Recurrence of hepatitis C virus infection after orthotopic liver transplantation: role of genotypes

In this study, we evaluated the correlation between alanine aminotrasferase levels and hepatitis C virus genotypes in liver transplant patients. We studied 18 patients who had undergone orthotopic liver transplantation because of end-stage cirrhosis (n = 9) or hepatocellular carcinoma (n = 9) hepatitis C virus related. Serum HCV-RNA testing was performed monthly on all the 18 series of serum samples from the first week after liver transplant until the end of the follow up, this period ranging from 1 to 39 months. After liver transplantation, serum HCV-RNA was detected in 14 patients (78%). Of the 8 patients infected with subtype 1b. 1 remained asymptomatic, 2 developed acute liver failure and 5 developed chronic hepatitis. In patients infected with types 1a (Choo et al., 1989), 2a (Choo et al., 1989), with a mixed infection 1b/3 (Kuo et al., 1989) or with an undetermined genotype, significant laboratory abnormalities were not observed. Recurrence of hepatitis C virus infection after liver transplantation is common, and recurrent hepatitis occurs in 50% of cases. Genotype 1b appears to be associated with a higher rate of recurrent hepatitis, compared to other genotypes.     

Proteomic analysis of the airway surface liquid: modulation by proinflammatory cytokines

The airway surface is covered by a fluid, the airway surface liquid, interposed between the mucous layer and the epithelium. The airway surface liquid contains proteins, secreted by different cell types, that may have pro-/anti-inflammatory or bactericidal functions or have a role in the mucociliary clearance. We have used a proteomics approach to identify the proteins secreted by an isolated in vitro model of human airway epithelium, at resting and under proinflammatory conditions, as a strategy to define the factors involved in epithelial barrier function. To this aim, we have analyzed the airway surface liquid from human bronchial epithelial cells grown as polarized monolayers in the presence and absence of inflammatory stimuli such as IL-4, IL-1beta, TNF-alpha, and IFN-gamma. Two-dimensional electrophoresis followed by mass spectrometry analysis has allowed the identification of approximately 175 secreted protein spots, among which are immune-related proteins, structural proteins, an actin severer, some protease inhibitors, and a metalloproteinase. Comparisons between treated and untreated conditions have shown that the expression of several proteins was significantly modified by the different cytokines. Our results indicate that the surface epithelium is an active player in the epithelial barrier function and that inflammatory conditions may modulate protein secretion.     

Is HSP70 upregulation crucial for cellular proliferative response in simulated microgravity?

Astronauts are susceptible to a variety of conditions such as motion sickness, muscular atrophy, bone demineralization and cardiovascular deconditioning. These findings suggest that the adaptation to the absence of gravity is due, at least in part, to the effects exerted by microgravity at the cellular level. Indeed, a number of studies have indicated that gravity affects mammalian cell growth and differentiation through the modulation of gene expression. We have characterized the behaviour of endothelial cells and of the human monocytic cell line U937 cultured in the NASA-developed bioreactor to simulate microgravity, the Rotating Wall Vessels (RWV). In simulated microgravity endothelial cells showed a different behavior which was dependent from the species and from the district of origin, while U937 in the RWV proliferated slower than the controls. All the effects we observed were promptly reversible upon return to normal culture conditions. It is noteworthy that all the cells which maintained the capability to proliferate in microgravity upregulated the stress protein HSP70. We therefore propose that only the cells which sense microgravity as a stressful condition and, consequently, overexpress HSP70 maintain their proliferative potential in simulated microgravity.     

Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach

Phagocytosis of human cells is a crucial activity for the virulence of the human parasite Entamoeba histolytica. This protozoan invades and destroys the intestine by killing and phagocytosing epithelial cells, erythrocytes and cells from the immune system. In this study, we used magnetic beads covered with proteins from human serum as a model system to study the early events involved in phagocytosis by E. histolytica. We validated the system showing that the beads uptake triggered the activation of the actin-myosin cytoskeleton and involved a PI3-kinase as previously described for erythrophagocytosis. We purified early phagosomes from wild-type (WT) amoeba and from parasites that overproduced myosin IB (MyoIB+), the unique unconventional myosin of E. histolytica. The MyoIB+ cells exhibit a slower and more synchronized uptake process than the WT strain. Proteomic analysis by liquid chromatography and tandem mass spectroscopy (LC-MS/MS) of the WT and MyoIB+ phagosomes allowed us to identify, for the first time, molecular actors involved in the early step of the uptake process. These include proteins involved in cytoskeleton activity, signalling, endocytosis, lytic activity and cell surface proteins. Interestingly, the proteins that we found specifically recruited on the phagosomes from the MyoIB+ strain were previously described in other eukarytotic cells, as involved in the regulation of cortical F-actin dynamics, such as alpha-actinin and formins. This proteomics approach allows a step further towards the understanding of the molecular mechanisms involved in phagocytosis in E. histolytica that revealed some interesting differences compared with phagocytosis in macrophages or Dictyostelium discoideum, and allowed to identify putative candidates for proteins linked to myosin IB activity during the phagocytic process.     

Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development

Cellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.     

B-raf alternative splicing is dispensable for development but required for learning and memory associated with the hippocampus in the adult mouse

The B-raf proto-oncogene exerts essential functions during development and adulthood. It is required for various processes, such as placental development, postnatal nervous system myelination and adult learning and memory. The mouse B-raf gene encodes several isoforms resulting from alternative splicing of exons 8b and 9b located in the hinge region upstream of the kinase domain. These alternative sequences modulate the biochemical and biological properties of B-Raf proteins. To gain insight into the physiological importance of B-raf alternative splicing, we generated two conditional knockout mice of exons 8b and 9b. Homozygous animals with a constitutive deletion of either exon are healthy and fertile, and survive up to 18 months without any visible abnormalities, demonstrating that alternative splicing is not essential for embryonic development and brain myelination. However, behavioural analyses revealed that expression of exon 9b-containing isoforms is required for B-Raf function in hippocampal-dependent learning and memory. In contrast, mice mutated on exon 8b are not impaired in this function. Interestingly, our results suggest that exon 8b is present only in eutherians and its splicing is differentially regulated among species.     

Dynamic Balancing of Isoprene Carbon Sources Reflects Photosynthetic and Photorespiratory Responses to Temperature Stress

The volatile gas isoprene is emitted in teragrams per annum quantities from the terrestrial biosphere and exerts a large effect on atmospheric chemistry. Isoprene is made primarily from recently fixed photosynthate; however, alternate carbon sources play an important role, particularly when photosynthate is limiting. We examined the relative contribution of these alternate carbon sources under changes in light and temperature, the two environmental conditions that have the strongest influence over isoprene emission. Using a novel real-time analytical approach that allowed us to examine dynamic changes in carbon sources, we observed that relative contributions do not change as a function of light intensity. We found that the classical uncoupling of isoprene emission from net photosynthesis at elevated leaf temperatures is associated with an increased contribution of alternate carbon. We also observed a rapid compensatory response where alternate carbon sources compensated for transient decreases in recently fixed carbon during thermal ramping, thereby maintaining overall increases in isoprene production rates at high temperatures. Photorespiration is known to contribute to the decline in net photosynthesis at high leaf temperatures. A reduction in the temperature at which the contribution of alternate carbon sources increased was observed under photorespiratory conditions, while photosynthetic conditions increased this temperature. Feeding [2-¹³C]glycine (a photorespiratory intermediate) stimulated emissions of [¹³C_1–5]isoprene and ¹³CO₂, supporting the possibility that photorespiration can provide an alternate source of carbon for isoprene synthesis. Our observations have important implications for establishing improved mechanistic predictions of isoprene emissions and primary carbon metabolism, particularly under the predicted increases in future global temperatures.Many plant species emit isoprene (2-methyl-1,3-butadiene [C₅H₈]) into the atmosphere at high rates (Sharkey and Yeh, 2001). With an estimated emission rate of 500 to 750 teragram per year by terrestrial ecosystems (Guenther et al., 2006), isoprene exerts a strong control over the oxidizing capacity of the atmosphere. Due to its high reactivity to oxidants, it fuels an array of atmospheric chemical and physical processes affecting air quality and climate, including the production of ground-level ozone in environments with elevated concentrations of nitrogen oxides (Atkinson and Arey, 2003; Pacifico et al., 2009) and the formation/growth of organic aerosols (Nguyen et al., 2011). At the plant level, isoprene provides protection from stress, through stabilizing membrane processes (Sharkey and Singsaas, 1995; Velikova et al., 2011) and/or reducing the accumulation of damaging reactive oxygen species in plant tissues under stress (Loreto et al., 2001; Vickers et al., 2009b; Velikova et al., 2012). While the mechanism(s) are still under investigation, isoprene may directly or indirectly stabilize hydrophobic interactions in membranes (Singsaas et al., 1997), minimize lipid peroxidation (Loreto and Velikova, 2001), and directly react with reactive oxygen species (Kameel et al., 2014), yielding first-order oxidation products methyl vinyl ketone and methacrolein (Jardine et al., 2012, 2013). The two main environmental drivers for global changes in isoprene fluxes are light and temperature (Guenther et al., 2006). Isoprene production is closely linked to net photosynthesis, and both isoprene emissions and net photosynthesis are controlled by light intensity (Monson and Fall, 1989). There is also a positive correlation between net photosynthesis and isoprene emissions as leaf temperatures increase up to the optimum temperature for net photosynthesis (Monson et al., 1992).Despite the close correlation between photosynthesis and isoprene emissions, plant enclosure observations and leaf-level analyses have both shown that the fraction of net photosynthesis dedicated to isoprene emissions is not constant. During stress events that decrease net photosynthetic rates, isoprene emissions are often less affected or even stimulated; this results in an increase in relative isoprene production from 1% to 2% of net photosynthesis under normal conditions to 15% to 50% under extreme stress (Goldstein et al., 1998; Fuentes et al., 1999; Kesselmeier et al., 2002; Harley et al., 2004). In severe stress conditions such as drought, isoprene emissions can even continue in the complete absence of photosynthesis (Fortunati et al., 2008). An uncoupling of isoprene emissions from net photosynthesis has also been observed in a number of other studies where the optimum temperature for isoprene emissions was found to be substantially higher than that of net photosynthesis; under the high-temperature conditions, isoprene emissions can account for more than 50% of net photosynthesis (Sharkey and Loreto, 1993; Lerdau and Keller, 1997; Harley et al., 2004; Magel et al., 2006).Analyses of carbon sources using ¹³CO₂ leaf labeling have revealed that under standard conditions (i.e. leaf temperature of 30°C and photosynthetically active radiation [PAR] levels of 1,000 µmol m^–2 s^–1), isoprene is produced primarily (70%–90%) using carbon directly derived from the Calvin cycle (Delwiche and Sharkey, 1993; Affek and Yakir, 2002; Karl et al., 2002) via the chloroplastic methylerythritol phosphate (MEP) isoprenoid pathway (Zeidler et al., 1997). The relative contributions of photosynthetic and alternate carbon sources for isoprene are now recognized as being variable under different environmental conditions. Changes in net photosynthesis rates under drought stress (Funk et al., 2004; Brilli et al., 2007), salt stress (Loreto and Delfine, 2000), and changes in ambient O₂ and CO₂ concentrations (Jones and Rasmussen, 1975; Karl et al., 2002; Trowbridge et al., 2012) alter their relative contributions. Under heat stress-induced photosynthetic limitation in Populus deltoides (a temperate species), an increase in the relative contribution of alternate carbon sources was also observed (Funk et al., 2004). However, our current understanding of the responses of isoprene carbon sources to changes in temperature and light levels is poor, and the connection(s) of these responses to changes in leaf primary carbon metabolism (e.g. photosynthesis, photorespiration, and respiration) remains to be determined.Studies over the last decade have shown or suggested that potential alternate carbon sources include refixation of respired CO₂ (Loreto et al., 2004), intermediates from the cytosolic mevalonate (MVA) isoprenoid pathway (Flügge and Gao, 2005; Lichtenthaler, 2010), and intermediates from central carbon metabolism, including pyruvate (Jardine et al., 2010), phosphoenolpyruvate (Rosenstiel et al., 2003), and Glc (Schnitzler et al., 2004). Over 40 years ago, it was also proposed that photorespiratory carbon could directly contribute to isoprene production in plants (Jones and Rasmussen, 1975); however, subsequent studies (Monson and Fall, 1989; Hewitt et al., 1990; Karl et al., 2002) have concluded that photorespiration does not contribute to isoprenoid production.In this study, we examined the carbon composition of isoprene emitted from tropical tree species under changes in light and temperature, the two key environmental variables that affect isoprene emissions. Using a novel real-time analytical approach, we were able to observe compensatory changes in carbon source contribution to isoprene during thermal ramping at high temperatures, despite the overall isoprene emissions remaining relatively stable. By conducting leaf temperature curves under variable ¹³CO₂ concentrations and applying [2-¹³C]Gly leaf labeling, we also reopen the discussion on the role of photorespiration as an alternate source of carbon for isoprenoid formation.     

The HIV Matrix Protein p17 Promotes the Activation of Human Hepatic Stellate Cells through Interactions with CXCR2 and Syndecan-2

Background

The human immunodeficiency virus type 1 (HIV-1) p17 is a matrix protein involved in virus life''s cycle. CXCR2 and Syndecan-2, the two major coreceptors for the p17 protein, are expressed in hepatic stellate cells (HSCs), a key cell type involved in matrix deposition in liver fibrotic disorders.

Aim

In this report we have investigated the in vitro impact of p17 on HSCs transdifferentiation and function and underlying signaling pathways involved in these processes.

Methods

LX-2 cells, a human HSC line, and primary HSC were challenged with p17 and expressions of fibrogenic markers and of p17 receptors were assessed by qRT-PCR and Western blot. Downstream intracellular signaling pathways were evaluated with qRT-PCR and Western blot as well as after pre-treatment with specific pathway inhibitors.

Results

Exposure of LX2 cells to p17 increases their contractile force, reshapes the cytoskeleton fibers and upregulates the expression of transdifferentiation markers including αSMA, COL1α1 and endothelin-1 through the activation of Jak/STAT and Rho signaling pathways. These effects are lost in HSCs pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide. Confocal laser microscopy studies demonstrates that CXCR2 and syndecan-2 co-associate at the plasma membrane after exposure to p17. Immunostaining of HIV/HCV liver biopsies from co-infected patients reveals that the progression of liver fibrosis correlates with a reduced expression of CXCR2.

Conclusions

The HIV matrix protein p17 is pro-fibrogenic through its interactions both with CXCR2 and syndecan-2 on activated HSCs.