The Glucose-Glutamine Metabolic Interplay in MCF-7 Cells, a Hormone-Sensitive Breast Cancer Model.

BACKGROUND: Selective deprivation of glutamine has been shown to accelerate the generation of reactive oxygen species (ROS) and to impair the activity of a specific pentose phosphate pathway (PPP) located within the endoplasmic reticulum (ER). The consequent oxidative damage suggests that glucose flux through this reticular pathway might contribute to the redox stress of breast cancer cells. We thus evaluated whether this response is reproduced when the glutamine shortage is coupled with the glucose deprivation. METHODS: Cancer growth, metabolic plasticity and redox status were evaluated under saturating conditions and after 48 h starvation (glucose 2.5 mM, glutamine 0.5 mM). The Seahorse technology was used to estimate adenosine triphosphate (ATP)-linked and ATP-independent oxygen consumption rate (OCR) as well as proton efflux rate (PER). 18F-fluoro-deoxy-glucose (FDG) uptake was evaluated through the LigandTracer device. Proliferation rate was estimated by the carboxyfluorescein-diacetate-succinimidyl ester (CFSE) staining, while cell viability by the propidium iodide exclusion assay. RESULTS: Starvation reduced the proliferation rate of MCF-7 cells without affecting their viability. It also decreased lactate release and PER. Overall OCR was left unchanged although ATP-synthase dependent fraction was increased under nutrient shortage. Glutaminolysis inhibition selectively impaired the ATP-independent and the oligomycin-sensitive OCR in control and starved cultures, respectively. The combined nutrient shortage decreased the cytosolic and mitochondrial markers of redox stress. It also left unchanged the expression of the reticular unfolded protein marker GRP78. By contrast, starvation decreased the expression of hexose-6P-dehydrogenase (H6PD) thus decreasing the glucose flux through the ER-PPP as documented by the profound impairment in the uptake rate of FDG. CONCLUSIONS: When combined with glucose deprivation, glutamine shortage does not elicit the expected enhancement of ROS generation in the studied breast cancer cell line. Combined with the decreased activity of ER-PPP, this observation suggests that glutamine interferes with the reticular glucose metabolism to regulate the cell redox balance.

Mandatory role of endoplasmic reticulum and its pentose phosphate shunt in the myocardial defense mechanisms against the redox stress induced by anthracyclines.

Anthracyclines' cardiotoxicity involves an accelerated generation of reactive oxygen species. This oxidative damage has been found to accelerate the expression of hexose-6P-dehydrogenase (H6PD), that channels glucose-6-phosphate (G6P) through the pentose phosphate pathway (PPP) confined within the endoplasmic/sarcoplasmic reticulum (SR). To verify the role of SR-PPP in the defense mechanisms activated by doxorubicin (DXR) in cardiomyocytes, we tested the effect of this drug in H6PD knockout mice (H6PD-/-). Twenty-eight wildtype (WT) and 32 H6PD-/- mice were divided into four groups to be treated with intraperitoneal administration of saline (untreated) or DXR (8 mg/Kg once a week for 3 weeks). One week thereafter, survivors underwent imaging of 18F-deoxyglucose (FDG) uptake and were sacrificed to evaluate the levels of H6PD, glucose-6P-dehydrogenase (G6PD), G6P transporter (G6PT), and malondialdehyde. The mRNA levels of SR Ca2+-ATPase 2 (Serca2) and ryanodine receptors 2 (RyR2) were evaluated and complemented with Hematoxylin/Eosin staining and transmission electron microscopy. During the treatment period, 1/14 DXR-WT and 12/18 DXR-H6PD-/- died. At microPET, DXR-H6PD-/- survivors displayed an increase in left ventricular size (p < 0.001) coupled with a decreased urinary output, suggesting a severe hemodynamic impairment. At ex vivo analysis, H6PD-/- condition was associated with an oxidative damage independent of treatment type. DXR increased H6PD expression only in WT mice, while G6PT abundance increased in both groups, mismatching a generalized decrease of G6PD levels. Switching-off SR-PPP impaired reticular accumulation of Ca2+ decelerating Serca2 expression and upregulating RyR2 mRNA level. It thus altered mitochondrial ultrastructure eventually resulting in a cardiomyocyte loss. The recognized vulnerability of SR to the anthracycline oxidative damage is counterbalanced by an acceleration of G6P flux through a PPP confined within the reticular lumen. The interplay of SR-PPP with the intracellular Ca2+ exchanges regulators in cardiomyocytes configure the reticular PPP as a potential new target for strategies aimed to decrease anthracycline toxicity.

Divergent Oxidative Stress in Normal Tissues and Inflammatory Cells in Hodgkin and Non-Hodgkin Lymphoma.

BACKGROUND: Previous studies reported mitochondrial and endoplasmic reticulum redox stress in peripheral blood mononucleated cells (PBMCs) of treatment-naïve Hodgkin lymphoma (HL) patients. Here, we assessed whether this response also applies to non-HL (NHL) patients, and whether the oxidative damage is a selective feature of PBMCs or, rather, also affects tissues not directly involved in the inflammatory response. METHODS: Isolated PBMCs of 28 HL, 9 diffuse large B cell lymphoma, 8 less aggressive-NHL, and 45 controls underwent flow cytometry to evaluate redox stress and uptake of the glucose analogue 2-NBDG. This analysis was complemented with the assay of malondialdehyde (MDA) levels and enzymatic activity of glucose-6P-dehydrogenase and hexose-6P-dehydrogenase (H6PD). In all lymphoma patients, 18F-fluoro-deoxyglucose uptake was estimated in the myocardium and skeletal muscles. RESULTS: Mitochondrial reactive oxygen species generation and MDA levels were increased only in HL patients as well as H6PD activity and 2-NBDG uptake. Similarly, myocardial FDG retention was higher in HL than in other groups as opposed to a similar tracer uptake in the skeletal muscle. CONCLUSIONS: Redox stress of PBMCs is more pronounced in HL with respect to both NHL groups. This phenomenon is coherent with an increased activity of H6PD that also extends to the myocardium.

Gene's expression underpinning the divergent predictive value of [18F]F-fluorodeoxyglucose and prostate-specific membrane antigen positron emission tomography in primary prostate cancer: a bioinformatic and experimental study.

BACKGROUND: Positron Emission Tomography (PET) imaging with Prostate-Specific Membrane Antigen (PSMA) and Fluorodeoxyglucose (FDG) represent promising biomarkers for risk-stratification of Prostate Cancer (PCa). We verified whether the expression of genes encoding for PSMA and enzymes regulating FDG cellular uptake are independent and additive prognosticators in PCa. METHODS: mRNA expression of genes involved in glucose metabolism and PSMA regulation obtained from primary PCa specimens were retrieved from open-source databases and analyzed using an integrative bioinformatics approach. Machine Learning (ML) techniques were used to create predictive Progression-Free Survival (PFS) models. Cellular models of primary PCa with different aggressiveness were used to compare [18F]F-PSMA-1007 and [18F]F-FDG uptake kinetics in vitro. Confocal microscopy, immunofluorescence staining, and quantification analyses were performed to assess the intracellular and cellular membrane PSMA expression. RESULTS: ML analyses identified a predictive functional network involving four glucose metabolism-related genes: ALDOB, CTH, PARP2, and SLC2A4. By contrast, FOLH1 expression (encoding for PSMA) did not provide any additive predictive value to the model. At a cellular level, the increase in proliferation rate and migratory potential by primary PCa cells was associated with enhanced FDG uptake and decreased PSMA retention (paralleled by the preferential intracellular localization). CONCLUSIONS: The overexpression of a functional network involving four glucose metabolism-related genes identifies a higher risk of disease progression since the earliest phases of PCa, in agreement with the acknowledged prognostic value of FDG PET imaging. By contrast, the prognostic value of PSMA PET imaging is independent of the expression of its encoding gene FOLH1. Instead, it is influenced by the protein docking to the cell membrane, regulating its accessibility to tracer binding.

Fundamental Role of Pentose Phosphate Pathway within the Endoplasmic Reticulum in Glutamine Addiction of Triple-Negative Breast Cancer Cells.

Cancer utilization of large glutamine equivalents contributes to diverging glucose-6-P flux toward the pentose phosphate shunt (PPP) to feed the building blocks and the antioxidant responses of rapidly proliferating cells. In addition to the well-acknowledged cytosolic pathway, cancer cells also run a largely independent PPP, triggered by hexose-6P-dehydrogenase within the endoplasmic reticulum (ER), whose activity is mandatory for the integrity of ER-mitochondria networking. To verify whether this reticular metabolism is dependent on glutamine levels, we complemented the metabolomic characterization of intermediates of the glucose metabolism and tricarboxylic acid cycle with the estimation of proliferating activity, energy metabolism, redox damage, and mitochondrial function in two breast cancer cell lines. ER-PPP activity and its determinants were estimated by the ER accumulation of glucose analogs. Glutamine shortage decreased the proliferation rate despite increased ATP and NADH levels. It depleted NADPH reductive power and increased malondialdehyde content despite a marked increase in glucose-6P-dehydrogenase. This paradox was explained by the deceleration of ER-PPP favored by the decrease in hexose-6P-dehydrogenase expression coupled with the opposite response of its competitor enzyme glucose-6P-phosphatase. The decreased ER-PPP activity eventually hampered mitochondrial function and calcium exchanges. These data configure the ER-PPP as a powerful, unrecognized regulator of cancer cell metabolism and proliferation.

Intramuscular injection of vectorized-scFvMC1 reduces pathological tau in two different tau transgenic models.

With evidence supporting the prion-like spreading of extracellular tau as a mechanism for the initiation and progression of Alzheimer's disease (AD), immunotherapy has emerged as a potential disease-modifying strategy to target tau. Many studies have proven effective to clear pathological tau species in animal models of AD, and several clinical trials using conventional immunotherapy with anti-tau native antibodies are currently active. We have previously generated a vectorized scFv derived from the conformation-dependent anti-tau antibody MC1, scFvMC1, and demonstrated that its intracranial injection was able to prevent tau pathology in adult tau mice. Here, we show that, in a prevention paradigm and in two different tau transgenic models (JNPL3 and P301S), a one-time intramuscular injection of AAV1-scFvMC1 generated a long-lasting peripheral source of anti-tau scFvMC1 and significantly reduced insoluble and soluble tau species in the brain. Moreover, our data showed that scFvMC1 was internalized by the microglia, in the absence of overt inflammation. This study demonstrates the efficacy of intramuscular delivery of vectorized scFv to target tau, and suggests a new potential application to treat AD and the other tauopathies.

Meat Production from Dairy Breed Lambs Due to Slaughter Age and Feeding Plan Based on Wheat Bran.

This experiment aimed to investigate the possibility to increase the carcass weight of dairy breed lambs and produce moderate-fat meat by applying inexpensive feeding strategies based on restriction and through the use of a fibrous byproduct such as the durum wheat bran (DWB). Sixty-five 45-day-old lambs of the Valle del Belice breed, divided into 6 groups, were fed alfalfa hay supplemented with concentrate feeds including DWB at 0% or 20% (DWB0, DWB20), supplied ad libitum (L) or restricted at 75% (R), and slaughtered at 90 or 120 days of age. The groups were as follows: DWB0-90L (n = 14), DWB20-90L (n = 14), DWB0-120R (n = 10), DWB20-120R (n = 9), DWB0-120L (n = 9), DWB20-120L (n = 9). The diet did not affect feed intake, growth or carcass weight of lambs fed ad libitum, whereas 120-day-old lambs fed DWB associated to restriction showed the lowest weight gain (105 vs. 170, 185 and 190 g/day in DWD20-120R, DWB0-120R, DWB0-120L and DWB20-120L; p = 0.04). The incidence of fat tissue in the hind leg increased (p < 0.0001) from 90L (5.82 and 5.45% with DWB0 and DWB20) to 120R (8.80 and 8.43% with DWB0 and DWB20) and 120L lambs (10.7 and 11.8% with DWB0 and DWB20). Older lambs' meat, compared to that of 90L lambs, showed analogous levels of intramuscular fat, higher water retention, tenderness and lightness, and a more intense red colour. In meat from 120-day-old lambs, DWB intake tended to reduce the fat level (p = 0.009) and increased polyphenol content (1.10 vs. 1.62, and 1.02 vs. 1.65 g GAE/kg dry matter (DM) in 120R and 120L lambs; p = 0.02), antioxidant capacity (12.8 vs. 14.9, and 12.8 vs. 15.7 mmol trolox eq/kg DM in 120R and 120L lambs; p = 0.02), and the presence of n-3 polyunsaturated fatty acids (FA) (1.61 vs. 2.81, and 1.43 vs. 2.61 g/100 g FA in 120R and 120L lambs; p = 0.007), thereby improving the meat's health properties. The panelists perceived the effects of DWB inclusion as well as the feeding level with triangle tests.

Improvement of Oxidative Status, Milk and Cheese Production, and Food Sustainability Indexes by Addition of Durum Wheat Bran to Dairy Cows' Diet.

Durum wheat bran (DWB) is a by-product mostly used in feeding ruminants, contributing to decrease in the utilization of feeds suitable as foods for human consumption, thus improving the sustainability of livestock production. However, the potential benefits of DWB, due to its content in phenolic acids, mainly consisting of ferulic acid with antioxidant properties, have not been well clarified yet. Accordingly, in this experiment, 36 lactating cows divided into three groups received, over a period of 100 days, one of three concentrates including DWB at 0% (DWB0), 10% (DWB10), or 20% (DWB20). The concentrates were formulated to be isoproteic and isoenergetic and, to balance the higher fiber content of the concentrates with DWB, the hay in the diets was slightly reduced. During the trial, the group feed intake and the individual milk production were monitored, and cheese was made with bulk milk from each group. Milk yield and microbiological characteristics of milk and cheese were similar among groups, indicating no DWB effect on cows performance and fermentation process. Milk from DWB20 group resulted slightly higher in casein and curd firmness (a2r). In cows fed DWB, the higher polyphenol intake was responsible for higher blood contents of these bioactive compounds, that seemed to have contributed in reducing the level of reactive oxygen metabolites (ROMs), which were higher in DWB0 cows. DWB20 cheeses showed a higher polyphenol content, lower number of peroxides, and higher antioxidant capacity than DWB0 cheeses. DWB20 and DWB10 diets resulted less expensive. In addition, the DWB20 group showed the best indexes heFCE (human edible feed conversion efficiency = milk/human edible feed) and NFP (net food production = milk - human edible food), expressed as crude protein or gross energy. In conclusion, the DWB fed to dairy cows at 12% of diet dry matter (DM) can lead to benefits, such as the improvement of oxidative status of cows, milk quality, shelf-life, and functional properties of cheese, and might contribute to reduce the feeding cost and limit the human-animal competition for feeding sources.

Anti-tau conformational scFv MC1 antibody efficiently reduces pathological tau species in adult JNPL3 mice.

Tau, the main component of the neurofibrillary tangles (NFTs), is an attractive target for immunotherapy in Alzheimer's disease (AD) and other tauopathies. MC1/Alz50 are currently the only antibodies targeting a disease-specific conformational modification of tau. Passive immunization experiments using intra-peritoneal injections have previously shown that MC1 is effective at reducing tau pathology in the forebrain of tau transgenic JNPL3 mice. In order to reach a long-term and sustained brain delivery, and avoid multiple injection protocols, we tested the efficacy of the single-chain variable fragment of MC1 (scFv-MC1) to reduce tau pathology in the same animal model, with focus on brain regional differences. ScFv-MC1 was cloned into an AAV delivery system and was directly injected into the hippocampus of adult JNPL3 mice. Specific promoters were employed to selectively target neurons or astrocytes for scFv-MC1 expression. ScFv-MC1 was able to decrease soluble, oligomeric and insoluble tau species, in our model. The effect was evident in the cortex, hippocampus and hindbrain. The astrocytic machinery appeared more efficient than the neuronal, with significant reduction of pathology in areas distant from the site of injection. To our knowledge, this is the first evidence that an anti-tau conformational scFv antibody, delivered directly into the mouse adult brain, is able to reduce pathological tau, providing further insight into the nature of immunotherapy strategies.

Airway management in obese patients.

The well-known difficulties in airway management in obese patients are caused by obesity-related airways and respiratory changes. Anesthesiologists confront a number of troubles, including rapid oxygen desaturation, difficulty with laryngoscopy/intubation and mask ventilation, and increased susceptibility to the respiratory depressant effects of anesthetic drugs. Preoperative assessment of the airways in the obese should include examination of specific predictors of difficult mask ventilation other than those for difficult intubation. Difficulties in airway management are decreased after providing optimal preoxygenation and positioning ("ramped"). Other strategies may include availability of alternative airway management devices, including new video laryngoscopes that significantly improve the visualization of the larynx and thereby facilitate intubation. If awake intubation is mandatory, it may be performed with fibrobronchoscope after providing an adequate topical anesthesia and sedation with short-acting drugs, such as remifentanil. Succinylcholine for rapid sequence induction might be replaced by rocuronium where sugammadex is available for reversal. A complete reversal of neuromuscular block, measured by train-of-four monitoring, should be obtained before extubation, which requires a fully awake patient in the same position with airway equipment used for intubation.

Prediction of poor graft function by means of gastric tonometry in patients undergoing liver transplantation.

UNLABELLED: INTRODUCTION. Splanchnic hypoperfusion appears to play a key role in the failure of functional recovery of the graft after orthotopic liver transplantation (LT). The aim of this study was to determine if alterations of tonometric parameters, which are related to splanchnic perfusion, could predict poor graft function in patients undergoing LT. MATERIALS AND METHODS: After Ethics Committee approval, 68 patients undergoing LT were enrolled. In all the patients, regional-arterial CO2 gradient (Pr-aCO2) was recorded; in addition, the difference between Pr-aCO2 recorded at anhepatic phase (T1) and at the end of surgery (T2) (T2- T1 = ΔPr-aCO2) was calculated. Poor graft function was determined on the basis of Toronto's classification 72 hours after LT. Student t-test and logistic regression analysis were used for statistical purpose. Results. ΔPr-aCO2 was significantly greater in patients with poor graft function (3.5 ± 13.2) compared to patients with good graft function (-5.8 ± 12.3) (p = 0.014). The logistic regression analysis showed that the ΔPr-aCO2 was able to predict the onset of poor graft function (p = 0.037). A value of ΔPr-aCO2 ≥ -4 was associated with poor graft function with a sensibility of 93.3% and a specificity of 42.3%. CONCLUSION. Our study suggests that the change of Pr-aCO2 may be a valuable index of graft dysfunction. Gastric tonometry might give early prognostic information on the graft outcome, and it may aid clinicians in planning a more strict follow-up and proper interventions in order to improve graft survival.

Carbon dioxide elimination pattern in morbidly obese patients undergoing laparoscopic surgery.

BACKGROUND: Hypercapnia can result from carbon dioxide pneumoperitoneum and adversely affect the postoperative period, particularly in morbidly obese patients. The purpose of the present study was to examine carbon dioxide homeostasis using a metabolic monitor in morbidly obese and normal weight patients during laparoscopic surgical procedures. The setting was a university hospital in Italy. METHODS: The data from 25 patients with a body mass index of 47.7 ± 5.5 kg/m2 undergoing laparoscopic gastric mini-bypass were compared with the data from 25 normal weight patients undergoing laparoscopic cholecystectomy. The minute ventilation was adjusted to maintain a normal arterial partial pressure of carbon dioxide and normal end-tidal partial pressure of carbon dioxide throughout surgical procedures. The arterial partial pressure of carbon dioxide, end-tidal partial pressure of carbon dioxide, total exhaled carbon dioxide per minute, and arterial blood gas analysis were obtained at 10-minute intervals, along with other cardiorespiratory parameters. RESULTS: The total exhaled carbon dioxide per minute increased by the same percentage in both groups (around 20%). In the laparoscopic cholecystectomy patients, a definite plateau in the total exhaled carbon dioxide per minute was observed within 20 minutes from the start of pneumoperitoneum but not in the morbidly obese patients. After desufflation, the total exhaled carbon dioxide per minute returned more rapidly to the baseline values in the laparoscopic cholecystectomy group than in the morbidly obese group (17.4 ± 6.2 and 24.1 ± 8.3 min, respectively). CONCLUSION: The results of our study have shown that the load of carbon dioxide insufflated is well tolerated in morbidly obese patients, as well as in normal patients, with proper intraoperative ventilation adjustments. However, after pneumoperitoneum, the return to a normal total exhaled carbon dioxide per minute required a longer period in the morbidly obese group. Prolonged mechanical ventilation is therefore advisable in morbidly obese patients.

Preinductive use of clonidine and ketamine improves recovery and reduces postoperative pain after bariatric surgery.

BACKGROUND: In obese patients, concomitant use of clonidine and ketamine might be suitable to reduce the doses and minimize the undesired side effects of anesthetic and analgesic drugs. In this study, we evaluated the perioperative effects of administration of clonidine and ketamine in morbidly obese patients undergoing weight loss surgery at a university hospital in Rome, Italy. METHODS: A total of 50 morbidly obese patients undergoing open biliopancreatic diversion for weight loss surgery were enrolled. The patients were randomly allocated into a study group (n = 23) receiving a slow infusion of ketamine-clonidine before anesthesia induction and a control group (n = 27) who received standard anesthesia. The hemodynamic profile, intraoperative end-tidal sevoflurane and opioid consumption, tracheal extubation time, Aldrete score, postoperative pain assessment by visual analog scale, and analgesic requirements were recorded. RESULTS: The patients in the study group required less end-tidal sevoflurane, lower total doses of fentanyl (3.8 +/- 0.3 gamma/kg actual body weight versus 5.0 +/- 0.2 gamma/kg actual body weight, respectively; P <.05) and had a shorter time to extubation (15.1 +/- 5 min versus 28.2 +/- 6 min, P <.05). The Aldrete score was significantly better in the postanesthesia care unit in the study group. The study group consumed less tramadol than did the control group (138 +/- 57 mg versus 252 +/- 78 mg, P <.05) and had a lower visual analog scale score postoperatively during the first 6 hours. CONCLUSION: The preoperative administration of low doses of ketamine and clonidine at induction appears to provide early extubation and diminished postoperative analgesic requirements in morbidly obese patients undergoing open bariatric surgery.