RESUMEN

Diagnostic markers are desperately needed for the early detection of pancreatic ductal adenocarcinoma (PDA). We describe sets of markers expressed in temporal order in mouse models during pancreatitis, PDA initiation and progression. Cell type specificity and the differential expression of PDA markers were identified by screening single cell (sc) RNAseq from tumor samples of a mouse model for PDA (KIC) at early and late stages of PDA progression compared to that of a normal pancreas. Candidate genes were identified from three sources: (1) an unsupervised screening of the genes preferentially expressed in mouse PDA tumors; (2) signaling pathways that drive PDA, including the Ras pathway, calcium signaling, and known cancer genes, or genes encoding proteins that were identified by differential mass spectrometry (MS) of mouse tumors and conditioned media from human cancer cell lines; and (3) genes whose expression is associated with poor or better prognoses (PAAD, oncolnc.org). The developmental progression of PDA was detected in the temporal order of gene expression in the cancer cells of the KIC mice. The earliest diagnostic markers were expressed in epithelial cancer cells in early-stage, but not late-stage, PDA tumors. Other early markers were expressed in the epithelium of both early- and late-state PDA tumors. Markers that were expressed somewhat later were first elevated in the epithelial cancer cells of the late-stage tumors, then in both epithelial and mesenchymal cells, or only in mesenchymal cells. Stromal markers were differentially expressed in early- and/or late-stage PDA neoplasia in fibroblast and hematopoietic cells (lymphocytes and/or macrophages) or broadly expressed in cancer and many stromal cell types. Pancreatitis is a risk factor for PDA in humans. Mouse models of pancreatitis, including caerulein treatment and the acinar-specific homozygous deletion of differentiation transcription factors (dTFs), were screened for the early expression of all PDA markers identified in the KIC neoplasia. Prognostic markers associated with a more rapid decline were identified and showed differential and cell-type-specific expression in PDA, predominately in late-stage epithelial and/or mesenchymal cancer cells. Select markers were validated by immunohistochemistry in mouse and human samples of a normal pancreas and those with early- and late-stage PDA. In total, we present 2165 individual diagnostic and prognostic markers for disease progression to be tested in humans from pancreatitis to late-stage PDA.

Asunto(s)

Biomarcadores de Tumor , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Pancreatitis , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/patología , Pancreatitis/metabolismo , Pancreatitis/genética , Pancreatitis/patología , Pancreatitis/diagnóstico , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Humanos , Pronóstico , Regulación Neoplásica de la Expresión Génica , Modelos Animales de Enfermedad , Línea Celular Tumoral , Progresión de la Enfermedad

RESUMEN

The cracking problem of asphalt concrete panels is a crucial consideration in the design of hydraulic asphalt concrete seepage control bodies. Panels experiencing uneven rises or falls of water levels during impoundment may exhibit loading rate effects. Investigating the fracture toughness value of asphalt concrete under varying loading rates is essential. This study employs a statistical method to calculate the fracture index KIC, using the semi-circular bending test (SCB) to examine the effect of loading rates on the Type I fracture mode of hydraulic asphalt concrete. The data are analyzed using the two-parameter Weibull distribution curve, offering insights into the minimum number of KIC test specimens. The results indicate an increase in KIC with loading rate, with greater data dispersion at faster rates. The Weibull distribution curve successfully fits the fracture behavior under different loading rates, providing valuable predictions. This study estimates the minimum number of SCB test specimens to be nine, based on a confidence level of 0.95 and a relative deviation not exceeding 5%.

RESUMEN

BACKGROUND: Ketamine is a novel and exciting putative antidepressant medication for patients with treatment-resistant depression. A complication commonly seen in frequent and heavy recreational use of ketamine is ulcerative cystitis, which presents with lower urinary tract symptoms (LUTS) and upper renal tract damage and can be seen in over 25% of regular users. Although Ketamine-induced cystitis (KIC) is a recognised complication in recreational use of ketamine, its occurrence in therapeutic use of ketamine in depression has so far not been reported. The exact pathogenesis of KIC is currently unknown, making treatment and prevention advice much more difficult. Early diagnosis of KIC and immediate cessation of ketamine has been shown to improve adverse urinary tract symptoms and prevent further damage. CASE PRESENTATION: We present a case of a 28-year-old female who was started on ketamine treatment for depression, and who then developed symptoms of KIC, which was confirmed by urine microscopy, culture and analysis. CONCLUSIONS: To our knowledge, this is the first reported case of KIC in a patient receiving treatment-dose ketamine as part of their antidepressant therapy.

Asunto(s)

Cistitis , Ketamina , Trastornos Relacionados con Sustancias , Femenino , Humanos , Adulto , Ketamina/efectos adversos , Depresión/tratamiento farmacológico , Microscopía , Trastornos Relacionados con Sustancias/complicaciones , Urinálisis , Cistitis/inducido químicamente , Cistitis/tratamiento farmacológico , Cistitis/complicaciones

RESUMEN

In order to provide guidance for furthering the balance of strength and toughness of AerMet 100 steel through tempering treatment, the effects of the tempering time on microstructure and mechanical properties are investigated. The microstructure evolution, especially M2C precipitates and austenite in AerMet 100 tempered at 482 °C for 1~20 h, was characterized, and its influences on the mechanical properties were studied. The tensile strength decreases gradually, the yield strength increases first and then decreases, and the fracture toughness KIC increases gradually with an increasing tempering time. The strength and toughness matching of AerMet 100 steel is achieved by tempering at 482 °C for 5~7 h. Without considering the martensitic size effect, the influence of the dislocation density on the tensile strength is more significant during tempering at 482 °C. The precipitation strengthening mechanism plays a dominant role in the yield strength when tempering for 5 h or less, and the combined influence of carbide coarsening and a sharp decrease in the dislocation density resulted in a significant decrease in tensile strength when tempering for 8 h or more. The fracture toughness KIC is primarily influenced by the reverted austenite, so that KIC increases gradually with the prolongation of the tempering time. However, a significant decrease in the dislocation density resulting from long-term tempering has a certain impact on KIC, giving rise to a decrease in the rising amplitude in KIC after tempering for 8 h or more.

RESUMEN

Robust yeast strains that are tolerant to multiple stress environments are desired for an efficient biorefinery. Our previous studies revealed that zinc sulfate serves as an important nutrient for stress tolerance of budding yeast Saccharomyces cerevisiae. Acetic acid is a common inhibitor in cellulosic hydrolysate, and the development of acetic acid-tolerant strains is beneficial for lignocellulosic biorefineries. In this study, comparative proteomic studies were performed using S. cerevisiae cultured under acetic acid stress with or without zinc sulfate addition, and novel zinc-responsive proteins were identified. Among the differentially expressed proteins, the protein kinase Kic1p and the small rho-like GTPase Cdc42p, which is required for cell integrity and regulation of cell polarity, respectively, were selected for further studies. Overexpression of KIC1 and CDC42 endowed S. cerevisiae with faster growth and ethanol fermentation under the stresses of acetic acid and mixed inhibitors, as well as in corncob hydrolysate. Notably, the engineered yeast strains showed a 12 h shorter lag phase under the three tested conditions, leading to up to 52.99% higher ethanol productivity than that of the control strain. Further studies showed that the transcription of genes related to stress response was significantly upregulated in the engineered strains under the stress condition. Our results in this study provide novel insights in exploring zinc-responsive proteins for applications of synthetic biology in developing a robust industrial yeast.

RESUMEN

The major industrial companies located in the Kwinana Industrial Area (KIA) produce many industrial, agricultural and mining chemicals and refined materials, for national and international markets. With over 150 documented product and by-product exchanges, Kwinana is considered to be one of the best examples of industrial symbiosis (IS) in the world. A new model of IS comprised of four dimensions is under development, whereby whilst each dimension is unique, collectively, they interact to characterise an industrial estate, thus contributing to the evolutionary understanding of IS. We investigate the basis for this model through an analysis of two water circular economy examples as they relate to Western Australia's premier industrial area, the KIA. Case studies will consider a managed aquifer recharge (MAR) project that failed and the process water interconnectedness of enterprises operating successfully as a sub-ecology within the industrial cluster. Apart from the traditional product and by-product dimension of IS, three additional dimensions seem to be playing a crucial role in the KIA, these being the skilled workforce, support industry and governance dimensions. We provide additional context for the water-related examples of the circular economy at Kwinana by exploring a new four-dimensional model for IS.

RESUMEN

Ti(C,N)-reinforced alumina-zirconia composites with different ratios of C to N in titanium carbonitride solid solutions, such as Ti(C0.3,N0.7) (C:N = 30:70) and Ti(C0.5,N0.5) (C:N = 50:50), were tested to improve their mechanical properties. Spark plasma sintering (SPS) with temperatures ranging from 1600 °C to 1675 °C and pressureless sintering (PS) with a higher temperature of 1720 °C were used to compare results. The following mechanical and physical properties were determined: Vickers hardness, Young's modulus, apparent density, wear resistance, and fracture toughness. A composite with the addition of Ti(C0.5,N0.5)n nanopowder exhibited the highest Vickers hardness of over 19.0 GPa, and its fracture toughness was at 5.0 Mpa·m1/2. A composite with the Ti(C0.3,N0.7) phase was found to have lower values of Vickers hardness (by about 10%), friction coefficient, and specific wear rate of disc (Wsd) compared to the composite with the addition of Ti(C0.5,N0.5). The Vickers hardness values slightly decreased (from 5% to 10%) with increasing sintering temperature. The mechanical properties of the samples sintered using PS were lower than those of the samples that were spark plasma sintered. This research on alumina-zirconia composites with different ratios of C to N in titanium carbonitride solid solution Ti(C,N), sintered using an unconventional SPS method, reveals the effect of C/N ratios on improving mechanical properties of tested composites. X-ray analysis of the phase composition and an observation of the microstructure was carried out.

Asunto(s)

Óxido de Aluminio/química , Materiales Biocompatibles/química , Cerámica/química , Gases em Plasma/química , Titanio/química , Circonio/química , Dureza , Ensayo de Materiales , Fenómenos Mecánicos , Propiedades de Superficie , Temperatura

RESUMEN

Cyanobacteria can be utilized as a platform for direct phototrophic conversion of CO2 to produce several types of carbon-neutral biofuels. One promising compound to be produced photobiologically in cyanobacteria is isobutene. As a volatile compound, isobutene will quickly escape the cells without building up to toxic levels in growth medium or get caught in the membranes. Unlike liquid biofuels, gaseous isobutene may be collected from the headspace and thus avoid the costly extraction of a chemical from culture medium or from cells. Here we investigate a putative synthetic pathway for isobutene production suitable for a photoautotrophic host. First, we expressed α-ketoisocaproate dioxygenase from Rattus norvegicus (RnKICD) in Escherichia coli. We discovered isobutene formation with the purified RnKICD with the rate of 104.6 â€‹± â€‹9 â€‹ng (mg protein)-1 min-1 using α-ketoisocaproate as a substrate. We further demonstrate isobutene production in the cyanobacterium Synechocystis sp. PCC 6803 by introducing the RnKICD enzyme. Synechocystis strain heterologously expressing the RnKICD produced 91 â€‹ng â€‹l-1 OD750 -1 â€‹h-1. Thus, we demonstrate a novel sustainable platform for cyanobacterial production of an important building block chemical, isobutene. These results indicate that RnKICD can be used to further optimize the synthetic isobutene pathway by protein and metabolic engineering efforts.

RESUMEN

Branched-chain amino acids (BCAA) and their metabolites the branched-chain keto acids (BCKA) and ß-hydroxy ß-methylbutyric acid (HMB) are involved in the regulation of key signaling pathways in the anabolic response to a meal. However, their (inter)organ kinetics remain unclear. Therefore, branched-chain amino acids (BCAA) [leucine (Leu), valine (Val), isoleucine (Ile)], BCKA [α-ketoisocaproic acid (KIC), 3-methyl-2-oxovaleric acid (KMV), 2-oxoisovalerate (KIV)], and HMB across organ net fluxes were measured. In multi-catheterized pigs (n = 12, ±25 kg), net fluxes across liver, portal drained viscera (PDV), kidney, and hindquarter (HQ, muscle compartment) were measured before and 4 h after bolus feeding of a complete meal (30% daily intake) in conscious state. Arterial and venous plasma were collected and concentrations were measured by LC- or GC-MS/MS. Data are expressed as mean [95% CI] and significance (P < 0.05) from zero by the Wilcoxon Signed Rank Test. In the postabsorptive state (in nmol/kg body wt/min), the kidney takes up HMB (3.2[1.3,5.0]) . BCKA is taken up by PDV (144[13,216]) but no release by other organs. In the postprandial state, the total net fluxes over 4 h (in µmol/kg body wt/4 h) showed a release of all BCKA by HQ (46.2[34.2,58.2]), KIC by the PDV (12.3[7.0,17.6]), and KIV by the kidney (10.0[2.3,178]). HMB was released by the liver (0.76[0.49,1.0]). All BCKA were taken up by the liver (200[133,268]). Substantial differences are present in (inter)organ metabolism and transport among the BCAA and its metabolites BCKA and HMB. The presented data in a translation animal model are relevant for the future development of optimized clinical nutrition.NEW & NOTEWORTHY Branched-chain amino acids (BCAA) and their metabolites the branched-chain keto acids (BCKA) and ß-hydroxy ß-methylbutyric acid (HMB) are involved in the regulation of key signaling pathways in the anabolic response to a meal. Substantial differences are present in (inter)organ metabolism and transport among the BCAA and its metabolites BCKA and HMB. The presented data in a translation animal model are relevant for the future development of optimized clinical nutrition.

Asunto(s)

Aminoácidos de Cadena Ramificada/farmacocinética , Cetoácidos/farmacocinética , Análisis de Flujos Metabólicos , Animales , Femenino , Hemiterpenos/farmacocinética , Riñón/metabolismo , Leucina/farmacocinética , Hígado/metabolismo , Análisis de Flujos Metabólicos/veterinaria , Redes y Vías Metabólicas/fisiología , Músculo Esquelético/metabolismo , Porcinos , Distribución Tisular , Valeratos/farmacocinética , Vísceras/metabolismo

RESUMEN

The fracture toughness KIc of 11 clinically used acrylic bone cements was studied in air at room temperature with single edge V-notched beam specimens. By driving the crack step-wise through the specimens, crack resistance curves ("R-curves") were recorded. One group of bone cements showed an increase of the fracture toughness with increasing crack length (including CMW1+G and several Palacos bone cements) whereas another group (including Simplex, SmartSet, Copal and some Palacos bone cements) did not exhibit an R-curve behavior. The plateau values for KIc ranged from 0.93 MPa√m (Simplex P) to 1.98 MPa√m (Palacos R+G). The observation of the crack growth with an optical microscope revealed some mechanisms influencing the crack growth like the formation of microcracks in the extended damage zone of the crack tip, the attraction of the crack by inclusions or the shielding of the crack tip by bridges in the wake of the crack. Furthermore, bone cements could be distinguished by the pattern of the path the crack followed during propagation. The crack pattern of CMW1+G provides a possible explanation of the distinct R-curve behavior of this cement.

Asunto(s)

Cementos para Huesos/química , Fracturas Óseas/fisiopatología , Polimetil Metacrilato/química , Fenómenos Biomecánicos , Humanos , Ensayo de Materiales , Metilmetacrilato/química , Modelos Biológicos , Resistencia a la Tracción

RESUMEN

Accumulating evidence show that exercise and diet interventions are associated with improved sleep quality. Studies investigating the effects of exercise and dieting on circulating metabolomics in people with sleep disorders, particularly insomnia, are scarce. This 6-month randomized study aimed to assess the effects of exercise and dietary interventions on serum metabolites in men with insomnia symptoms. Seventy-two Finnish men (age: 51.6 ±â€¯10.1 years) with chronic insomnia symptoms who were assigned to different intervention groups completed this study (exercise, n = 24; diet, n = 27; and control, n = 21). The Shapiro-Wilk W-test, Levene test, Spearman correlation analysis, and analysis of variance were used for data analysis. We found that exercise and diet intervention were associated with improved sleep quality and with a number of metabolites across different biochemical pathways. Although we could not show causality, our findings provide new insight into the biological mechanisms underlying the health effects of physical activity, diet, and sleep quality. Further investigation is needed to better understand the link among lifestyle, sleep quality, and metabolic health.

RESUMEN

Modern quantitative mass spectrometry (MS)-based proteomics enables researchers to unravel signaling networks by monitoring proteome-wide cellular responses to different stimuli. MS-based analysis of signaling systems usually requires an integration of multiple quantitative MS experiments, which remains challenging, given that the overlap between these datasets is not necessarily comprehensive. In a previous study we analyzed the impact of the yeast mitogen-activated protein kinase (MAPK) Hog1 on the hyperosmotic stress-affected phosphorylome. Using a combination of a series of hyperosmotic stress and kinase inhibition experiments, we identified a broad range of direct and indirect substrates of the MAPK. Here we re-evaluate this extensive MS dataset and demonstrate that a combined analysis based on two software packages, MaxQuant and Proteome Discoverer, increases the coverage of Hog1-target proteins by 30%. Using protein-protein proximity assays we show that the majority of new targets gained by this analysis are indeed Hog1-interactors. Additionally, kinetic profiles indicate differential trends of Hog1-dependent versus Hog1-independent phosphorylation sites. Our findings highlight a previously unrecognized interconnection between Hog1 signaling and the RAM signaling network, as well as sphingolipid homeostasis.

Asunto(s)

Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteómica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Programas Informáticos , Células HeLa , Humanos , Fosforilación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo

RESUMEN

We present the first good evidence for exocomet transits of a host star in continuum light in data from the Kepler mission. The Kepler star in question, KIC 3542116, is of spectral type F2V and is quite bright at Kp = 10. The transits have a distinct asymmetric shape with a steeper ingress and slower egress that can be ascribed to objects with a trailing dust tail passing over the stellar disk. There are three deeper transits with depths of ≃ 0.1% that last for about a day, and three that are several times more shallow and of shorter duration. The transits were found via an exhaustive visual search of the entire Kepler photometric data set, which we describe in some detail. We review the methods we use to validate the Kepler data showing the comet transits, and rule out instrumental artefacts as sources of the signals. We fit the transits with a simple dust-tail model, and find that a transverse comet speed of ∼35-50 km s-1 and a minimum amount of dust present in the tail of ∼ 1016 g are required to explain the larger transits. For a dust replenishment time of ∼10 days, and a comet lifetime of only ∼300 days, this implies a total cometary mass of ≳ 3 × 1017 g, or about the mass of Halley's comet. We also discuss the number of comets and orbital geometry that would be necessary to explain the six transits detected over the four years of Kepler prime-field observations. Finally, we also report the discovery of a single comet-shaped transit in KIC 11084727 with very similar transit and host-star properties.

RESUMEN

Branched-chain amino acids (BCAA) have been clearly demonstrated to have anabolic effects on muscle protein synthesis. However, little is known about their roles in the regulation of net AA fluxes across skeletal muscle in vivo. This study was aimed to investigate the effect and related mechanisms of dietary supplementation of BCAA on muscle net amino acid (AA) fluxes using the hindlimb flux model. In all fourteen 4-week-old barrows were fed reduced-protein diets with or without supplemental BCAA for 28 d. Pigs were implanted with carotid arterial, femoral arterial and venous catheters, and fed once hourly with intraarterial infusion of p-amino hippurate. Arterial and venous plasma and muscle samples were obtained for the measurement of AA, branched-chain α-keto acids (BCKA) and 3-methylhistidine (3-MH). Metabolomes of venous plasma were determined by HPLC-quadrupole time-of-flight-MS. BCAA-supplemented group showed elevated muscle net fluxes of total essential AA, non-essential AA and AA. As for individual AA, muscle net fluxes of each BCAA and their metabolites (alanine, glutamate and glutamine), along with those of histidine, methionine and several functional non-essential AA (glycine, proline and serine), were increased by BCAA supplementation. The elevated muscle net AA fluxes were associated with the increase in arterial and intramuscular concentrations of BCAA and venous metabolites including BCKA and free fatty acids, and were also related to the decrease in the intramuscular concentration of 3-MH. Correlation analysis indicated that muscle net AA fluxes are highly and positively correlated with arterial BCAA concentrations and muscle net BCKA production. In conclusion, supplementing BCAA to reduced-protein diet increases the arterial concentrations and intramuscular catabolism of BCAA, both of which would contribute to an increase of muscle net AA fluxes in young pigs.

Asunto(s)

Aminoácidos de Cadena Ramificada/administración & dosificación , Anabolizantes/administración & dosificación , Dieta con Restricción de Proteínas/veterinaria , Desarrollo de Músculos , Proteínas Musculares/biosíntesis , Músculo Esquelético/metabolismo , Regulación hacia Arriba , Aminoácidos/sangre , Aminoácidos/metabolismo , Aminoácidos de Cadena Ramificada/sangre , Aminoácidos de Cadena Ramificada/metabolismo , Anabolizantes/sangre , Anabolizantes/metabolismo , Animales , China , Cruzamientos Genéticos , Dieta con Restricción de Proteínas/efectos adversos , Ácidos Grasos no Esterificados/sangre , Ácidos Grasos no Esterificados/metabolismo , Miembro Posterior , Técnicas de Dilución del Indicador , Cetoácidos/sangre , Cetoácidos/metabolismo , Masculino , Metabolómica/métodos , Metilhistidinas/sangre , Metilhistidinas/metabolismo , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/crecimiento & desarrollo , Orquiectomía/veterinaria , Flujo Sanguíneo Regional , Sus scrofa , Aumento de Peso

RESUMEN

OBJECTIVE: Glucose-stimulated insulin secretion in pancreatic beta cells requires metabolic signals including the generation of glucose-derived short chain acyl-CoAs in the cytosol from mitochondrially-derived metabolites. One concept of insulin secretion is that ATP citrate lyase generates short chain acyl-CoAs in the cytosol from mitochondrially-derived citrate. Of these, malonyl-CoA, is believed to be an important signal in insulin secretion. Malonyl-CoA is also a precursor for lipids. Our recent evidence suggested that, in the mitochondria of beta cells, glucose-derived pyruvate can be metabolized to acetoacetate that is exported to the cytosol and metabolized to the same short chain acyl-CoAs and fatty acids that can be derived from citrate. We tested for redundancy of the citrate pathway. METHODS: We inhibited ATP citrate lyase activity using hydroxycitrate as well as studying a stable cell line generated with shRNA knockdown of ATP citrate lyase in the pancreatic beta cell line INS-1 832/13. RESULTS: In both instances glucose-stimulated insulin release was not inhibited. Mass spectrometry analysis showed that the flux of carbon from [U-(13)C]glucose and/or [U-(13)C]α-ketoisocaproic acid (KIC) into short chain acyl-CoAs in cells with hydroxycitrate-inhibited ATP citrate lyase or in the cell line with stable severe (>90%) shRNA knockdown of ATP citrate lyase was similar to the controls. Both (13)C-glucose and (13)C-KIC introduced substantial (13)C labeling into acetyl-CoA, malonyl-CoA, and HMG-CoA under both conditions. Glucose flux into fatty acids was not affected by ATP citrate lyase knockdown. CONCLUSION: The results establish the involvement of the acetoacetate pathway in insulin secretion in pancreatic beta cells.

RESUMEN

In connection with the increasing interest in metabolic regulation of the immune response, this review discusses current advances in understanding the role of leucine and leucine metabolism in T lymphocyte (T cell) activation. T cell activation during the development of an immune response depends on metabolic reprogramming to ensure that sufficient nutrients and energy are taken up by the highly proliferating T cells. Leucine has been described as an important essential amino acid and a nutrient signal that activates complex 1 of the mammalian target of rapamycin (mTORC1), which is a critical regulator of T cell proliferation, differentiation, and function. The role of leucine in these processes is further discussed in relation to amino acid transporters, leucine-degrading enzymes, and other metabolites of leucine metabolism. A new model of T cell regulation by leucine is proposed and outlines a chain of events that leads to the activation of mTORC1 in T cells.

Asunto(s)

Inmunidad/fisiología , Leucina/metabolismo , Activación de Linfocitos/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Transducción de Señal/fisiología , Linfocitos T/inmunología , Animales , Humanos , Factores Inmunológicos , Leucina/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Transaminasas/metabolismo

RESUMEN

A direct, quantitative, and confirmatory method based on stable isotope dilution liquid chromatography-mass spectrometry was developed and validated for the analysis of leucine and metabolites ß-hydroxy-ß-methylbutyric acid (HMB), α-ketoisocaproic acid (KIC), and α-hydroxyisocaproic acid (HICA) in human breast milk. Chromatographic resolution was achieved between isobaric leucine and isoleucine. Accuracy and intermediate precision were 89-117% and <10% relative standard deviation (RSD) across three validation runs. Limits of quantitation for HMB, KIC, HICA, and leucine in human breast milk were 20 µg/L, 20 µg/L, 10 µg/L, and 1 mg/L. Measured concentrations of HMB, KIC, HICA, and free leucine in human breast milk from six donors at various stages of lactation were 42-164 µg/L, < 20-1057 µg/L, < 10 µg/L, and 2.1-88.5 mg/L. HMB and KIC were confirmed in human breast milk by orthogonal hydrophilic interaction chromatography (HILIC). This work provides a tool for further study of human breast milk composition and its effect on protein turnover in developing infants.

Asunto(s)

Butiratos/química , Caproatos/química , Cromatografía Liquida/métodos , Hidroxiácidos/química , Cetoácidos/química , Leucina/química , Espectrometría de Masas/métodos , Leche Humana/química , Butiratos/metabolismo , Caproatos/metabolismo , Femenino , Humanos , Hidroxiácidos/metabolismo , Cetoácidos/metabolismo , Leucina/metabolismo , Leche Humana/metabolismo , Estructura Molecular

RESUMEN

OBJECTIVE: α/ß-Hydrolase domain-6 (ABHD6) is a newly identified monoacylglycerol (MAG) lipase. We recently reported that it negatively regulates glucose stimulated insulin secretion (GSIS) in the ß cells by hydrolyzing lipolysis-derived MAG that acts as a metabolic coupling factor and signaling molecule via exocytotic regulator Munc13-1. Whether ABHD6 and MAG play a role in response to all classes of insulin secretagogues, in particular various fuel and non-fuel stimuli, is unknown. METHODS: Insulin secretion in response to various classes of secretagogues, exogenous MAG and pharmacological agents was measured in islets of mice deficient in ABHD6 specifically in the ß cell (BKO). Islet perifusion experiments and determinations of glucose and fatty acid metabolism, cytosolic Ca(2+) and MAG species levels were carried out. RESULTS: Deletion of ABHD6 potentiated insulin secretion in response to the fuels glutamine plus leucine and α-ketoisocaproate and to the non-fuel stimuli glucagon-like peptide 1, carbamylcholine and elevated KCl. Fatty acids amplified GSIS in control and BKO mice to the same extent. Exogenous 1-MAG amplified insulin secretion in response to fuel and non-fuel stimuli. MAG hydrolysis activity was greatly reduced in BKO islets without changes in total diacylglycerol and triacylglycerol lipase activity. ABHD6 deletion induced insulin secretion independently from KATP channels and did not alter the glucose induced rise in intracellular Ca(2+). Perifusion studies showed elevated insulin secretion during second phase of GSIS in BKO islets that was not due to altered cytosolic Ca(2+) signaling or because of changes in glucose and fatty acid metabolism. Glucose increased islet saturated long chain 1-MAG species and ABHD6 deletion caused accumulation of these 1-MAG species at both low and elevated glucose. CONCLUSION: ABHD6 regulates insulin secretion in response to fuel stimuli at large and some non-fuel stimuli by controlling long chain saturated 1-MAG levels that synergize with other signaling pathways for secretion.

RESUMEN

Estimating skeletal muscle (finger) forces using surface Electromyography (sEMG) signals poses many challenges. In general, the sEMG measurements are based on single sensor data. In this paper, two novel hybrid fusion techniques for estimating the skeletal muscle force from the sEMG array sensors are proposed. The sEMG signals are pre-processed using five different filters: Butterworth, Chebychev Type II, Exponential, Half-Gaussian and Wavelet transforms. Dynamic models are extracted from the acquired data using Nonlinear Wiener Hammerstein (NLWH) models and Spectral Analysis Frequency Dependent Resolution (SPAFDR) models based system identification techniques. A detailed comparison is provided for the proposed filters and models using 18 healthy subjects. Wavelet transforms give higher mean correlation of 72.6 ± 1.7 (mean ± SD) and 70.4 ± 1.5 (mean ± SD) for NLWH and SPAFDR models, respectively, when compared to the other filters used in this work. Experimental verification of the fusion based hybrid models with wavelet transform shows a 96% mean correlation and 3.9% mean relative error with a standard deviation of ± 1.3 and ± 0.9 respectively between the overall hybrid fusion algorithm estimated and the actual force for 18 test subjects' k-fold cross validation data.

Asunto(s)

Electromiografía/métodos , Modelos Estadísticos , Músculo Esquelético/fisiología , Análisis de Ondículas , Adulto , Algoritmos , Femenino , Dedos/fisiología , Antebrazo/fisiología , Humanos , Masculino

RESUMEN

OBJECTIVE: The ß-cell metabolism of glucose and of some other fuels (e.g. α-ketoisocaproate) generates signals triggering and acutely amplifying insulin secretion. As the pathway coupling metabolism with amplification is largely unknown, we aimed to narrow down the putative amplifying signals. MATERIALS/METHODS: An experimental design was used which previously prevented glucose-induced, but not α-ketoisocaproate-induced insulin secretion. Isolated mouse islets were pretreated for one hour with medium devoid of fuels and containing the sulfonylurea glipizide in high concentration which closed all ATP-sensitive K(+) channels. This concentration was also applied during the subsequent examination of fuel-induced effects. In perifused or incubated islets, insulin secretion and metabolic parameters were measured. RESULTS: The pretreatment decreased the islet ATP/ADP ratio. Whereas glucose and α-ketoisovalerate were ineffective or weakly effective, respectively, when tested separately, their combination strongly enhanced the insulin secretion. Compared with glucose, the strong amplifier α-ketoisocaproate caused less increase in NAD(P)H-fluorescence and less mitochondrial hyperpolarization. Compared with α-ketoisovalerate, α-ketoisocaproate caused greater increase in NAD(P)H-fluorescence and greater mitochondrial hyperpolarization. Neither α-ketoacid anion enhanced the islet ATP/ADP ratio during onset of the insulin secretion. α-Ketoisocaproate induced a higher pyruvate content than glucose, slowly elevated the citrate content which was not changed by glucose and generated a much higher acetoacetate content than other fuels. α-Ketoisovalerate alone or in combination with glucose did not increase the citrate content. CONCLUSIONS: In ß-cells, mitochondrial energy generation does not mediate acute metabolic amplification, but mitochondrial production of acetyl-CoA and supplemental acetoacetate supplies cytosolic metabolites which induce the generation of specific amplifying signals.

Asunto(s)

Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Mitocondrias/metabolismo , Acetoacetatos/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Ácido Cítrico/metabolismo , Femenino , Flavina-Adenina Dinucleótido/metabolismo , Fluorescencia , Glipizida/farmacología , Glucosa/metabolismo , Hemiterpenos , Cetoácidos/metabolismo , Ratones , NADP/metabolismo , Ácido Pirúvico/metabolismo , Compuestos de Sulfonilurea/farmacología