Biogenesis and Traffic of the Potassium Channel

Excitability of nerve and muscle cells depends on the number and the types of ion channels expressed at the plasma membrane. This work examines aspects of biogenesis and traffic of the Shaker voltage-gated potassium channel. Shaker is an oligomeric, polytopic membrane protein and, as such, its biogenesis begins at the endoplasmic reticulum (ER). I have studied (i) targeting of Shaker to the ER and stable integration into the lipid bilayer, (ii) N-linked glycosylation, assembly and folding Shaker in the ER, and (iii) export of Shaker from the ER and subsequent traffic to the surface. Targeting to the endoplasmic reticulum, integration into the lipid bilayer and assembly into tetramers occurs efficiently for Shaker translated in vitro. The first transmembrane domain (TM1) is most likely the earliest ER targeting signal on the growing Shaker polypeptide. TM1 that has adequately emerged from the ribosome is sufficient to initiate targeting to the ER in the absence of additional transmembrane domains. Further, efficient integration of Shaker into the bilayer is promoted by a glycoprotein fraction of ER microsomes, in which the active component was the translocon associated membrane protein (TRAM). Shaker is N-glycosylated on two consensus sites in the first extracellular loop. The importance of glycosylation at this location for Shaker biogenesis has not been previously studied. Elimination of the two consensus sites for N-linked glycosylation yields a channel that targets to the ER, integrates and tetramerizes normally, but is transported at a reduced rate to the surface the cell. This is due at least in part to a retardation of the unglycosylated channel early (i.e. pre-Golgi) step in its secretory traffic. Lastly, we attempted to develop assays to determine the efficiency at which the Shaker channel acquires its final, folded, conduction-competent state in the endoplasmic reticulum

The effect of n-acetylcysteine on hepatic histomorphology during hypothermic preservation

PURPOSE:To evaluate the NAC effects on liver hypothermic preservation at different time intervals.METHODS:For this, we used livers of male Wistar rats weighing between 250 and 300g, undergoing preservation in Ringer solution at 4°C for up to 24 hours. Tissue samples were obtained at four moments of preservation for histological examination by hematoxylin and eosin staining: T0 = beginning of preservation, T12 = 12 hours, T18 = 18 hours and T24 = 24 hours. Will be analyzed vacuolation, hepatic apoptosis by optical microscopy and parenchymal.RESULTS: The results showed a progressive increase in hepatic injury in both groups and showed that NAC was effective at T0. The parenchyma preservation was better in the NAC group and no difference when vacuolization of the cells.CONCLUSION: Hypothermic preservation, over time, causes changes in the hepatic parenchyma with increased apoptosis, loss of architecture, vacuolization, culminating in severe injury. The administration of N-acetylcysteine protects against preservation liver injury.University City of São PauloSão Paulo University Medical SchoolFederal University of São Paulo Paulista Medicine SchoolUNIFESP, Paulista Medicine SchoolSciEL

Dentro, fora, antes ou depois? – A política espaço-temporal de Notícias da América, de Paulo Nazareth

This article investigates the performance Notícias da América, by the artist Paulo Nazareth. It identifies discussions (and actions) that handle a plurality of pasts (ancestral, colonial) and, at the same time, open relational perspectives a-typical feature of contemporary art since the 1960s. In that performance, as Nazareth moves through Latin American cities, the images and discourses produced forge a common space between himself, his interlocutors and the landscape. Simultaneously, a multiple temporality emerges in which peripheral subjectivities become visible. It is also from this space-time relationship that other of his artistic productions (such as the series Aqui é arte) can be unfolded.Este artigo investiga a performance Notícias da América, do artista Paulo Nazareth. Nela são identificadas discussões (e ações) que manejam uma pluralidade de passados (ancestrais, coloniais) e, ao mesmo tempo, abrem perspectivas relacionais, característica típica da arte contemporânea desde os anos 1960. Na performance, à medida que Nazareth se desloca por cidades latino-americanas, as imagens e os discursos produzidos forjam um espaço comum entre ele próprio, seus interlocutores e a paisagem. Simultaneamente, emerge uma temporalidade múltipla em que subjetividades periféricas se tornam visíveis. É também a partir dessa relação espaço-temporal que outras de suas produções artísticas (como a série Aqui é arte), podem ser desdobradas

Cancer-associated fibroblast classification in single-cell and spatial proteomics data

Cancer-associated fibroblasts (CAFs) are a diverse cell population within the tumour microenvironment, where they have critical effects on tumour evolution and patient prognosis. To define CAF phenotypes, we analyse a single-cell RNA sequencing (scRNA-seq) dataset of over 16,000 stromal cells from tumours of 14 breast cancer patients, based on which we define and functionally annotate nine CAF phenotypes and one class of pericytes. We validate this classification system in four additional cancer types and use highly multiplexed imaging mass cytometry on matched breast cancer samples to confirm our defined CAF phenotypes at the protein level and to analyse their spatial distribution within tumours. This general CAF classification scheme will allow comparison of CAF phenotypes across studies, facilitate analysis of their functional roles, and potentially guide development of new treatment strategies in the future

Cancer-associated fibroblast phenotypes are associated with patient outcome in non-small cell lung cancer

Despite advances in treatment, lung cancer survival rates remain low. A better understanding of the cellular heterogeneity and interplay of cancer-associated fibroblasts (CAFs) within the tumor microenvironment will support the development of personalized therapies. We report a spatially resolved single-cell imaging mass cytometry (IMC) analysis of CAFs in a non-small cell lung cancer cohort of 1,070 patients. We identify four prognostic patient groups based on 11 CAF phenotypes with distinct spatial distributions and show that CAFs are independent prognostic factors for patient survival. The presence of tumor-like CAFs is strongly correlated with poor prognosis. In contrast, inflammatory CAFs and interferon-response CAFs are associated with inflamed tumor microenvironments and higher patient survival. High density of matrix CAFs is correlated with low immune infiltration and is negatively correlated with patient survival. In summary, our data identify phenotypic and spatial features of CAFs that are associated with patient outcome in NSCLC

Multiplex imaging of breast cancer lymph node metastases identifies prognostic single-cell populations independent of clinical classifiers

Although breast cancer mortality is largely caused by metastasis, clinical decisions are based on analysis of the primary tumor and on lymph node involvement but not on the phenotype of disseminated cells. Here, we use multiplex imaging mass cytometry to compare single-cell phenotypes of primary breast tumors and matched lymph node metastases in 205 patients. We observe extensive phenotypic variability between primary and metastatic sites and that disseminated cell phenotypes frequently deviate from the clinical disease subtype. We identify single-cell phenotypes and spatial organizations of disseminated tumor cells that are associated with patient survival and a weaker survival association for high-risk phenotypes in the primary tumor. We show that p53 and GATA3 in lymph node metastases provide prognostic information beyond clinical classifiers and can be measured with standard methods. Molecular characterization of disseminated tumor cells is an untapped source of clinically applicable prognostic information for breast cancer

A comprehensive single-cell map of T cell exhaustion-associated immune environments in human breast cancer

Immune checkpoint therapy in breast cancer remains restricted to triple negative patients, and long-term clinical benefit is rare. The primary aim of immune checkpoint blockade is to prevent or reverse exhausted T cell states, but T cell exhaustion in breast tumors is not well understood. Here, we use single-cell transcriptomics combined with imaging mass cytometry to systematically study immune environments of human breast tumors that either do or do not contain exhausted T cells, with a focus on luminal subtypes. We find that the presence of a PD-1high exhaustion-like T cell phenotype is associated with an inflammatory immune environment with a characteristic cytotoxic profile, increased myeloid cell activation, evidence for elevated immunomodulatory, chemotactic, and cytokine signaling, and accumulation of natural killer T cells. Tumors harboring exhausted-like T cells show increased expression of MHC-I on tumor cells and of CXCL13 on T cells, as well as altered spatial organization with more immature rather than mature tertiary lymphoid structures. Our data reveal fundamental differences between immune environments with and without exhausted T cells within luminal breast cancer, and show that expression of PD-1 and CXCL13 on T cells, and MHC-I - but not PD-L1 - on tumor cells are strong distinguishing features between these environments

The embryo splitting as an alternative to improve gestation indices from embryo transfer in horses

A transferência de embriões (TE) já vem sendo utilizada em eqüinos há pelo menos duas décadas, sempre a partir de ovulação simples. Para aumentar a eficiência reprodutiva, este estudo avaliou a bipartição embrionária como uma alternativa para melhorar os índices de gestação na transferência de embriões em eqüinos. Foram utilizadas 21 éguas de diferentes padrões raciais, com idade variando entre 4 e 15 anos de idade, pesando entre 270 a 480kg. A partir da identificação do cio (rufiação), os animais foram monitorados através de exames ultra-sonográficos trans-retais até o momento da ovulação, sendo as receptoras, uma vez ao dia e as doadoras três vezes ao dia. As receptoras utilizadas ovularam um dia antes ou até três dias depois das doadoras. As doadoras foram coletadas entre 144 e 156 horas após a ovulação (D0). Foram recuperados 20 embriões (mórulas) em 29 coletas (68,96%), sendo que 10 embriões foram transferidos inteiros (T1), e 10 embriões foram bipartidos (T2), originando 20 hemi-embriões e transferidos para 20 receptoras. Não houve diferença na taxa de prenhez entre os grupos, T1, 70% (7/10), e T2, 50% (10/20) (P>0,05). Em relação ao número inicial de embriões em cada grupo (10), houve diferença na taxa de prenhez entre os grupos, T1, 70% (7/10) e T2, 100% (10/10) (P0.05). Looking at the original number of embryos, there was a significant difference in pregnancy rate (70% vs 100% for T1 and T2 respectively P<0.05). These results show the possibility of increasing the number of gestations using embryo splitting and transfer in horses per embryo collected. This technique may improve pregnancy indices for ET in horses

Three-dimensional imaging mass cytometry for highly multiplexed molecular and cellular mapping of tissues and the tumor microenvironment

A holistic understanding of tissue and organ structure and function requires the detection of molecular constituents in their original three-dimensional (3D) context. Imaging mass cytometry (IMC) enables simultaneous detection of up to 40 antigens and transcripts using metal-tagged antibodies but has so far been restricted to two-dimensional imaging. Here we report the development of 3D IMC for multiplexed 3D tissue analysis at single-cell resolution and demonstrate the utility of the technology by analysis of human breast cancer samples. The resulting 3D models reveal cellular and microenvironmental heterogeneity and cell-level tissue organization not detectable in two dimensions. 3D IMC will prove powerful in the study of phenomena occurring in 3D space such as tumor cell invasion and is expected to provide invaluable insights into cellular microenvironments and tissue architecture

Hypertonic saline and pentoxifylline enhance survival, reducing apoptosis and oxidative stress in a rat model of strangulated closed loop small bowel obstruction

OBJECTIVES: Intestinal obstruction has a high mortality rate when therapeutic treatment is delayed. Resuscitation in intestinal obstruction requires a large volume of fluid, and fluid combinations have been studied. Therefore, we evaluated the effects of hypertonic saline solution (HS) with pentoxifylline (PTX) on apoptosis, oxidative stress and survival rate. METHODS: Wistar rats were subjected to intestinal obstruction and ischemia through a closed loop ligation of the terminal ileum and its vessels. After 24 hours, the necrotic bowel segment was resected, and the animals were randomized into four groups according to the following resuscitation strategies: Ringer’s lactate solution (RL) (RL-32 ml/kg); RL+PTX (25 mg/kg); HS+PTX (HS, 7.5%, 4 ml/kg), and no resuscitation (IO-intestinal obstruction and ischemia). Euthanasia was performed 3 hours after resuscitation to obtain kidney and intestine samples. A malondialdehyde (MDA) assay was performed to evaluate oxidative stress, and histochemical analyses (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling [TUNEL], Bcl-2 and Bax) were conducted to evaluate kidney apoptosis. Survival was analyzed with another series of animals that were observed for 15 days. RESULTS: PTX in combination with RL or HS reduced the MDA levels (nmol/mg of protein), as follows: kidney IO=0.42; RL=0.49; RL+PTX=0.31; HS+PTX=0.34 (po0.05); intestine: IO=0.42; RL=0.48; RL+PTX=0.29; HS +PTX=0.26 (po0.05). The number of labeled cells for TUNEL and Bax was lower in the HS+PTX group than in the other groups (po0.05). The Bax/Bcl-2 ratio was lower in the HS+PTX group than in the other groups (po0.05). The survival rate on the 15th day was higher in the HS+PTX group (77%) than in the RL+PTX group (11%). CONCLUSION: PTX in combination with HS enhanced survival and attenuated oxidative stress and apoptosis. However, when combined with RL, PTX did not reduce apoptosis or mortalit