likely to be suitable for a range of host–nematode systems, including varying degrees of infection and treatment with different trace elements.

Heart failure is associated with pump dysfunction and remodeling but it is not yet known if the condition affects different transmural regions of the heart in the same way. We tested the hypotheses that the left ventricles of non-failing human hearts exhibit transmural heterogeneity of cellular level contractile properties, and that heart failure produces transmural region-specific changes in contractile function. Permeabilized samples were prepared from the sub-epicardial, mid-myocardial, and sub-endocardial regions of the left ventricular free wall of non-failing (n=6) and failing (n=10) human hearts. Power, an in vitro index of systolic function, was higher in non-failing mid-myocardial samples (0.59±0.06μWmg(-1)) than in samples from the sub-epicardium (p=0.021) and the sub-endocardium (p=0.015). Non-failing mid-myocardial samples also produced more isometric force (14.3±1.33kNm(-2)) than samples from the sub-epicardium (p=0.008) and the sub-endocardium (p=0.026). Heart failure reduced power (p=0.009) and force (p=0.042) but affected the mid-myocardium more than the other transmural regions. Fibrosis increased with heart failure (p=0.021) and mid-myocardial tissue from failing hearts contained more collagen than matched sub-epicardial (p0.001) and sub-endocardial (p=0.043) samples. Power output was correlated with the relative content of actin and troponin I, and was also statistically linked to the relative content and phosphorylation of desmin and myosin light chain-1. Non-failing human hearts exhibit transmural heterogeneity of contractile properties. In failing organs, region-specific fibrosis produces the greatest contractile deficits in the mid-myocardium. Targeting fibrosis and sarcomeric proteins in the mid-myocardium may be particularly effective therapies for heart failure.

Heart failure is a progressive condition in which the ventricles can no longer pump enough blood to meet the body's basal demands. Our laboratory is investigating whether the transmural variation in cellular contractile properties that occurs in normal hearts (and which is thought to be important for ventricular function) is altered in heart failure. We procured through wall samples of failing left ventricle from patients receiving transplants at the University of Kentucky and non-failing samples from brain dead organ donors. The tissue was divided into epicardial, midmyocardial and endocardial layers and frozen in liquid nitrogen within 30 minutes. Multicellular chemically permeabilized preparations were subsequently obtained from these samples by mechanical homogenization and triton treatment. The samples were attached between a force transducer and a motor and subjected to two mechanical protocols: 1) a stretch-restretch protocol in solutions with different activating Ca2+ concentrations and 2) a force-velocity protocol in which maximally-activated preparations were allowed to shorten against pre-set loads. Parameters including steady-state force, short-range stiffness, short-range force and maximum power output were measured using these two protocols. The results suggested a 30% decrease in maximum power output (p-value = 0.01) and steady-state force (p-value=0.005) in heart failure patients (n=8, total of 72 preparations) as compared to non-failing (n=4, total of 36 preparations). Short-range stiffness (p-value=0.003) and short-range force (p-value=0.002) also significantly decreased in heart failure vs. non-failing. Transmurally there was a significant difference in maximum power output between the regions (p-value=0.02). The data suggests that mechanically the mid myocardium maybe affected the most in heart failure. Further studies need to be done to understand the protein modifications that may be responsible for these variations.

Myocardial power output is an important determinant of ventricular function but few measurements of this parameter have been made using human tissue samples. Our group has been measuring force-velocity curves, and thus power output, in left ventricular samples obtained from patients undergoing cardiac surgery at the University of Kentucky. Multicellular preparations are prepared from previously-frozen tissue samples by mechanical homogenization and chemical permeabilization. They are then connected between a force transducer and a motor and maximally activated in a saturating [Ca2+] solution. Once force has reached steady-state, the samples are allowed to shorten against pre-set loads imposed using SLControl software. Panel A in the figure shows superposed force and muscle-length traces for an isolated myocardial preparation obtained from the heart removed from a 32 year old male during a transplant procedure. Panel B shows force-velocity and power curves calculated from these records. One of the goals of our ongoing study is to determine whether maximal power output in isolated left ventricular samples improves after patients are fitted with left ventricular assist devices. Our study will, to our knowledge, be the first to measure force-velocity and power output in preparations isolated from these patients.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Diabetes mellitus is often associated with abnormalities in active relaxation and passive stiffness of the left ventricle but the molecular mechanisms responsible for the dysfunction are not yet clear. This study was designed to identify the molecular components that are responsible for the increased myocardial stiffness associated with diabetes. Multicelluar myocardial preparations were isolated from control Sprague-Dawley rats and an experimental group of rats injected 4 weeks previously with streptozotocin (model of Type I Diabetes). Preparations were subjected to paired ramp stretches/releases imposed under fiber length control in a series of calcium activations (pCa 4.5 - 9.0). The relative short-range force and elastic limits were substantially higher in the diabetic groups. The rate of tension recovery (ktr) was considerably lower in the diabetic groups. Short range stiffness values did not differ in the control and diabetic animals. Gel electrophoresis showed that the relative content of slower beta Myosin heavy chain increased from 34±15% in control hearts to 100% in the diabetic rat hearts. These results support the hypothesis that pathological changes in the mechanical properties of diabetic rat myocardium are mostly due to alterations in the active component (cycling cross-bridges) of ventricular stiffness.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Patients in heart failure are sometimes implanted with a left ventricular assist device (LVAD) to bridge them to transplant. Clinical data shows that ∼15% of these patients recover pump function and can subsequently have their LVAD removed. The goal of our study was to determine whether cellular level maximal power output of the left ventricle improves after patients are fitted with LVADs. Myocardial samples were obtained (1) from the left ventricle apex of patients when they were implanted with a LVAD and (2) from the left ventricle free wall when they subsequently received a heart transplant. Non failing cardiac samples were also obtained from organ donors for comparison. All samples were snap frozen in liquid nitrogen and stored in the vapor phase of liquid nitrogen. Multicellular preparations were then obtained by chemically permeabilizing the samples in 1% Triton solution after mechanical homogenization. These preparations were connected between a force transducer and a motor and maximally activated in a saturating Ca2+ solution. Once force had reached steady state, the preparations were allowed to shorten against pre-set loads imposed using SLControl software. Maximum power output was determined from the force-velocity curves. Preliminary results suggest that LVAD treatment improved cellular level maximum power output and isometric force. There was also a trend towards a lower maximum shortening velocity post-LVAD. Further studies, will determine whether intrinsic ventricular function is improved by LVAD implantation as well as the molecular mechanisms that may mediate any observed responses.

The effect of dietary cobalt from three different sources on uninfected andAscaridia galli-infected Hisex chickens, has been studied. The chicken diet was supplemented with 0.06 Co2+kg-1food either in the form of two glycine–cobalt compounds or mixed zinc–cobalt basic salt. An excess of dietary cobalt in small doses increases the gain of body weight and decreases host mortality. A greater bioefficiency of cobalt was established in infected chickens. A mathematical model has been used to provide a quantitative interpretation of the observed results. The model solutions of the kinetics of worm numbers and body weight are in a good agreement with experimental data. The model is valid for different degrees ofA. galliinfections and for treatment with different trace elements. The value of the kinetic parameter, regarded as a phenomenological constant of the host immune response, depends on the degree of infection.

Female astronauts are more likely than male astronauts to develop presyncope and orthostatic intolerance when they return to normal gravity after space-flights. The underlying mechanisms for this are unclear but could include myocardial adaptations to micro-gravity conditions. This study was designed to test whether there are sex-specific changes in the mechanical properties of myocardial tissue in rats subjected to a period of simulated microgravity. Multicellular myocardial preparations were isolated from control male and female Sprague-Dawley rats and corresponding experimental animals that were hind-limb unloaded (HLU) for 14 days. Preparations were subjected to standard mechanical tests imposed under muscle length control in solutions with pCa values ranging from 9.0 to 4.5. The rates of tension recovery (ktr) measured in pCa 4.5 solution were significantly higher (p

Fatty Acid Synthase (FASN), a key enzyme of lipid biogenesis, is significantly up regulated in colorectal cancer (CRC) and its activity is associated with poor prognosis. Mitochondrial metabolism plays a crucial role in cancer cells proliferation and survival. However, very little is known about the role of de novo fatty acid synthesis in production of ATP via beta-oxidation and oxidative phosphorylation in CRC. The purpose of the present study was to determine: (i) the role of aberrant activation of FASN in cellular respiration, (ii) contribution of endogenously synthesized lipids to fatty acid oxidation, and (iii) consequences of FASN inhibition on mitochondrial properties and redox homeostasis in CRC. METHODS. We utilized KM20, HT29, and HCT116 CRC cells with stable knockdown of FASN. Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using XF Cell Mito Stress and XF Glycolysis Stress tests, respectively. The XF Palmitate-BSA conjugate was used to assess oxidation of fatty acids (FAO). OCR, ECAR and FAO experiments were performed using the Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). Intracellular ROS and mitochondrial membrane potential was measured by DCFDA and TMRE assays. RESULTS. We found that stable inhibition of FASN is associated with a significant decrease in glycolysis in all tested CRC cell lines. The glycolytic capacity and glycolytic reserve were significantly decreased in HCT116 cell line; however, no significant differences in these parameters were observed in HT29 and KM20 cells. Knock-down of FASN led to a significant decrease in OCR in CRC cells that is consistent with a decrease in cellular proliferation. Treatment with etomoxir demonstrated that CRC cell lines have the different level of dependence on fatty acid oxidation. Furthermore, using the Palmitate-BSA substrate we showed that CRC cells primarily utilize endogenously synthesized fatty acids and knockdown of FASN significantly decreases the capacity of cells to oxidize fatty acids. Finally, our data suggest that FASN regulates mitochondrial membrane potential and ROS production in CRC. CONCLUSIONS. Fatty acid oxidation is a dominant bioenergetic pathway in many cancers. This study suggests that up regulation of FASN and an increase in the level of de novo synthesized lipids are crucial for modulation of mitochondrial oxidative capacity in CRC cells. Furthermore, our results proposes that inhibition of FASN and a decrease in mitochondrial ATP production impair glycolysis in CRC cells. Together, our work demonstrates an importance of FASN in re-programming cellular energy metabolism, in particular, beta-oxidation. Further investigations into the mechanisms of regulation of mitochondrial function by FASN and interconnection with other metabolic pathways will uncover new therapeutic approaches in CRC. Citation Format: Yekaterina Zaytseva, Mihail Mitov, D. Allan Butterfield, Tianyan Gao, B. Mark Evers. Inhibition of fatty acid synthase reduces mitochondrial respiration and addiction of colorectal cancer cells to glycolysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4343. doi:10.1158/1538-7445.AM2014-4343

Blebbistatin (BLEB) and 2,3-butanedione monoxime (BDM) are well-know inhibitors of myofilament force production and useful tools in structural and functional studies of cell motility and muscle contraction. In this study, we investigated the effects of BLEB and BDM on the short-range mechanical properties of single chemically permeabilized murine diaphragm fibers. BLEB and BDM were used in separate sets of experiments to reduce isometric force in saturating Ca2+ solution to approximately 50% of the control value. Muscle fibers were subjected to repeated triangular length changes (paired ramp stretches/releases, 0.04 l0, 0.33 l0 s−1) imposed under fiber length control in solutions with free Ca2+ concentrations ranging from pCa 9.0 to pCa 4.5. Short-range stiffness values were calculated from the slopes of regression lines fitted to the first 15 ms of XY plots of force against muscle length for each stretch response and expressed as Young's Moduli. Analysis of results obtained in control Ca2+ solutions (without BLEB or BDM) showed that short-range stiffness increased proportionately with the level of isometric force. Experiments performed with BLEB showed that short-range stiffness declined in proportion with the reduction in isometric force (no change in the stiffness/force ratio). In contrast, BDM produced a disproportionately large decrease in isometric force (that is, the stiffness/force ratio increased in the presence of BDM, ANACOVA test, p

Understanding how cellular level mechanical properties change as failing hearts remodel may help identify the protein modifications that produce contractile dysfunction associated with this condition. We therefore procured through wall left ventricular samples from patients undergoing heart transplants at the University of Kentucky and from organ donors who did not have heart failure. Multicellular chemically permeabilized preparations were subsequently obtained from sub epicardial, midwall and sub endocardial regions of these samples by mechanical homogenization and triton treatment. The mechanical properties of the preparations were assessed by attaching them between a force transducer and a motor and subjecting them to a standard series of mechanical assays. There was a 30% decrease in maximum power output (p-value = 0.01) and steady-state force (p-value=0.005) in patients with end-stage heart failure (n=8, total of 72 preparations) as compared to samples from nonfailing organ donors (n=4, total of 36 preparations). The phosphorylation of two cardiac sarcomeric proteins- myosin binding protein-C (cMyBP-C) and troponin-I (cTnI) which have been previously associated with contractile dysfunction were investigated using phospho specific antibodies. Western blot analysis of three major phosphorylated sites of cMyBP-C, PSer273, PSer282 and PSer302 were not significantly different between heart failure and non failing groups but cTnI phosphorylation at PSer22/23 decreased significantly in heart failure (p=0.01). This study suggests that the cellular level mechanical dysfunction seen in heart failure may be in part due to phosphorylation modifications.