Phosphatidylinositol 3-Kinase/AKT pathway regulates the endoplasmic reticulum to Golgi traffic of ceramide in glioma cells : a link between lipid signaling pathways involved in the control of cell survival

Different lines of evidence indicate that both aberrant activation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt survival pathway and down-regulation of the death mediator ceramide play a critical role in the aggressive behavior, apoptosis resistance, and adverse clinical outcome of glioblastoma multiforme. Furthermore, the inhibition of the PI3K/Akt pathway and the up-regulation of ceramide have been found functional to the activity of many cytotoxic treatments against glioma cell lines and glioblastomas as well. A reciprocal control between PI3K/Akt and ceramide signaling in glioma cell survival/death is suggested by data demonstrating a protective role of PI3K/Akt on ceramide-induced cell death in glial cells. In this study we investigated the role of the PI3K/Akt pathway in the regulation of the ceramide metabolism in C6 glioma cells, a cell line in which the PI3K/Akt pathway is constitutively activated. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids and microscopy studies with fluorescent ceramides demonstrated that the chemical inhibition of PI3K and the transfection with a dominant negative Akt strongly inhibited ceramide utilization for the biosynthesis of complex sphingolipids by controlling the endoplasmic reticulum (ER) to Golgi vesicular transport of ceramide. These findings constitute the first evidence for a PI3K/Akt-dependent regulation of vesicle-mediated movements of ceramide in the ER-Golgi district. Moreover, the findings also suggest the activation of the PI3K/Akt pathway as crucial to coordinate the biosynthesis of membrane complex sphingolipids with cell proliferation and growth and/or to maintain low ceramide levels, especially as concerns those treatments that promote ceramide biosynthesis in the ER

Tuberculose Pulmonar associada ao Tabagismo

Objetivos: Analisar a associação entre tabagismo e variáveis sócio demográficas, epidemiológicas e clínicas de pacientes com TB pulmonar encaminhados ao Centro de Pesquisa Clínica do Hospital Cassiano Moraes. Metodologia: Estudo descritivo analítico de corte transversal. Foi realizado no Hospital Universitário Cassiano Antônio de Moraes. A pesquisa foi realizada com analise de fichas de pacientes participantes de uma pesquisa multicêntrica, somente com pacientes de Vitória-ES, sendo o único polo no Brasil. Atendidos no Centro de Pesquisas Clínicas nos anos de 2003 a 2006. Resultados: Foram analisados fichas de 537 pacientes. Destes 225 (41,9%) eram não fumantes, 183 (34,1%) fumantes e 129 (24%) ex-fumantes. O sexo masculino foi o mais prevalente entre todos os grupos, sendo que entre os não fumantes eram 58,7% (132), entre os fumantes 73,2% (134) e entre ex-fumantes 66,7% (86). A maioria dos pacientes atendidos não possuiam nenhuma escolaridade. A presença de tosse, entre os não fumantes foi de 175 (78,1%) pessoas, já em fumantes foi 165 (90,2%) e no grupo de ex-fumantes 109 (84,5). A presença de escarro purulento, entre os não fumantes foi de 122 (54,2%) fumantes 142 (77,6%) e ex-fumantes 82 (63,6%). Quando analisado a ingestão de bebida alcoólica entre não fumantes eram 24 (10,7%), em fumantes 62 (33,9%) e em ex-fumantes 18 (14%) pacientes. A mediana em anos de tabagismo foi de 20 (IIQ: 20) entre fumantes, ex-fumantes 12 (IIQ:15). Em relação ao número de cigarros fumados por dia entre fumantes a mediana foi de 20 cigarros/dia (IIQ: 10), nos ex-fumantes de 10 cigarros/dia (IIQ: 16). Quanto a lesões cavitárias, não fumantes eram 110 (49,1%), fumantes 94 (51,9%) e ex-fumantes 79 (61,2%). Conclusão: Este estudo confirmou que há diferenças entre pacientes que possuem histórico de tabagismo e aqueles que nunca fumaram, principalmente no tempo de aparecimento dos sinais e sintomas clínicos comuns da TB, podendo se tornar um fator confundidor na detecção de casos da TB retardando o diagnóstico

Detection of Synaptic Proteins in Microglia by Flow Cytometry.

A growing body of evidence indicates that microglia actively remove synapses in vivo, thereby playing a key role in synaptic refinement and modulation of brain connectivity. This phenomenon was mainly investigated in immunofluorescence staining and confocal microscopy. However, a quantification of synaptic material in microglia using these techniques is extremely time-consuming and labor-intensive. To address this issue, we aimed to quantify synaptic proteins in microglia using flow cytometry. With this approach, we first showed that microglia from the healthy adult mouse brain contain a detectable level of VGLUT1 protein. Next, we found more than two-fold increased VGLUT1 immunoreactivity in microglia from the developing brain (P15) as compared to adult microglia. These data indicate that microglia-mediated synaptic pruning mostly occurs during the brain developmental period. We then quantified the VGLUT1 staining in microglia in two transgenic models characterized by pathological microglia-mediated synaptic pruning. In the 5xFAD mouse model of Alzheimer's disease (AD) microglia exhibited a significant increase in VGLUT1 immunoreactivity before the onset of amyloid pathology. Moreover, conditional deletion of TDP-43 in microglia, which causes a hyper-phagocytic phenotype associated with synaptic loss, also resulted in increased VGLUT1 immunoreactivity within microglia. This work provides a quantitative assessment of synaptic proteins in microglia, under homeostasis, and in mouse models of disease

S-Thiolation Targets Albumin in Heart Failure

Human serum albumin (HSA) is associated with several physiological functions, such as maintaining oncotic pressure and microvascular integrity, among others. It also represents the major and predominant antioxidant in plasma due to the presence of the Cys34 sulfhydryl group. In this study, we assessed qualitative and quantitative changes in HSA in patients with heart failure (HF) and their relationship with the severity of the disease. We detected by means of mass spectrometry a global decrease of the HSA content in the plasma of HF patients in respect to control subjects, a significant increase of thio-HSA with a concomitant decrease in the reduced form of albumin. Cysteine and, at a lesser extent, homocysteine represent the most abundant thiol bound to HSA. A strong inverse correlation was also observed between cysteine-HSA and peak VO2/kg, an index of oxygen consumption associated with HF severity. Moreover, in HL-1 cardiomyocytes incubated with H2O2, we showed a significant decrease of cell viability in cells treated with thio-HSA in respect to restored native-HSA. In conclusion, we found for the first time that S-thiolation of albumin is increased in the plasma of HF patients and induced changes in the structure and antioxidant function of HSA, likely contributing to HF progression

In vivo acquisition and risk of inter-species spread of blaKPC-3-plasmid from Klebsiella pneumoniae to Serratia marcescens in the lower respiratory tract

In recent years, Serratia marcescens has emerged as an important agent of hospital-acquired infections, such as pneumonia, urinary tract infection, septicaemia and meningitis, particularly in vulnerable patients. Compared to Klebsiella pneumoniae and Escherichia coli, S. marcescens is less commonly associated with blaKPC genes, yet few cases of plasmid transmission at the gastrointestinal level from K. pneumoniae carbapenemase (KPC)-producing Enterobacterales to S. marcescens have been described. Here we report a case of in vivo acquisition, during a 3-month period of hospitalization in the intensive care unit, of a blaKPC-3 gene carried by a pKpQIL-IT plasmid, and its probable transmission at the bronchial level among different species of Enterobacterales, including K. pneumoniae and S. marcescens. By using whole genome sequence analyses we were able provide insight into the dynamics of carbapenem-resistance determinants acquisition in the lower respiratory tract, a novel anatomical region for such plasmid transmission events, that usually involve the gastrointestinal tract. The co-presence at the same time of both wild-type and resistant Enterobacterales could have been the critical factor leading to the spread of plasmids harbouring carbapenem-resistance genes, of particular importance during surveillance screenings. The possibility of such an event may have significant consequences in terms of antimicrobial treatment, with a potential limitation of therapeutic options, thereby further complicating the clinical management of high-risk critically ill patients