Molecular profiling of hepatocellular carcinoma in mice with a chronic deficiency of hepatic s-adenosylmethionine: relevance in human liver diseases

S-adenosylmethionine arises as a central molecule in the preservation of liver homeostasis as a chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. In the present work, we have attempted a comprehensive analysis of proteins associated with hepatocarcinogenesis in MAT1A knock out mice using a combination of two-dimensional electrophoresis and mass spectrometry, to then apply the resulting information to identify hallmarks of human HCC. Our results suggest the existence of individual-specific factors that might condition the development of preneoplastic lesions. Proteomic analysis allowed the identification of 151 differential proteins in MAT1A-/- mice tumors. Among all differential proteins, 27 changed in at least 50% of the analyzed tumors, and some of these alterations were already detected months before the development of HCC in the KO liver. The expression level of genes coding for 13 of these proteins was markedly decreased in human HCC. Interestingly, seven of these genes were also found to be down-regulated in a pretumoral condition such as cirrhosis, while depletion of only one marker was assessed in less severe liver disorders

Non-motor symptom burden in patients with Parkinson's disease with impulse control disorders and compulsive behaviours : results from the COPPADIS cohort

The study was aimed at analysing the frequency of impulse control disorders (ICDs) and compulsive behaviours (CBs) in patients with Parkinson's disease (PD) and in control subjects (CS) as well as the relationship between ICDs/CBs and motor, nonmotor features and dopaminergic treatment in PD patients. Data came from COPPADIS-2015, an observational, descriptive, nationwide (Spain) study. We used the validated Questionnaire for Impulsive-Compulsive Disorders in Parkinson's Disease-Rating Scale (QUIP-RS) for ICD/CB screening. The association between demographic data and ICDs/CBs was analyzed in both groups. In PD, this relationship was evaluated using clinical features and treatment-related data. As result, 613 PD patients (mean age 62.47 ± 9.09 years, 59.87% men) and 179 CS (mean age 60.84 ± 8.33 years, 47.48% men) were included. ICDs and CBs were more frequent in PD (ICDs 12.7% vs. 1.6%, p < 0.001; CBs 7.18% vs. 1.67%, p = 0.01). PD patients had more frequent previous ICDs history, premorbid impulsive personality and antidepressant treatment (p < 0.05) compared with CS. In PD, patients with ICDs/CBs presented younger age at disease onset, more frequent history of previous ICDs and premorbid personality (p < 0.05), as well as higher comorbidity with nonmotor symptoms, including depression and poor quality of life. Treatment with dopamine agonists increased the risk of ICDs/CBs, being dose dependent (p < 0.05). As conclusions, ICDs and CBs were more frequent in patients with PD than in CS. More nonmotor symptoms were present in patients with PD who had ICDs/CBs compared with those without. Dopamine agonists have a prominent effect on ICDs/CBs, which could be influenced by dose

Hypermethioninaemia due to methionine adenosyltransferase I/III (MAT I/III) deficiency: diagnosis in an expanded neonatal screening programme

The Expanded Newborn Screening Program (MS/MS) in the region of Galicia (NW Spain) was initiated in 2000 and includes the measurement of methionine levels in dried blood spots. Between June 2000 and June 2007, 140 818 newborns were analysed, and six cases of persistent hypermethioninaemia were detected: one homocystinuria due to cystathionine β-synthase (CβS) deficiency, and five methionine adenosyltransferase I/III (MAT I/III) deficiencies. The five cases of MAT I/III deficiency represent an incidence of 1/28 163 newborns. In these five patients, methionine levels in dried blood spots ranged from 50 to 147 μmol/L. At confirmation of the persistence of the hypermethioninaemia in a subsequent plasma sample, plasma methionine concentrations were moderately elevated in 4 of the 5 patients (mean 256 μmol/L), while total homocysteine (tHcy) was normal; the remaining patient showed plasma methionine of 573 μmol/L and tHcy of 22.8 μmol/L. All five patients were heterozygous for the same dominant mutation, R264H in the MAT1A gene. With a diet not exceeding recommended protein requirements for their age, all patients maintained methionine levels below 300 μmol/L. Currently, with a mean of 2.5 years since diagnosis, the patients are asymptomatic and show developmental quotients within the normal range. Our results show a rather high frequency of hypermethioninaemia due to MAT I/III deficiency in the Galician neonatal population, indicating a need for further studies to evaluate the impact of persistent isolated hypermethioninaemia in neonatal screening programmes

Proteome of the early embryo-maternal dialogue in the cattle uterus

We analyzed embryo-maternal interactions in the bovine uterus on day 8 of development. Proteomic profiles were obtained by two-dimensional difference gel electrophoresis from 8 paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus. Results were contrasted with UF obtained after artificial insemination. We detected 50 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify 38 proteins, obtaining for first time the earliest evidence of involvement of the down-regulated NFkB system in cattle as a pregnancy signature pathway. Embryos enhanced the embryotrophic ability of UF and decreased uterine protein, while blood progesterone was unaltered. Twinfilin, hepatoma-derived growth factor, and synaptotagmin-binding cytoplasmic RNA interacting protein have not previously been identified in the mammalian uterus. TNFα and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNFα, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNFα were up-regulated. The embryonic signaling agents involved could be TNFα and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development

Proteomic analysis of chemonaive pediatric osteosarcomas and corresponding normal bone reveals multiple altered molecular targets

With a view to identify the proteins involved in transformation, metastasis or chemoresistance in pediatric osteosarcoma, we carried out a new experimental approach based on comparison of the proteomic profile of paired samples of osteosarcoma and normal bone tissues from the same patient. The proteomic profiles of five pairs of cell lines (normal vs tumoral) were obtained by two-dimensional difference gel electrophoresis. We detected 56 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify some of these proteins, 16 of which were chosen on the basis of the change of their relative abundance between osteosarcomas and paired normal bones and also because their involvement was supported by the genomic analysis. Two of the 16 proteins, Alpha-crystallin B chain (CRYAB) and ezrin (EZR1), were selected for further studies: an immunohistochemical analysis of a TMA (tissue microarray) and real-time PCR for a set of 14 osteosarcoma/normal-bone pairs. The results of this second tier of studies confirmed that there were significant increases in the amounts of CRYAB and ezrin, especially in advanced stages of the disease. Our overall conclusion is that proteomic profiling of paired samples of osteosarcoma and normal bone tissues from the same patient is a practicable and potentially powerful way of initiating and proceeding with a search for proteins and genes involved in pediatric osteosarcoma

79 a dimorphic response to early male and female embryos in the bovine uterus

Sexual dimorphism has been reported in early mammalian embryos. However, it is unknown whether in utero signalling at early stages differs between male and female embryos. In this work, we used bovine embryos produced with sex-sorted spermatozoa to analyse embryo-maternal interactions measured as changes in uterine fluid (UF). Male (M) or female (F), Day-5 in vitro-produced embryos (E) (n=23-51) were non-surgically transferred into the uteri of well-nourished heifers (body condition score=3 in a scale 0-5). All recipients (n=8) received male and female embryos within non-consecutive oestrous cycles (4 recipients with male embryos first and 4 with female embryos first). On Day 8, embryos and their corresponding diluted MUF and FUF were recovered. Proteins were extracted from a first-flushed fraction of 45mL PBS containing protease inhibitor, while flushing continued for embryo recovery. Data were analysed by ANOVA and Duncan's test. Total embryo recovery rates (RR) tended to differ (P=0.06) between ME and FE (18.6±2.5 vs 27.7±3.3). However, blastocysts RR (11.6±1.7 vs 14.2±2.3; P=0.56) and flushed volume RR (57.8±2.9 vs 60.9±2.3) did not vary between ME and FE. Recoverable protein was lower in FUF than MUF (9.0±1.2 vs 13.2±1.5μg/100μL [P<0.05] and 2580±102 vs 3450±131μg total [P<0.001], respectively). Proteomic profiles were obtained in concentrated UFs by 2-D fluorescence difference gel electrophoresis and protein characterisation by nano-LC-ESI-MS/MS. After dialyzation against SOFaaci, factors ≥3kDa contained in MUF and FUF were used in culture (1mgmL(-1)) with Day-5 male and female embryos in a 2×2 factorial design. Blastocyst development, cell counts and caspase-3 positive embryonic cells were analysed in 5 replicates. FUF and MUF differed in 41 protein spots (t-test; P<0.05), out of which 35 proteins were identified. Up-regulated proteins (n=34; in FUF) represented an increased carbohydrate metabolism activity combined with anti-stress responses, involving the NFkB system, insulin and oestradiol. PARK7, a protein not previously identified in the bovine uterus is also diferentially expressed in FUF and MUF. MUF+ME tended to show (P<0.06) higher expansion rates in vitro than MUF+FE, FUF+FE and FUF+ME (51.4±5.2 vs 30.0±5.2, 24.5±5.7 and 35.7±5.7, respectively). Trophoblast cell counts tended to be higher (P<0.10) in MUF+ME (98.7±9.5) than in FUF+FE (85.7±10.6) and MUF+FE (81.0±9.8). In the inner cell mass, caspase-positive cells percentage in MUF+ME (9.8±1.5) differed (P<0.03) from FUF+FE (15.6±1.5) (groups omitted did not show significant differences). Embryonic sex is maternally detectable at early stages, leading to a favourable uterine environment specifically induced by males, but not by females. This could be associated with a sex-selection mechanism for male embryos in well-nourished females

79 a dimorphic response to early male and female embryos in the bovine uterus

Sexual dimorphism has been reported in early mammalian embryos. However, it is unknown whether in utero signalling at early stages differs between male and female embryos. In this work, we used bovine embryos produced with sex-sorted spermatozoa to analyse embryo-maternal interactions measured as changes in uterine fluid (UF). Male (M) or female (F), Day-5 in vitro-produced embryos (E) (n=23-51) were non-surgically transferred into the uteri of well-nourished heifers (body condition score=3 in a scale 0-5). All recipients (n=8) received male and female embryos within non-consecutive oestrous cycles (4 recipients with male embryos first and 4 with female embryos first). On Day 8, embryos and their corresponding diluted MUF and FUF were recovered. Proteins were extracted from a first-flushed fraction of 45mL PBS containing protease inhibitor, while flushing continued for embryo recovery. Data were analysed by ANOVA and Duncan's test. Total embryo recovery rates (RR) tended to differ (P=0.06) between ME and FE (18.6±2.5 vs 27.7±3.3). However, blastocysts RR (11.6±1.7 vs 14.2±2.3; P=0.56) and flushed volume RR (57.8±2.9 vs 60.9±2.3) did not vary between ME and FE. Recoverable protein was lower in FUF than MUF (9.0±1.2 vs 13.2±1.5μg/100μL [P<0.05] and 2580±102 vs 3450±131μg total [P<0.001], respectively). Proteomic profiles were obtained in concentrated UFs by 2-D fluorescence difference gel electrophoresis and protein characterisation by nano-LC-ESI-MS/MS. After dialyzation against SOFaaci, factors ≥3kDa contained in MUF and FUF were used in culture (1mgmL(-1)) with Day-5 male and female embryos in a 2×2 factorial design. Blastocyst development, cell counts and caspase-3 positive embryonic cells were analysed in 5 replicates. FUF and MUF differed in 41 protein spots (t-test; P<0.05), out of which 35 proteins were identified. Up-regulated proteins (n=34; in FUF) represented an increased carbohydrate metabolism activity combined with anti-stress responses, involving the NFkB system, insulin and oestradiol. PARK7, a protein not previously identified in the bovine uterus is also diferentially expressed in FUF and MUF. MUF+ME tended to show (P<0.06) higher expansion rates in vitro than MUF+FE, FUF+FE and FUF+ME (51.4±5.2 vs 30.0±5.2, 24.5±5.7 and 35.7±5.7, respectively). Trophoblast cell counts tended to be higher (P<0.10) in MUF+ME (98.7±9.5) than in FUF+FE (85.7±10.6) and MUF+FE (81.0±9.8). In the inner cell mass, caspase-positive cells percentage in MUF+ME (9.8±1.5) differed (P<0.03) from FUF+FE (15.6±1.5) (groups omitted did not show significant differences). Embryonic sex is maternally detectable at early stages, leading to a favourable uterine environment specifically induced by males, but not by females. This could be associated with a sex-selection mechanism for male embryos in well-nourished females

HSV-1 Cgal+ infection promotes quaking RNA binding protein production and induces nuclear-cytoplasmic shuttling of quaking I-5 isoform in human hepatoma cells

Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal(+) infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal(+) induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27(Kip1) protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16-24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator

HSV-1 Cgal+ infection promotes quaking RNA binding protein production and induces nuclear-cytoplasmic shuttling of quaking I-5 isoform in human hepatoma cells

Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal(+) infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal(+) induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27(Kip1) protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16-24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator

Identification of replication-competent HSV-1 Cgal+ strain targets in a mouse model of human hepatocarcinoma xenograft

Recent studies based on animal models have shown the advantages and potential of oncolytic viral therapy using HSV-1 -based replication-competent vectors in the treatment of liver tumors, but little is known about the cellular targets that are modulated during viral infection. In the present work, we have studied the effects of intratumoral injections of HSV-1 Cgal(+) strain in a murine model of human hepatoma xenografts. Viral replication was assessed for more than 1month, leading to a significant reduction of tumor growth rate mediated, in part, by a cyclin B dependent cell proliferation arrest. Early events resulting in this effect were analyzed using a proteomic approach. Protein extracts from xenografted human hepatomas treated with saline or HSV-1 Cgal(+) strain during 24h were compared by 2-D DIGE and differential spots were identified by nanoLC-ESI-MS/MS. Alterations on glutathione S transferase 1 Omega, and ERp29 suggest novel HSV-1 Cgal(+) targets in solid liver tumors. Additionally, ERp29 showed a complex differential isoform pattern upon HSV-1 Cgal(+) infection, suggesting regulatory mechanisms based on post-translational modification events