Dysregulation in CD39/CD73 Axis May Trigger the Upsurge of the Immune Suppressive Agent Adenosine in OSA Patients

Introduction Although higher incidence of cancer represents a major burden for obstructive sleep apnea (OSA) patients, the molecular pathways driving this association are not completely understood. Interestingly, adenosinergic signaling has emerged as a powerful immune checkpoint driving tumor development and progression. Methods Here, we explored the expression of the adenosinergic ecto-enzymes CD39 and CD73 in T-lymphocytes of OSA patients without any evidence of cancer, as well as their soluble forms in plasma (sCD39 and sCD73), along with adenosine. In addition, we explored the role of intermittent hypoxia (IH) in this context by in vitro models. Results Our results showed that CD39 is upregulated while CD73 is downregulated in OSA T-cells’ membrane. Moreover, our findings suggest that IH, through HIF-1, mediates the upregulation of both CD39 and CD73; and that CD73 downregulation could be mediated by a higher release of sCD73 by OSA T-lymphocytes. Importantly, we found that both sCD39 and sCD73 are upregulated in OSA plasma, suggesting T-lymphocytes as a potential source for plasmatic sCD73. Finally, our data propose the alterations in CD39/CD73 axis could underlie the upsurge of adenosine levels in the plasma of OSA patients. Conclusion Our study reveals a hypoxia-mediated alteration of the CD39/CD73 axis in OSA patients, which could trigger ADO upregulation, thus potentially contributing to the immune suppressive environment and ultimately facilitating tumor development and progression. Therefore, our data highlights the need for new longitudinal studies evaluating CD39 and/or CD73 as potential cancer-risk prognostic biomarkers in OSA patients. (AU)

Dysregulation in CD39/CD73 Axis May Trigger the Upsurge of the Immune Suppressive Agent Adenosine in OSA Patients.

INTRODUCTION: Although higher incidence of cancer represents a major burden for obstructive sleep apnea (OSA) patients, the molecular pathways driving this association are not completely understood. Interestingly, adenosinergic signaling has emerged as a powerful immune checkpoint driving tumor development and progression. METHODS: Here, we explored the expression of the adenosinergic ecto-enzymes CD39 and CD73 in T-lymphocytes of OSA patients without any evidence of cancer, as well as their soluble forms in plasma (sCD39 and sCD73), along with adenosine. In addition, we explored the role of intermittent hypoxia (IH) in this context by in vitro models. RESULTS: Our results showed that CD39 is upregulated while CD73 is downregulated in OSA T-cells' membrane. Moreover, our findings suggest that IH, through HIF-1, mediates the upregulation of both CD39 and CD73; and that CD73 downregulation could be mediated by a higher release of sCD73 by OSA T-lymphocytes. Importantly, we found that both sCD39 and sCD73 are upregulated in OSA plasma, suggesting T-lymphocytes as a potential source for plasmatic sCD73. Finally, our data propose the alterations in CD39/CD73 axis could underlie the upsurge of adenosine levels in the plasma of OSA patients. CONCLUSION: Our study reveals a hypoxia-mediated alteration of the CD39/CD73 axis in OSA patients, which could trigger ADO upregulation, thus potentially contributing to the immune suppressive environment and ultimately facilitating tumor development and progression. Therefore, our data highlights the need for new longitudinal studies evaluating CD39 and/or CD73 as potential cancer-risk prognostic biomarkers in OSA patients.

Multiplex real-time PCR FilmArray performance in the diagnosis of meningoencephalitis: lights and shadows.

PURPOSE: We aimed to evaluate the performance of the FilmArray (FA) meningitis/encephalitis (ME) panel. Secondarily, we analyzed the false positive (FP) and false negative (FN) results, as well as the predictive values of the technique, regarding the cerebrospinal fluid (CSF) characteristics. METHODS: FA is a multiplex real-time PCR detecting 14 of the most common ME pathogens in CSF. All FA performed at our hospital (2018-2022) were retrospectively reviewed. FA was compared to conventional techniques and its performance was assessed based on the final diagnosis of the episode. RESULTS: FA was performed in 313 patients with suspicion of ME. Most patients had altered mental status (65.2%) and fever (61%). Regarding CSF characteristics, 49.8% and 53.7% presented high CSF proteins and pleocytosis, respectively. There were 84 (26.8%) positive FA results, mainly for HSV-1 (10.9%), VZV (5.1%), Enterovirus (2.6%), and S. pneumoniae (1.9%). In the 136 cases where both FA and routine methods were performed, there was a 25.7% lack of agreement. We identified 6.6% FN results, but 28.6% FP, mainly due to HSV-1. This resulted in a high negative predictive value (NPV) of 93.4%, but a positive predictive value (PPV) of 73%. Remarkably, PPV as low as 36.9%, and 70.2%, were found in cases without pleocytosis, or lack of high CSF protein levels, respectively. CONCLUSION: FA was associated with high NPV, but frequent FP results and low PPV, particularly for HSV-1, and especially in patients without high CSF protein levels or pleocytosis.

Enhancement of biogas production rate from bioplastics by alkaline pretreatment.

The effect of alkali-based pretreatment on the methanization of bioplastics was investigated. The tested bioplastics included PHB [poly(3-hydroxybutyrate)], PHBH [poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)], PHBV [poly(3-hydroxybutyrate-co-3-hydroxyvalerate], PLA (polylactic acid), and a PLA/PCL [poly(caprolactone)] 80/20 blend. Prior to methanization tests, the powdered polymers (500-1000 µm) at a concentration of 50 g/L were subjected to alkaline pretreatment using NaOH 1 M for PLA and PLA/PCL, and NaOH 2 M for PHB-based materials. Following 7 days of pretreatment, the amount of solubilized carbon for PLA and its blend accounted for 92-98% of the total initial carbon, while lower carbon recoveries were recorded for most PHB-based materials (80-93%), as revealed by dissolved total organic carbon analysis. The pretreated bioplastics were then tested for biogas production by means of mesophilic biochemical methane potential tests. Compared to unpretreated PHBs, methanization rates of pretreated PHBs were accelerated by a factor of 2.7 to 9.1 with comparable (430 NmL CH4/g material feed) or slightly lower (15% in the case of PHBH) methane yields, despite featuring a 1.4-2.3 times longer lag phases. Both materials, PLA and the PLA/PCL blend, were only extensively digested when pretreated, yielding about 360-380 NmL CH4 per gram of material fed. Unpretreated PLA-based materials showed nearly zero methanization under the timeframe and experimental conditions tested. Overall, the results suggested that alkaline pretreatment can help to enhance the methanization kinetics of bioplastics.

Screening Enzymes That Can Depolymerize Commercial Biodegradable Polymers: Heterologous Expression of Fusarium solani Cutinase in Escherichia coli.

In recent years, a number of microbial enzymes capable of degrading plastics have been identified. Biocatalytic depolymerization mediated by enzymes has emerged as a potentially more efficient and environmentally friendly alternative to the currently employed methods for plastic treatment and recycling. However, the functional and systematic study of depolymerase enzymes with respect to the degradation of a series of plastic polymers in a single work has not been widely addressed at present. In this study, the ability of a set of enzymes (esterase, arylesterase and cutinase) to degrade commercial biodegradable polymers (PBS, PBAT, PHB, PHBH, PHBV, PCL, PLA and PLA/PCL) and the effect of pre-treatment methods on their degradation rate was assessed. The degradation products were identified and quantified by HPLC and LC-HRMS analysis. Out of the three enzymes, Fusarium solani cutinase (FsCut) showed the highest activity on grinded PBAT, PBS and PCL after 7 days of incubation. FsCut was engineered and heterologous expressed in Escherichia coli, which conferred the bacterium the capability of degrading solid discs of PBAT and to grow in PBS as the sole carbon source of the medium.

Production of volatile fatty acids (VFAs) from five commercial bioplastics via acidogenic fermentation.

The feasibility of producing volatile fatty acids (VFAs) from five commercial bioplastics via acidogenic fermentation by a non-pretreated anaerobic sludge was investigated. Mesophilic, anaerobic, acidogenic batch assays at 1, 10 and 20 g/L feed concentrations revealed the feasibility of producing VFAs from polyhydroxyalkanoates (PHA), i.e., PHB and PHBV, but not from PBS, PCL and PLA under the test conditions and time. However, only high PHA substrate concentrations (10-20 g/L) resulted in organic overloading and decreasing the pH of the culture broth down to 4-5, which in turn induced the accumulation of VFAs via kinetic imbalance between acidogenesis and methanogenesis. Gaseous carbon (C-CO2 and C-CH4) accounted for 8-35% of the total initial carbon, while C-VFAs represented 10-18%, mainly as acetate and butyrate. This study represents the first systematically assessed proof-of-concept to produce VFAs from PHA, which is key for the design of bioplastic-to-bioplastic recycling (bio)technologies.

Longitudinal study of changes observed in quality of life, psychological state cognition and pulmonary and functional capacity after COVID-19 infection: A six- to seven-month prospective cohort.

AIMS: To investigate the health-related quality of life (HRQoL), symptoms, psychological and cognitive state and pulmonary and physical function of nonhospitalised COVID-19 patients at long-term, and to identify factors to predict a poor HRQoL in this follow-up. BACKGROUND: Studies have focused on persistent symptoms of hospitalised COVID-19 patients in the medium term. Thus, long-term studies of nonhospitalised patients are urgently required. DESIGN: A longitudinal cohort study. METHODS: In 102 nonhospitalised COVID-19 patients, we collected symptoms at 3 months (baseline) and at 6-7 months (follow-up) from diagnosis (dyspnoea, fatigue/muscle weakness and chest/joint pain), HRQoL, psychological state, cognitive function, pulmonary and physical function. This study adhered to the STROBE statement. RESULTS: HRQoL was impaired in almost 60% of the sample and remained impaired 6-7 months. At 3 months, more than 60% had impaired physical function (fatigue/muscle weakness and reduced leg and inspiratory muscle strength). About 40%-56% of the sample showed an altered psychological state (post-traumatic stress disorder (PTSD), anxiety/depression), cognitive function impairment and dyspnoea. At 6-7-months, only a slight improvement in dyspnoea and physical and cognitive function was observed, with a very high proportion of the sample (29%-55%) remained impaired. Impaired HRQoL at 6-7 months was predicted with 82.4% accuracy (86.7% sensitivity and 83.3% specificity) by the presence at 3 months of muscle fatigue/muscle weakness (OR = 5.7 (1.8-18.1)), PTSD (OR = 6.0 (1.7-20.7)) and impaired HRQoL (OR = 11.7 (3.7-36.8)). CONCLUSION: A high proportion of nonhospitalised patients with COVID-19 experience an impaired HRQoL, cognitive and psychological function at long-term. HRQoL, PTSD and dyspnoea at 3 months can identify the majority of patients with COVID-19 who will have impaired quality of life at long-term. RELEVANCE TO CLINICAL PRACTICE: Treatments aimed at improving psychological state and reducing the fatigue/muscle weakness of post-COVID-19 patients could be necessary to prevent the patients' HRQoL from being impaired at 6-7 months after their reported recovery.

Biodegradation of bioplastics under aerobic and anaerobic aqueous conditions: Kinetics, carbon fate and particle size effect.

The biodegradation of PHB, PHBV, PBS, PBAT, PCL, PLA, and a PLA-PCL blend was compared under aerobic and anaerobic aqueous conditions assessing biodegradation kinetics, extent, carbon fate and particle size influence (in the range of 100-1000 µm). Under standard test conditions, PHB and PBHV were biodegraded anaerobically (83.9 ± 1.3% and 81.2 ± 1.7%, respectively) in 77 days or aerobically (83.0 ± 1.6% and 87.4 ± 7.5%) in 117 days, while PCL was only biodegraded (77.6 ± 2.4%) aerobically in 177 days. Apparent biomass growth accounted for 10 to 30.5% of the total initial carbon depending on the bioplastic and condition. Maximum aerobic and anaerobic biodegradation rates were improved up to 331 and 405%, respectively, at the lowest particle size tested (100-250 µm). This study highlights the usefulness of analysing biodegradation kinetics and carbon fate to improve both the development and testing of biodegradable materials, and waste treatments in the context of a circular bioeconomy.

The autophagic marker p62 highlights Alzheimer type II astrocytes in metabolic/hepatic encephalopathy.

Metabolic/hepatic encephalopathy is neuropathologically characterized by the presence of Alzheimer type II astrocytes (AA II) with large and clear nuclear morphology. To date, there is no good immunohistochemical marker to better identify these cells. Here, we assessed cases of hepatic encephalopathy of different etiologies by immunohistochemistry using an anti-p62 antibody. We observed peripheral or diffuse nuclear staining of variable intensity in AA II in all cases but not in normal controls or reactive astrocytes. We conclude that p62 is a useful immunohistochemical marker for the identification of AA II and may be helpful for the neuropathological diagnosis of metabolic/hepatic encephalopathy in difficult or equivocal cases.

Cápsulas de arte: memoria frente al Alzheimer / Art capsule: memory to fight Alzheimer's / Cápsula de arte: memória contra a doença de Alzheimer

El presente trabajo analiza, a través de una técnica de panel, denominada "de cohorte", las reflexiones de las investigadoras españolas en el campo del Arte y del Alzheimer, con el fin de estudiar su trayectoria profesional y de relacionar las conclusiones de sus trabajos, con los resultados del Informe Mundial de la Organización Mundial de la Salud (OMS), el cual establece, por vez primera, una correlación positiva entre las variables arte y salud, situando el compromiso ético del arte, en la vanguardia de la investigación. Las conclusiones derivadas del estudio muestran, cómo al hablar de sí mismas, las investigadoras se definen no sólo como educadoras artísticas, sino también como cuidadoras, reconociendo el papel del contexto, esto es de su propia bibliografía, como determinante en las investigaciones que han realizado en el campo de la Educación Artística aplicada al Alzheimer.(AU)

The objective of this study is to analyze—through a panel technique called "cohort"—the authors' reflections on the fields of Arts and Alzheimer's in order to study their professional path and correlate the conclusions of their work with the results presented on the Health Evidence Network synthesis report 67 of the World Health Organization (WHO). For the first time, the report establishes a positive correlation between the arts and health, highlighting the importance of the ethical commitment of the arts to the research. The conclusions of this study show how the researchers refer to themselves as both "arts educators" and "caregivers," acknowledging the decisive role of context, i.e. their won biographies, to the research conducted in the field of Arts Education applied to Alzheimer's.(AU)

O presente trabalho analisa, por meio de uma técnica de painel denominada "coorte", as reflexões das investigadoras espanholas no campo da Arte e do Alzheimer, com o fim de estudar o seu percurso profissional e de relacionar as conclusões dos seus trabalhos com os resultados do Relatório Mundial da Organização Mundial da Saúde (OMS), que estabelece, pela primeira vez, uma correlação positiva entre as variáveis arte e saúde, situando o compromisso ético da arte na vanguarda da investigação. As conclusões resultantes do estudo mostram como, ao falar de si mesmas, as pesquisadoras definem-se não só como educadoras artísticas, mas também como cuidadoras, reconhecendo o papel do contexto, isto é, de suas próprias biografias como determinantes nas investigações que realizaram no domínio da Educação Artística aplicada ao Alzheimer.(AU)

Circulating levels of plasminogen activator inhibitor-1 are associated with metabolic syndrome rather than with menopause.

The plasminogen activator inhibitor type 1 (PAI-1) is the major determinant of fibrinolytic activity. PAI-1 concentrations are elevated in obesity, type 2 diabetes and metabolic syndrome (MetS). On the other hand, during menopause, fibrinolytic activity decreases and, consequently, PAI-1 concentration increases; however, it is debated whether menopause is an independent determinant factor of PAI-1 levels. The objective of this study was to evaluate the effect of hormonal and metabolic status on the concentration of PAI-1 in pre-and post-menopausal women. A case-control study was conducted in ninety pre-and post-menopausal women aged 45 to 55 years, matched by body mass index (BMI). Anthropometric measurements and biochemical determinations were performed on all participants. The fibrinolytic activity was determined by measuring PAI-1 by ELISA. Of all the women, 30% presented MetS. Women with MetS had higher values of PAI-1 (36.0 ± 19.1 vs 19.3 ± 14.8 ng/mL, p < .001); in contrast, no differences were observed when compared by hormonal status (20.7 ± 18.10 vs 20.2 ± 17.0 ng/mL, NS). The results of this study suggest that in women, MetS plays a more important role in the deterioration of the fibrinolytic mechanisms rather than their hormonal status. Therefore, the identification of cardio-metabolic factors is relevant to reduce the presence of thrombosis in post-menopausal women.

Home Delivered Meals to Older Adults: A Critical Review of the Literature.

The United States is witnessing a growing aging population stemming in part from medical advancements allowing people to live decades longer than previous generations. Simultaneously, food insecurity among older adults has increased, and is projected to get worse as the Baby Boomer generation ages. This review focuses on an assistance program for older adults: home-delivered meals. Specifically, we focus on the effects of Meals on Wheels (MOW) on the physical and emotional well-being of older adults, and the wide variety of procedural and operational issues that various MOW programs around the country experience. Findings from the literature highlight the positive outcomes these programs have on their clients. Although there have been recent budget cut threats from the federal government, evidence suggests that more funding should be allocated so these programs can provide services to everyone in need, and even expand what they are able to offer to older adults.

Training small producers in Good Manufacturing Practices for the development of goat milk cheese

Abstract Training in Good Manufacturing Practices enhances quality during food processing. This paper evaluates GMP training aimed at improving the chemical, sensory and microbiological quality of goat milk cheese. We worked with 26 families that produce cheese as their main source of income. Semi-structured interviews and observation were conducted to select relevant topics. The manufacturing processes were compared and samples were analyzed before and after GMP training. We trained 80% of the producers. Before receiving training, they used to make cheese from raw milk in unhygienic conditions and with little equipment. The products obtained had bad sensory characteristics, cracks, eyes on the pasta, a high number of aerobic mesophilic bacteria and total coliforms. After training, the producers pasteurized the milk and standardized processing procedures, resulting in final products that contained higher protein and calcium content, suitable sensory characteristics, and a significant reduction in microorganisms, with total coliforms falling to ≤ 5.103 UFC/g. Therefore, this study shows that the manufacturing process and the chemical, sensory and microbiological parameters of goat milk cheese improved after GMP training.

Similar to Adiponectin, Serum Levels of Osteocalcin are Associated with Mammographic Breast Density in Postmenopausal Women.

OBJECTIVE: Breast cancer is the most common type of cancer in Canadian women and worldwide. Mammographic density is a well-established breast cancer risk. Recent evidence suggested inverse correlations among adiponectin, osteocalcin, and the risk developing breast cancer. The objective of the study was to evaluate the relationship between breast density and adiponectin and osteocalcin concentrations. METHODS: A cross-sectional study was performed in 239 women, age range 40 to 60. Mammographic density, serum adiponectin, and osteocalcin levels were measured. According to the Wolfe method, participants were divided into those with low-risk and high-risk pattern mammograms. RESULTS: The study population included 107 premenopausal and 132 postmenopausal women. Parameters were no different between women with low-risk and high-risk patterns. In obese postmenopausal women, the high-risk pattern mammogram group had significantly higher values of adiponectin and osteocalcin compared with the low-risk pattern group. Multiple linear regression analyses showed that adiponectin and osteocalcin levels were associated with high-risk pattern mammograms. CONCLUSION: Adiponectin and osteocalcin levels were directly associated with high-risk pattern mammograms in obese postmenopausal women. These results do not support the use of adipokines as biomarkers; nevertheless, the most important factor is to assess the risk through breast density.

Association of mSin1 with mTORC2 Ras and Akt reveals a crucial domain on mSin1 involved in Akt phosphorylation.

mSin1 is a unique component within the mammalian target of rapamycin (mTOR) complex 2 (mTORC2), which is responsible for cellular morphology and glucose metabolism. The association between mSin1 and other mTORC2 components, as well as their functions, has been explored previously; nevertheless, the mapping of the various binding domains of the components is lacking. Based on an evolutionary analysis of the gene, we constructed various fragments and truncated-forms of mSin1. We characterized the individual binding sites of mSin1 with its various partners, including mTOR, Rictor, Ras, and Akt. mTOR and Rictor bind to the amino acid (aa) 100-240 region of mSin1, which is different to the Ras binding site, the aa 260-460 region. A reciprocal examination found that mSin1 associated with the aa 2148-2300 region of mTOR, which is within the kinase domain, and with the carboxyl terminus of Rictor. Interestingly, Akt was found to associate with mSin1 in a region that slightly overlapped with the mTOR/Rictor complex binding site, namely aa 220-260. When only the Akt binding site was deleted from mSin1, phosphorylation of Akt S473 was greatly reduced. Furthermore, the association between Akt and mTOR can be regulated by serum, insulin and LY294002, but not by rapamycin or MAPK kinase inhibitors. Taken together, mSin1 would seem to act as a hub that allows mTORC2 to phosphorylate Akt S473. Our findings should facilitate future proteomic and crystallographic studies, help the development of dominant inhibitors and promote the identification of new drug targets.

Bacteriemia por Listeria monocytogenes / Bacteremia for Listeria monocytogenes

Se realiza un estudio en el Laboratorio de Microbiología del Centro Provincial de Higiene de Guantánamo, del 23 al 30 de mayo de 2011, con el objetivo de identificar el agente etiológico de bacteriemia en un paciente con insuficiencia renal. El universo de estudio estuvo formado por una cepa procedente del Laboratorio de Microbiología del Hospital General Docente de Guantánamo, aislada de un hemocultivo, a la que se le realizaron diferentes pruebas para su identificación. La cepa se identifica como Listeria monocytogenes. Se describen las características morfológicas, culturales, bioquímicas y se realizó antibiograma por el método de Kirby Bauer, informando al hospital, donde el paciente tuvo una evolución satisfactoria. Se concluye sobre la importancia de la correcta limpieza de los vegetales previo al consumo y ser oportunos en el estudio microbiológico ante la sospecha de sepsis(AU)

A study is done in the Laboratory of Microbiology of the Provincial Center of Hygiene of Guantanamo, from May 23 to May 30, 2011, with the target to identify the etilogical agent of bacteremia in a patient with renal insufficiency. The studied universe was formed by a stump proceeding from the Laboratory of Microbiology of the Hospital Guantanamo, isolated of a culture, to whom different tests were done for its identification. The stump is identified as Listeria monocytogenes. The morphologic, cultural, biochemical characteristics are described and it was done antibiotic tests by the method of Kirby Bauer, reporting to the hospital, where the patient had a satisfactory evolution. It ends on the importance of the correct cleanliness of the vegetables before the consumption and to be opportune in the microbiological study before the sepsis suspicion

Resistencia antimicrobiana de cepas aisladas en muestras de esputo / Antimicrobial resistance of strains isolated from sputum samples

Se realiza una investigación en el Laboratorio de Microbiología del Centro Provincial de Higiene Epidemiología y Microbiología (CPHEM) en el período de enero-marzo de 2009, con el objetivo de determinar la resistencia a antimicrobianos en 73 cepas aisladas de microorganismos e identificadas a partir de las muestras de esputo de pacientes con infecciones respiratorias bajas. Se determina la sensibilidad y resistencia a los antimicrobianos in vitro por el método de Bauer Kirby. Los resultados se expresan en tabla bidimensional y la medida de resumen utilizada fue el porcentaje. El mayor número de bacterias identificadas corresponde a bacilos gram negativos. La mayor resistencia lo muestrael Pseudomona aeruginosa, seguido de Staphylococcus aureus y Klebsiella pneumoniae. Se concluye que la resistencia a los antimicrobianos es un problema emergente, que exige su selección adecuada e identificación del agente etiológico (AU)

A research is done at the Microbiology lab of the Provincial Center of Hygiene Epidemiology and Microbiology (CPHEM) from January to March 2009 with the aim to determine antimicrobial resistance in 73 strains of microorganisms isolated and identified from sputum samples of patients with lower respiratory infections. Determine the sensitivity and resistance to antimicrobials in vitro by the Kirby Bauer method. The results are expressed in two-dimensional table and the summary measure was used by percentage. The highest number of bacteria was identified gram-negative bacilli. The greatest resistance is shown by the Pseudomonas aeruginosa, Staphylococcus aureus, followed by Klebsiella pneumoniae. As a conclusion antimicrobial resistance is an emerging problem, which requires the proper selection and identification of the etiologic agent

The mechanism of insulin-stimulated 4E-BP protein binding to mammalian target of rapamycin (mTOR) complex 1 and its contribution to mTOR complex 1 signaling.

Insulin activation of mTOR complex 1 is accompanied by enhanced binding of substrates. We examined the mechanism and contribution of this enhancement to insulin activation of mTORC1 signaling in 293E and HeLa cells. In 293E, insulin increased the amount of mTORC1 retrieved by the transiently expressed nonphosphorylatable 4E-BP[5A] to an extent that varied inversely with the amount of PRAS40 bound to mTORC1. RNAi depletion of PRAS40 enhanced 4E-BP[5A] binding to ∼70% the extent of maximal insulin, and PRAS40 RNAi and insulin together did not increase 4E-BP[5A] binding beyond insulin alone, suggesting that removal of PRAS40 from mTORC1 is the predominant mechanism of an insulin-induced increase in substrate access. As regards the role of increased substrate access in mTORC1 signaling, RNAi depletion of PRAS40, although increasing 4E-BP[5A] binding, did not stimulate phosphorylation of endogenous mTORC1 substrates S6K1(Thr(389)) or 4E-BP (Thr(37)/Thr(46)), the latter already ∼70% of maximal in amino acid replete, serum-deprived 293E cells. In HeLa cells, insulin and PRAS40 RNAi also both enhanced the binding of 4E-BP[5A] to raptor but only insulin stimulated S6K1 and 4E-BP phosphorylation. Furthermore, Rheb overexpression in 293E activated mTORC1 signaling completely without causing PRAS40 release. In the presence of Rheb and insulin, PRAS40 release is abolished by Akt inhibition without diminishing mTORC1 signaling. In conclusion, dissociation of PRAS40 from mTORC1 and enhanced mTORC1 substrate binding results from Akt and mTORC1 activation and makes little or no contribution to mTORC1 signaling, which rather is determined by Rheb activation of mTOR catalytic activity, through mechanisms that remain to be fully elucidated.