The Potential Utilization of Machine Perfusion to Increase Transplantation of Macrosteatotic Livers.

BACKGROUND: The demand for liver transplantation has led to the utilization of marginal grafts including moderately macrosteatotic livers (macrosteatosis ≥30% [Mas30]), which are associated with an elevated risk of graft failure. Machine perfusion (MP) has emerged as a technique for organ preservation and viability testing; however, little is known about MP in Mas30 livers. This study evaluates the utilization and outcomes of Mas30 livers in the era of MP. METHODS: The Organ Procurement and Transplantation Network database was queried to identify biopsy-proven Mas30 deceased donor liver grafts between June 1, 2016, and June 23, 2023. Univariable and multivariable models were constructed to study the association between MP and graft utilization and survival. RESULTS: The final cohort with 3317 Mas30 livers was identified, of which 72 underwent MP and were compared with 3245 non-MP livers. Among Mas30 livers, 62 (MP) and 1832 (non-MP) were transplanted (utilization of 86.1% versus 56.4%, P < 0.001). Donor and recipient characteristics were comparable between MP and non-MP groups. In adjusted analyses, MP was associated with significantly increased Mas30 graft utilization (odds ratio, 7.89; 95% confidence interval [CI], 3.76-16.58; P < 0.001). In log-rank tests, MP was not associated with 1- and 3-y graft failure (hazard ratio, 0.49; 95% CI, 0.12-1.99; P = 0.319 and hazard ratio 0.43; 95% CI, 0.11-1.73; P = 0.235, respectively). CONCLUSIONS: The utilization rate of Mas30 grafts increases with MP without detriment to graft survival. This early experience may have implications for increasing the available donor pool of Mas30 livers.

Efecto de extractos de frutas y hortalizas en la incorporación de glucosa en células musculares in vitro / Effect of fruit and vegetable extracts on the incorporation of glucose in muscle cells "in vitro"

Skeletal muscle appears to play a central role in the development of insulin resistance (IR) and consequently the metabolic syndrome due to high-fat diets, obesity, and aging. Recent evidence suggests that some bioactive compounds present in natural products can affect blood glucose, possibly due to interactions between the compounds and glucose transporters. As an objective, to evaluate the effect of the extract of the green bean (PV, Phaseolus vulgaris) and apple of small fruit of thinning (Malus domestica, MAF and MIT extracts) on the incorporation of glucose in C2C12 muscle cells. For this, the cytotoxic effect of the extracts on the cells was determined by detecting formazan. Subsequently, glucose incorporation was determined using a fluorescent glucose analog in cells treated with the extracts. Finally, the effect of the extracts on IL-6 and TNFα production was evaluated by ELISA. Results: PV and MAF decreased 50% of viability at 1000µg / mL while MIT only decreased 10% at that concentration. PV had no significant effect on glucose incorporation and the MAF and MIT extract extracts significantly increased glucose incorporation at 100 µg / mL (13500 and 18000 URF respectively). PV increases the secretion of IL-6 and TNF-α, MAF and MIT only increase the expression of IL-6. Conclusion: These results make it possible to establish natural extracts derived from thinning small fruit apple can be used as a possible treatment for pathologies with high blood glucose levels.

Spontaneous splenic rupture in a patient with chronic myeloid leukemia: A case report.

INTRODUCTION: Spontaneous splenic rupture is an atraumatic event that represents a rare and life-threatening acute complication in which the spleen is damaged producing internal hemorrhage in the abdominal cavity. Its association with hematologic malignancies, although a rare occurrence, has been previously described. Among this subset of patients, chronic myeloid leukemia is one of the main causes. PRESENTATION OF CASE: A 26-year-old male with history of chronic myeloid leukemia presented with acute intense abdominal right lower quadrant pain. Computed tomography showed a wedge in the lower third of the spleen (probably associated with infarction), active bleeding, and hemoperitoneum. Laparotomy and splenectomy were performed. DISCUSSION: The most common symptom of spontaneous splenic rupture is acute abdominal pain, sometimes radiating to the left shoulder. It can also be associated with nausea, emesis and signs of hypovolemia or shock. Splenomegaly may be absent. Diagnostic methods of choice are computed tomography and ultrasound. Management of splenic rupture is divided in surgical and conservative. The former is reserved for patients with extensive splenic injury that is accompanied by hemodynamic instability or other trauma that warrants surgical treatment. Patients who do not meet these criteria and respond to initial stabilization strategies can be offered clinical and laboratory monitoring. Stable patients with moderate to severe splenic injuries can be offered angioembolization. CONCLUSION: It is important to include splenic rupture as a differential diagnosis for acute abdominal pain, especially in patients with hematologic malignancy, since early recognition and treatment increases patient survival and improves prognosis.

Introduction of Application of Gini Coefficient to Heart Rate Variability Spectrum for Mental Stress Evaluation / Introdução da Aplicação do Coeficiente de Gini ao Espectro de Variabilidade da Frequência Cardíaca para Avaliação do Estresse Mental

Abstract Background: The Gini coefficient is a statistical tool generally used by economists to quantify income inequality. However, it can be applied to any kind of data with unequal distribution, including heart rate variability (HRV). Objectives: To assess the application of the Gini coefficient to measure inequality in power spectral density of RR intervals, and to use this application as a psychophysiological indicator of mental stress. Methods: Thirteen healthy subjects (19 ± 1.5 years) participated in this study, and their RR intervals were obtained by electrocardiogram during rest (five minutes) and during mental stress (arithmetic challenge; five minutes). These RR intervals were used to obtain the estimates of power spectral densities (PSD). The limits for the PSD bands were defined from 0.15 to 0.40 Hz for high frequency band (HF), from 0.04 to 0.15 Hz for low frequency band (LF), from 0.04 to 0.085 Hz for first low frequency sub-band (LF1) and from 0.085 to 0.15 Hz for second low frequency sub-band (LF2). The spectral Gini coefficient (SpG) was proposed to measure the inequality in the power distribution of the RR intervals in each of above-mentioned HRV bands. SpG from each band was compared with its respective traditional index of HRV during the conditions of rest and mental stress. All the differences were considered statistically significant for p < 0.05. Results: There was a significant decrease in HF power (p = 0.046), as well as significant increases in heart rate (p = 0.004), LF power (p = 0.033), LF2 power (p = 0.019) and LF/HF (p = 0.002) during mental stress. There was also a significant increase in SpG(LF) (p = 0.009) and SpG(LF2) (p = 0.033) during mental stress. Coefficient of variation showed SpG has more homogeneity compared to the traditional index of HRV during mental stress. Conclusions: This pilot study suggested that spectral inequality of Heart Rate Variability analyzed using the Gini coefficient seems to be an independent and homogeneous psychophysiological indicator of mental stress. Also, HR, LF/HF, SpG(LF) of HRV are possibly important, reliable and valid indicators of mental stress.

Resumo Fundamento: O coeficiente de Gini é um instrumento estatístico geralmente usado por economistas para quantificar a desigualdade de renda. No entanto, ele pode ser aplicado a qualquer tipo de dados com distribuição desigual, incluindo a variabilidade da frequência cardíaca (VFC). Objetivos: Avaliar a aplicação do coeficiente de Gini para medir a desigualdade na densidade espectral de potência de intervalos RR, e usar esta aplicação como um indicador psicofisiológico do estresse mental. Métodos: Treze indivíduos saudáveis (19 ± 1,5 anos) participaram deste estudo, e seus intervalos RR foram obtidos por eletrocardiograma durante repouso (cinco minutos) e durante estresse mental (desafio aritmético; cinco minutos). Esses intervalos RR foram utilizados para obter as estimativas de densidades espectrais de potência (PSD). Os limites para as bandas PSD foram definidos de 0,15 a 0,40 Hz para banda de alta frequência (HF), de 0,04 a 0,15 Hz para banda de baixa frequência (LF), de 0,04 a 0,085 Hz para a primeira sub-banda de baixa frequência (LF1) e de 0,085 a 0,15 Hz para a segunda sub-banda de baixa frequência (LF2). O coeficiente de Gini espectral (SpG) foi proposto para medir a desigualdade na distribuição de potência dos intervalos RR em cada uma das bandas de VFC mencionadas acima. O SpG de cada banda foi comparado com seu respectivo índice tradicional de VFC durante as condições de repouso e de estresse mental. Todas as diferenças foram consideradas estatisticamente significativas para p < 0,05. Resultados: Houve uma diminuição significativa no poder de FC (p=0,046), bem como aumentos significativos na frequência cardíaca (p = 0,004), potência da LF (p = 0,033), potência da LF2 (p = 0,019) e LF/HF (p = 0,002) durante estresse mental. Houve também um aumento significativo de SpG(LF) (p = 0,009) e SpG(LF2) (p = 0,033) durante estresse mental. O coeficiente de variação mostrou que o SpG tem mais homogeneidade em comparação com o índice tradicional de VFC durante o estresse mental. Conclusões: Este estudo piloto sugeriu que a desigualdade espectral da VFC analisada pelo coeficiente de Gini parece ser um indicador psicofisiológico independente e homogêneo de estresse mental. Além disso, FC, LF/HF, SpG(LF) da VFC são possivelmente indicadores importantes, confiáveis e válidos de estresse mental.

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription.

Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications.IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets.

Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly.

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites.IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly.

Morphometric study of five constant skull base foramina in the Muisca population of the Tibanica anthropological collection of the Universidad de Los Andes / Estudio morfométrico de cinco forámenes constantes de la base del cráneo en la población Muisca de la colección antropológica Tibanica de la Universidad de Los Andes

Among osteological anatomic variations are those of the skull base foramina. These openings have conventionally been classified as either constant or variant. Their presence and dimensions have been associated with certain pathologies and procedural complications. Additionally, variability in these foramina between different ethnic groups has been observed, and it is sometimes possible to identify particular patterns of variability in certain populations. This anthropometric cross-sectional study seeks to determine the principal dimensions (bilateral anteroposterior and lateromedial diameters) of five constant skull base foramina in the adult Muisca population of the Tibanica anthropological collection at Universidad de los Andes. The studied foramina were magnum, jugular, ovale, spinosum, and external opening of the carotid canal. Only dimensions of the external openings of the foramina were recorded, owing to the preservation state of the skulls in the collection. The mean left and right anteroposterior and lateromedial diameters were 3.48 mm, 6.16 mm and 3.25 mm, 6.26 mm for the foramen ovale; 2.38 mm, 2.65 mm and 2.39 mm, 2.66 mm for foramen spinosum; 8.36 mm, 15.41 mm and 8.55 mm, 15.10 mm for the jugular foramen; 5.28 mm, 6.75 mm and 5.48 mm, 6.97 mm for the external opening of the carotid canal; and 33.90 mm, 29.47 mm for the foramen magnum. All foramina were measured twice, no important differences were observed between the results obtained in the first and second measurements. The skull base foramina of the sample studied did not suggest high variability within the population regarding these characteristics. Moreover, we can state that the morphometric profile displayed by the Tibanica collection at Universidad de los Andes is different from the one observed in other populations. Additional studies of anatomic variations in indigenous populations may be needed to make possible similarities and/or differences and their causes evident.

Entre las variaciones anatómicas óseas, se consideran aquellas referentes a los forámenes de la base del cráneo. Convencionalmente, estos orificios han sido clasificados como variantes o constantes. Su presencia y dimensiones se asocian a ciertas patologías y complicaciones procedimentales. Adicionalmente, se ha observado variabilidad en estos entre distintos grupos étnicos y en algunas ocasiones es posible identificar patrones particulares en poblaciones específicas. Este estudio morfométrico de corte transversal busca determinar las dimensiones principales (diámetros anteroposterior y lateromedial) de cinco forámenes constantes de la base del cráneo en la población adulta Muisca de la colección antropológica Tibanica de la Universidad de los Andes. Los forámenes estudiados fueron: magno, yugular, ovale, espinoso y la apertura externa del canal carotideo. Debido al estado de preservación de los cráneos, se registraron las dimensiones de las aperturas externas de los forámenes. La media de los diámetros izquierdo y derecho anteroposterior y lateromedial fue 3,48 mm, 6,16 mm y 3,25 mm, 6,26 mm para el foramen ovale; 2,38 mm, 2,65 mm y 2,39 mm, 2,66 mm para el espinoso; 8,36 mm, 15,41 mm y 8,55 mm, 15,10 mm para el yugular; 5,28 mm, 6,75 mm y 5,48 mm, 6,97 mm para la apertura externa del canal carotideo; y 33,90 mm, 29,47 mm para el foramen magno. Todos los diámetros se registraron dos veces, no se observó ninguna diferencia importante entre los registros de la primera y segunda medición. En general, las dimensiones de los forámenes estudiados no varían mucho en la colección ósea Muisca de Tibanica. En cuanto a estas características anatómicas, la población utilizada es diferente con respecto a algunas modernas. Por otro lado, se requieren investigaciones adicionales de este tipo con el fin de evidenciar posibles similitudes y diferencias entre poblaciones (prehispánicas y modernas) y determinar sus causas.

Hantavirus Gn and Gc envelope glycoproteins: key structural units for virus cell entry and virus assembly.

In recent years, ultrastructural studies of viral surface spikes from three different genera within the Bunyaviridae family have revealed a remarkable diversity in their spike organization. Despite this structural heterogeneity, in every case the spikes seem to be composed of heterodimers formed by Gn and Gc envelope glycoproteins. In this review, current knowledge of the Gn and Gc structures and their functions in virus cell entry and exit is summarized. During virus cell entry, the role of Gn and Gc in receptor binding has not yet been determined. Nevertheless, biochemical studies suggest that the subsequent virus-membrane fusion activity is accomplished by Gc. Further, a class II fusion protein conformation has been predicted for Gc of hantaviruses, and novel crystallographic data confirmed such a fold for the Rift Valley fever virus (RVFV) Gc protein. During virus cell exit, the assembly of different viral components seems to be established by interaction of Gn and Gc cytoplasmic tails (CT) with internal viral ribonucleocapsids. Moreover, recent findings show that hantavirus glycoproteins accomplish important roles during virus budding since they self-assemble into virus-like particles. Collectively, these novel insights provide essential information for gaining a more detailed understanding of Gn and Gc functions in the early and late steps of the hantavirus infection cycle.

Caveolin-1 is a risk factor for postsurgery metastasis in preclinical melanoma models.

Melanomas are highly lethal skin tumours that are frequently treated by surgical resection. However, the efficacy of such procedures is often limited by tumour recurrence and metastasis. Caveolin-1 (CAV1) has been attributed roles as a tumour suppressor, although in late-stage tumours, its presence is associated with enhanced metastasis. The expression of this protein in human melanoma development and particularly how the presence of CAV1 affects metastasis after surgery has not been defined. CAV1 expression in human melanocytes and melanomas increases with disease progression and is highest in metastatic melanomas. The effect of increased CAV1 expression can then be evaluated using B16F10 murine melanoma cells injected into syngenic immunocompetent C57BL/6 mice or human A375 melanoma cells injected into immunodeficient B6Rag1-/- mice. Augmented CAV1 expression suppresses tumour formation upon a subcutaneous injection, but enhances lung metastasis of cells injected into the tail vein in both models. A procedure was initially developed using B16F10 melanoma cells in C57BL/6 mice to mimic better the situation in patients undergoing surgery. Subcutaneous tumours of a defined size were removed surgically and local tumour recurrence and lung metastasis were evaluated after another 14 days. In this postsurgery setting, CAV1 presence in B16F10 melanomas favoured metastasis to the lung, although tumour suppression at the initial site was still evident. Similar results were obtained when evaluating A375 cells in B6Rag1-/- mice. These results implicate CAV1 expression in melanomas as a marker of poor prognosis for patients undergoing surgery as CAV1 expression promotes experimental lung metastasis in two different preclinical models.

Hantavirus Gn and Gc glycoproteins self-assemble into virus-like particles.

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.

Postoperative chemotherapy followed by conformal concomitant chemoradiotherapy in high-risk gastric cancer.

PURPOSE: To analyze the efficacy, toxicity, and pattern of relapse after adjuvant cisplatin-based chemotherapy followed by three-dimensional irradiation and concomitant LV5FU2 chemotherapy (high-dose leucovorin and 5-fluorouracil bolus plus continuous infusion) in the treatment of completely resected high-risk gastric cancer. METHODS AND MATERIALS: This was a retrospective analysis of 52 patients with high-risk gastric cancer initially treated by total/partial gastrectomy and lymphadenectomy between January 2002 and June 2007. Median age was 54 years (range, 36-75 years). Postoperative treatment consisted of 5-fluorouracil and cisplatin chemotherapy. Adjuvant chemotherapy was followed by three-dimensional conformal radiotherapy in the tumor bed and regional lymph nodes at 4500 cGy/25 fractions in association with concomitant chemotherapy. Concomitant chemotherapy consisted of a 2-h infusion of leucovorin (200 mg/m²) followed by a bolus of 5-fluorouracil (400 mg/m²) and then a 44-h continuous infusion of 5-fluorouracil (2400-3600 mg/m²) given every 14 days, for three cycles (LV5FU2 protocol). RESULTS: Five-year overall and disease-free survival were 50% and 48%, respectively. Distant metastases and peritoneal spread were the most frequent sites of relapse (37% each). After multivariate analysis, only pathologic nodal status was significantly associated with disease-free and overall survival. Acute toxicities were essentially gastrointestinal and hematologic. One myocardial infarction and one pulmonary embolism were also reported. Eighteen patients had a radiotherapy program interruption because of acute toxicity. All patients but 2 have completed radiotherapy. CONCLUSION: Postoperative cisplatin-based chemotherapy followed by conformal radiotherapy in association with concurrent 5-fluorouracil seemed to be feasible and resulted in successful locoregional control.

ATP release due to Thy-1-integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation.

Thy-1, an abundant mammalian glycoprotein, interacts with αvß3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvß3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

Aromatic and polar residues spanning the candidate fusion peptide of the Andes virus Gc protein are essential for membrane fusion and infection.

Hantaviruses infect human cells through cell attachment and subsequent fusion of viral and cellular membranes at low pH. This largely unknown entry process is mediated by the Gn and Gc glycoproteins, anchored at the viral envelope membrane. Performing bioinformatic analysis and peptide-liposome-binding assays we suggested in a former report that Gc of Andes virus (ANDV) and other hantaviruses corresponds to the viral fusion protein sharing characteristics with class II fusion proteins. To gain insights into the fusion protein of hantaviruses, residues within the previously predicted fusion peptide of ANDV Gc were substituted and mutant proteins tested in fusion and infection assays. To ensure proper folding of mutant proteins, they were first characterized for trafficking to the plasma membrane and incorporation on to ANDV Gn/Gc-pseudotyped lentiviral particles. Cell attachment of these particles was assessed using a newly developed binding assay and their subsequent entry properties determined by FACS analysis of transduced cells expressing the GFP reporter gene. Furthermore, a three-colour-based cell-cell fusion assay of ANDV Gn/Gc expressing cells was performed. The results indicate an essential role of conserved Gc residues W115 and N118 in membrane fusion. Conversely, substitutions of the non-conserved Gc residue G116 did not considerably affect fusion and infection. Altogether, the findings are fully consistent with our earlier prediction suggesting Gc residues 115-121 as an internal fusion peptide and further emphasize the importance of aromatic and polar residues in hantavirus-cell membrane fusion.

Development of a lentiviral vector system to study the role of the Andes virus glycoproteins.

To infect target cells, enveloped viruses use their virion surface proteins to direct cell attachment and subsequent entry via virus-cell membrane fusion. How hantaviruses enter cells has been largely unexplored. To study early steps of Andes virus (ANDV) cell infection, a lentiviral vector system was developed based on a Simian immunodeficiency virus (SIV) vector pseudotyped with the ANDV-Gn/Gc envelope glycoproteins. The incorporation of Gn and Gc onto SIV-derived vector particles was assessed using newly generated monoclonal antibodies against ANDV glycoproteins. In addition, sera of ANDV infected humans were able to block cell entry of the SIV vector pseudotyped with ANDV glycoproteins, suggesting that their antigenic conformation is similar to that in the native virus. The use of such SIV vector pseudotyped with ANDV-Gn/Gc glycoproteins should facilitate studies on ANDV cell entry. Along this line, it was found that depletion of cholesterol from target cells strongly diminished cell infection, indicating a possible role of lipid rafts in ANDV cell entry. The Gn/Gc pseudotyped SIV vector has several advantages, notably high titer vector production and easy quantification of cell infection by monitoring GFP reporter gene expression by flow cytometry. Such pseudotyped SIV vectors can be used to identify functional domains in the Gn/Gc glycoproteins and to screen for potential hantavirus cell entry inhibitors.

Neuronal Thy-1 induces astrocyte adhesion by engaging syndecan-4 in a cooperative interaction with alphavbeta3 integrin that activates PKCalpha and RhoA.

Clustering of alphavbeta3 integrin after interaction with the RGD-like integrin-binding sequence present in neuronal Thy-1 triggers formation of focal adhesions and stress fibers in astrocytes via RhoA activation. A putative heparin-binding domain is present in Thy-1, raising the possibility that this membrane protein stimulates astrocyte adhesion via engagement of an integrin and the proteoglycan syndecan-4. Indeed, heparin, heparitinase treatment and mutation of the Thy-1 heparin-binding site each inhibited Thy-1-induced RhoA activation, as well as formation of focal adhesions and stress fibers in DI TNC(1) astrocytes. These responses required both syndecan-4 binding and signaling, as evidenced by silencing syndecan-4 expression and by overexpressing a syndecan-4 mutant lacking the intracellular domain, respectively. Furthermore, lack of RhoA activation and astrocyte responses in the presence of a PKC inhibitor or a dominant-negative form of PKCalpha implicated PKCalpha and RhoA activation in these events. Therefore, combined interaction of the astrocyte alphavbeta3-integrin-syndecan-4 receptor pair with Thy-1, promotes adhesion to the underlying matrix via PKCalpha- and RhoA-dependent pathways. Importantly, signaling events triggered by such receptor cooperation are shown here to be the consequence of cell-cell rather than cell-matrix interactions. These observations are likely to be of widespread biological relevance because Thy-1-integrin binding is reportedly relevant to melanoma invasion, monocyte transmigration through endothelial cells and host defense mechanisms.

Caveolin-1-mediated suppression of cyclooxygenase-2 via a beta-catenin-Tcf/Lef-dependent transcriptional mechanism reduced prostaglandin E2 production and survivin expression.

Augmented expression of cyclooxygenase-2 (COX-2) and enhanced production of prostaglandin E(2) (PGE(2)) are associated with increased tumor cell survival and malignancy. Caveolin-1 is a scaffold protein that has been proposed to function as a tumor suppressor in human cancer cells, although mechanisms underlying this ability remain controversial. Intriguingly, the possibility that caveolin-1 regulates the expression of COX-2 has not been explored. Here we show that augmented caveolin-1 expression in cells with low basal levels of this protein, such as human colon cancer (HT29, DLD-1), breast cancer (ZR75), and embryonic kidney (HEK293T) cells reduced COX-2 mRNA and protein levels and beta-catenin-Tcf/Lef and COX-2 gene reporter activity, as well as the production of PGE(2) and cell proliferation. Moreover, COX-2 overexpression or PGE(2) supplementation increased levels of the inhibitor of apoptosis protein survivin by a transcriptional mechanism, as determined by PCR analysis, survivin gene reporter assays and Western blotting. Furthermore, addition of PGE(2) to the medium prevented effects attributed to caveolin-1-mediated inhibition of beta-catenin-Tcf/Lef-dependent transcription. Finally, PGE(2) reduced the coimmunoprecipitation of caveolin-1 with beta-catenin and their colocalization at the plasma membrane. Thus, by reducing COX-2 expression, caveolin-1 interrupts a feedback amplification loop involving PGE(2)-induced signaling events linked to beta-catenin/Tcf/Lef-dependent transcription of tumor survival genes including cox-2 itself and survivin.