VRK1 Regulates Sensitivity to Oxidative Stress by Altering Histone Epigenetic Modifications and the Nuclear Phosphoproteome in Tumor Cells.

The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.

A metagenomic study identifies a Prevotella copri enriched microbial profile associated with non-alcoholic steatohepatitis in subjects with obesity.

BACKGROUND AND AIM: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease. Increasing evidence indicates that the gut microbiota can play an important role in the pathophysiology of NAFLD. Recently, several studies have tested the predictive value of gut microbiome profiles in NAFLD progression; however, comparisons of microbial signatures in NAFLD or non-alcoholic steatohepatitis (NASH) have produced discrepant results, possibly due to ethnic and environmental factors. Thus, we aimed to characterize the gut metagenome composition of patients with fatty liver disease. METHODS: Gut microbiome of 45 well-characterized patients with obesity and biopsy-proven NAFLD was evaluated using shot-gun sequencing: 11 non-alcoholic fatty liver controls (non-NAFL), 11 with fatty liver, and 23 with NASH. RESULTS: Our study showed that Parabacteroides distasonis and Alistipes putredenis were enriched in fatty liver but not in NASH patients. Notably, in a hierarchical clustering analysis, microbial profiles were differentially distributed among groups, and membership to a Prevotella copri dominant cluster was associated with a greater risk of developing NASH. Functional analyses showed that although no differences in LPS biosynthesis pathways were observed, Prevotella-dominant subjects had higher circulating levels of LPS and a lower abundance of pathways encoding butyrate production. CONCLUSIONS: Our findings suggest that a Prevotella copri dominant bacterial community is associated with a greater risk for NAFLD disease progression, probably linked to higher intestinal permeability and lower capacity for butyrate production.

Changes in Cerebral Hemodynamics in Patients With Cirrhosis After Liver Transplantation.

Improvement in cognitive function after orthotopic liver transplantation (LT) has been demonstrated in the acute setting immediately after LT and in acute liver failure. However, the longterm changes in cerebral hemodynamics after LT remain unexplored. Therefore, we aimed to evaluate the longterm changes in cerebral hemodynamics of patients with cirrhosis after LT. In this prospective cohort study, we performed transcranial Doppler ultrasonography (TCD) measuring the pulsatility index (PI), resistance index (RI), and breath-holding index (BHI) to evaluate cerebrovascular structural integrity and reactivity, respectively, in both middle cerebral arteries before and after LT. Neuropsychometric tests and West-Haven criteria were used for hepatic encephalopathy (HE) characterization. Interleukin 6 and tumor necrosis factor α plasma levels were measured. Descriptive statistics and Wilcoxon's test were used. There were 27 patients who were included. Median follow-up after LT was 6 months, mean age before LT was 46.3 ± 10.3 years, the main etiology was hepatitis C virus (59%), and most of the patients were Child-Pugh B (15/27). Model for End-Stage Liver Disease (MELD) score was 16 ± 7.5, MELD-Na was 19.3 ± 7.1, Psychometric Hepatic Encephalopathy Score was -3.48 ± 3.66, and critical flicker fusion (CFF) was 40.28 ± 5.70 Hz. Before LT, 17/27 patients had HE and 11/27 ascites. A decrease of 20.8% and 13.5% in PI and RI was observed after LT (P < 0.001, both), together with an increase in BHI (32.4%, P = 0.122). These changes in cerebral hemodynamics paralleled those in systemic inflammation. Clinical improvement in cognition was observed in all patients with overt HE after LT. In conclusion, these results show a significant improvement in cerebral hemodynamics after LT, obtained through TCD, indicating less arterial cerebral vasoconstriction together with a decrease in systemic inflammation. Changes in cerebral vasoconstriction can be the basis for the improvement in cognitive function after LT in the long term.

Genetic variants in COL13A1, ADIPOQ and SAMM50, in addition to the PNPLA3 gene, confer susceptibility to elevated transaminase levels in an admixed Mexican population.

Non-alcoholic fatty liver disease (NAFLD) is the accumulation of extra fat in liver cells not caused by alcohol. Elevated transaminase levels are common indicators of liver disease, including NAFLD. Previously, we demonstrated that PNPLA3 (rs738409), LYPLAL1 (rs12137855), PPP1R3B (rs4240624), and GCKR (rs780094) are associated with elevated transaminase levels in overweight/obese Mexican adults. We investigated the association between 288 SNPs identified in genome-wide association studies and risk of elevated transaminase levels in an admixed Mexican-Mestizo sample of 178 cases of NAFLD and 454 healthy controls. The rs2896019, rs12483959, and rs3810622 SNPs in PNPLA3 and rs1227756 in COL13A1 were associated with elevated alanine aminotransferase (ALT, ≥40IU/L). A polygenic risk score (PRS) based on six SNPs in the ADIPOQ, COL13A1, PNPLA3, and SAMM50 genes was also associated with elevated ALT. Individuals carrying 9-12 risk alleles had 65.8% and 48.5% higher ALT and aspartate aminotransferase (AST) levels, respectively, than those with 1-4 risk alleles. The PRS showed the greatest risk of elevated ALT levels, with a higher level of significance than the individual variants. Our findings suggest a significant association between variants in COL13A1, ADIPOQ, SAMM50, and PNPLA3, and risk of NAFLD/elevated transaminase levels in Mexican adults with an admixed ancestry. This is the first study to examine high-density single nucleotide screening for genetic variations in a Mexican-Mestizo population. The extent of the effect of these variations on the development and progression of NAFLD in Latino populations requires further analysis.

Hepatic miR-33a/miR-144 and their target gene ABCA1 are associated with steatohepatitis in morbidly obese subjects.

BACKGROUND AND AIM: Abnormal cholesterol metabolism may contribute to the pathogenesis of non-alcoholic steatohepatitis (NASH) and fibrosis. miR-33 and miR-144 regulate adenosine triphosphate binding cassette transporter (ABCA1) and other target genes involved in cholesterol efflux, fatty acid oxidation and inflammation. We explored relationships between non-alcoholic fatty liver disease (NAFLD) and the hepatic expression of ABCA1/ABCG1, as well as other target genes regulated by miR-33 (carnitine O-octanoyltransferase, CROT and hydroxyacyl-CoA-dehydrogenase ß-subunit, HADHB) and miR-144 (toll-like receptor-2, TLR2). Moreover, we evaluated whether the expression of these genes is correlated with miR-33a/b and miR-144 expression in Mexican individuals with morbid obesity. METHODS: Eighty-four morbidly obese subjects undergoing bariatric surgery were included in this study. Liver biopsies were obtained to measure hepatic triglyceride and free cholesterol contents, as well as ABCA1, ABCG1, CROT, HADHB, TLR2, miR-33a/b and miR-144 expression. RESULTS: Hepatic free cholesterol content was significantly increased in NASH as compared to non-NASH subjects, while ABCA1 and ABCG1 protein levels significantly decreased with NASH and fibrosis progression. The relative expression of miR-33a and miR-144 correlated inversely with ABCA1 but not with ABCG1 protein levels. Moreover, both miRNAs increased significantly in NASH individuals. miR-33 target genes CROT and HADHB correlated inversely with miR-33a. However, the expression of these genes was not associated with NASH. CONCLUSIONS: miR-33a/144 and their target gene ABCA1 may contribute to the pathogenesis of NASH in morbidly obese subjects.

A genetic risk score is associated with hepatic triglyceride content and non-alcoholic steatohepatitis in Mexicans with morbid obesity.

BACKGROUND AND AIMS: Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) near/in PNPLA3, NCAN, LYPLAL1, PPP1R3B, and GCKR genes associated with non-alcoholic fatty liver disease (NAFLD) mainly in individuals of European ancestry. The aim of the study was to test whether these genetic variants and a genetic risk score (GRS) are associated with elevated liver fat content and non-alcoholic steatohepatitis (NASH) in Mexicans with morbid obesity. METHODS: 130 morbidly obese Mexican individuals were genotyped for six SNPs in/near PNPLA3, NCAN, LYPLAL1, PPP1R3B, and GCKR genes. Hepatic fat content [triglyceride (HTG) and total cholesterol (HTC)] was quantified directly in liver biopsies and NASH was diagnosed by histology. A GRS was tested for association with liver fat content and NASH using logistic regression models. In addition, 95 ancestry-informative markers were genotyped to estimate population admixture proportions. RESULTS: After adjusting for age, sex and admixture, PNPLA3, LYPLAL1, GCKR and PPP1R3B polymorphisms were associated with higher HTG content (P < 0.05 for PNPLA3, LYPLAL1, GCKR polymorphisms and P = 0.086 for PPP1R3B). The GRS was significantly associated with higher HTG and HTC content (P = 1.0 × 10(-4) and 0.048, respectively), steatosis stage (P = 0.029), and higher ALT levels (P = 0.002). Subjects with GRS ≥ 6 showed a significantly increased risk of NASH (OR = 2.55, P = 0.045) compared to those with GRS ≤ 5. However, the GRS did not predict NASH status, as AUC of ROC curves was 0.56 (P = 0.219). CONCLUSION: NAFLD associated loci in Europeans and a GRS based on these loci contribute to the accumulation of hepatic lipids and NASH in morbidly obese Mexican individuals.

SFRP5 hepatic expression is associated with non-alcoholic liver disease in morbidly obese women.

BACKGROUND AND AIMS: Secreted frizzled-related protein 5 (SFRP5) was recently described as a new adipokine protective for hepatic steatosis and other obesity-related complications in the mouse model. To date, SFRP5 expression in non-alcoholic fatty liver disease (NAFLD) has not been fully assessed in humans. We measured circulating SFRP5 levels and its expression in liver and adipose tissue, and evaluated its association with NAFLD in morbidly obese women. MATERIAL AND METHODS: Fifty-four morbidly obese women undergoing bariatric surgery were included in the study. Liver biopsies were used for histology and hepatic triglyceride content quantification. Circulating SFRP5 levels were measured through enzyme-linked immunoabsorbent assay, and SFRP5 expression was performed in hepatic and adipose tissue (subcutaneous and visceral). RESULTS: Although circulating SFRP5 levels showed a tendency to decrease with NAFLD progression, no significant differences were observed among non-alcoholic steatosis, steatohepatitis, and control subjects. Hepatic SFRP5 expression showed a negative correlation with hepatic triglyceride content (r = -0.349, P = 0.016 for mRNA and r = -0.291, P = 0.040 for SRFP5 protein) and ALT serum levels (r = -0.437, P = 0.001 for SRFP5 protein). In addition, hepatic SFRP5 protein levels were significantly lower in NASH than in control subjects (P = 0.006). CONCLUSION: This is the first study reporting an association of hepatic SFRP5 expression with NAFLD in humans.

[Second Consensus of the Chilean Society of Endocrinology and Diabetes about insulin resistance]. / II Consenso de la Sociedad Chilena de Endocrinología y Diabetes sobre resistencia a la insulina.

Insulin resistance is a prevalent condition commonly associated with unhealthy lifestyles. It affects several metabolic pathways, increasing risk of abnormalities at different organ levels. Thus, diverse medical specialties should be involved in its diagnosis and treatment. With the purpose of unifying criteria about this condition, a scientific-based consensus was elaborated. A questionnaire including the most important topics such as cardio-metabolic risk, non-alcoholic fatty liver disease and polycystic ovary syndrome, was designed and sent to national experts. When no agreement among them was achieved, the Delphi methodology was applied. The main conclusions reached are that clinical findings are critical for the diagnosis of insulin resistance, not being necessary blood testing. Acquisition of a healthy lifestyle is the most important therapeutic tool. Insulin-sensitizing drugs should be prescribed to individuals at high risk of disease according to clinically validated outcomes. There are specific recommendations for pregnant women, children, adolescents and older people.

PD-L1 gene polymorphisms and low serum level of PD-L1 protein are associated to type 1 diabetes in Chile.

INTRODUCTION: Type 1 diabetes (T1D) has a complex etiology in which genetic and environmental factors are involved, whose interactions have not yet been completely clarified. In this context, the role in PD-1 pathway and its ligands 1 and 2 (PD-L1 and PD-L2) have been proposed as candidates in several autoimmune diseases. The aim of this work was to determine the allele and haplotype frequency of six gene polymorphisms of PD-ligands (PD-L1 and PD-L2) in Chilean T1D patients and their effect on serum levels of PD-L1 and autoantibody profile (GAD65 and IA2). METHODS: This study cohort comprised 205 T1D patients and 205 normal children. We performed genotypic analysis of PD-L1 and PD-L2 genes by TaqMan method. Determination of anti-GAD65 and anti-IA-2 autoantibodies was performed by ELISA. The PD-L1 serum levels were measured. RESULTS: The allelic distribution of PD-L1 variants (rs2297137 and rs4143815) showed differences between T1D patients and controls (p = 0.035 and p = 0.022, respectively). No differences were detected among the PD-L2 polymorphisms, and only the rs16923189 showed genetic variation. T1D patients showed decreased serum levels of PD-L1 compared to controls: 1.42 [0.23-7.45] ng/mL versus 3.35 [0.49-5.89] ng/mL (p < 0.025). In addition, the CGG haplotype in PD-L1 associated with T1D (constructed from rs822342, rs2297137 and rs4143815 polymorphisms) showed an OR = 1.44 [1.08 to 1.93]. Finally, no association of these genetic variants was observed with serum concentrations of PD ligands or auto-antibody profile, although a correlation between PD-L1 ligand serum concentration and the age at disease onset was detected. CONCLUSION: Two polymorphism of PD-L1 are presented in different allelic variants between T1D and healthy subjects, also PDL-1 serum levels are significantly lowered in diabetics patients. Moreover, the age of onset of the disease determine differences between serum ligand levels in diabetics, being lower in younger. These results points to a possible establishment of PDL-1 as a genetic and biochemical marker for T1D onset, at least in Chilean population.

PNPLA3 I148M polymorphism is associated with elevated alanine transaminase levels in Mexican Indigenous and Mestizo populations.

The patatin like phospholipase domain-containing (PNPLA3) I148M variant is the strongest genetic factor associated with elevated alanine transaminase (ALT) levels in different populations, particularly in Hispanics who have the highest 148M risk allele frequency reported to date. It has been suggested that Indigenous ancestry is associated with higher ALT levels in Mexicans. The aim of the present study was to assess the frequency of the PNPLA3 148M risk allele in Mexican indigenous and Mestizo individuals, and to examine its association with serum ALT levels. The study included a total of 1624 Mexican individuals: 919 Indigenous subjects from five different native groups and 705 Mexican Mestizo individuals (141 cases with ALT levels ≥ 40 U/L and 564 controls with ALT <40 U/L). The I148M polymorphism was genotyped by TaqMan assays. The frequency of elevated ALT levels in Indigenous populations was 18.7%, and varied according to obesity status: 14.4% in normal weight, 19.9% in overweight and 24.5% in obese individuals. The Mexican indigenous populations showed the highest reported frequency of the PNPLA3 148M risk allele (mean 0.73). The M148M genotype was significantly associated with elevated ALT levels in indigenous individuals (OR = 3.15, 95 % CI 1.91-5.20; P = 7.1 × 10(-6)) and this association was confirmed in Mexican Mestizos (OR = 2.24, 95% CI 1.50-3.33; P = 8.1 × 10(-5)). This is the first study reporting the association between M148M genotype and elevated ALT levels in Indigenous Mexican populations. The 148M allele risk may be considered an important risk factor for liver damage in Mexican indigenous and Mestizo populations.

Modelling the low temperature growth boundaries of Salmonella Enteritidis in raw and pasteurized egg yolk, egg white and liquid whole egg: Influence of the initial concentration.

Salmonella is the most frequently reported cause of foodborne outbreaks with known origin in Europe, with eggs and egg products standing out as the most frequent food source (when it was known). The growth and survival of Salmonella in eggs and egg products have been extensively studied and, recently, it has been reported that factors such as the initial concentration and thermal history of the egg product can also influence its growth capability. Therefore, the objective of this study was to define the boundary zones of the growth/no growth domain of Salmonella Enteritidis (4 strains) as a function of temperature (low temperature boundary) and the initial concentration in different egg products. A series of polynomial logistic regression equations were successfully adjusted, allowing the study of these factors and their interaction on the probability of growth of S. Enteritidis in these products. Results obtained indicate that the minimum growth temperatures of Salmonella Enteritidis are higher in egg white (9.5-18.3 °C) than in egg yolk (7.1-7.8 °C) or liquid whole egg (7.2-7.9 °C). Results also demonstrate that in raw liquid whole egg and raw and pasteurized egg white, the minimum growth temperature of Salmonella Enteritidis does depend on the initial concentration. Similarly, the previous thermal history of the egg product only influenced the minimum growth temperature in some of them. On the other hand, large differences in the minimum growth temperatures among strains were observed in some products (up to approx. 6 °C in egg white). Finally, it should be noted that none of the strains grew at 5 °C under any of the conditions assayed. Therefore, storage of egg products (particularly whole liquid egg and egg yolk) below this temperature might be regarded/proposed as a good management approach. Our experimental approach has allowed us to provide a more accurate prediction of S. Enteritidis minimum growth temperatures in egg products by taking into account additional factors (initial concentration and thermal history) while also providing a quantification of the intra-specie variability. This would be of high relevance for improving the safety of egg products.

Loss of VRK1 alters the nuclear phosphoproteome in the DNA damage response to doxorubicin.

Dynamic chromatin remodeling requires regulatory mechanisms for its adaptation to different nuclear function, which are likely to be mediated by signaling proteins. In this context, VRK1 is a nuclear Ser-Thr kinase that regulates pathways associated with transcription, replication, recombination, and DNA repair. Therefore, VRK1 is a potential regulatory, or coordinator, molecule in these processes. In this work we studied the effect that VRK1 depletion has on the basal nuclear and chromatin phosphoproteome, and their associated pathways. VRK1 depletion caused an alteration in the pattern of the nuclear phosphoproteome, which is mainly associated with nucleoproteins, ribonucleoproteins, RNA splicing and processing. Next, it was determined the changes in proteins associated with DNA damage that was induced by doxorubicin treatment. Doxorubicin alters the nuclear phosphoproteome affecting proteins implicated in DDR, including DSB repair proteins NBN and 53BP1, cellular response to stress and chromatin organization proteins. In VRK1-depleted cells, the effect of doxorubicin on protein phosphorylation was reverted to basal levels. The nuclear phosphoproteome patterns induced by doxorubicin are altered by VRK1 depletion, and is enriched in histone modification proteins and chromatin associated proteins. These results indicate that VRK1 plays a major role in processes requiring chromatin remodeling in its adaptation to different biological contexts.

Vimentin single cysteine residue acts as a tunable sensor for network organization and as a key for actin remodeling in response to oxidants and electrophiles.

Cysteine residues can undergo multiple posttranslational modifications with diverse functional consequences, potentially behaving as tunable sensors. The intermediate filament protein vimentin has important implications in pathophysiology, including cancer progression, infection, and fibrosis, and maintains a close interplay with other cytoskeletal structures, such as actin filaments and microtubules. We previously showed that the single vimentin cysteine, C328, is a key target for oxidants and electrophiles. Here, we demonstrate that structurally diverse cysteine-reactive agents, including electrophilic mediators, oxidants and drug-related compounds, disrupt the vimentin network eliciting morphologically distinct reorganizations. As most of these agents display broad reactivity, we pinpointed the importance of C328 by confirming that local perturbations introduced through mutagenesis provoke structure-dependent vimentin rearrangements. Thus, GFP-vimentin wild type (wt) forms squiggles and short filaments in vimentin-deficient cells, the C328F, C328W, and C328H mutants generate diverse filamentous assemblies, and the C328A and C328D constructs fail to elongate yielding dots. Remarkably, vimentin C328H structures resemble the wt, but are strongly resistant to electrophile-elicited disruption. Therefore, the C328H mutant allows elucidating whether cysteine-dependent vimentin reorganization influences other cellular responses to reactive agents. Electrophiles such as 1,4-dinitro-1H-imidazole and 4-hydroxynonenal induce robust actin stress fibers in cells expressing vimentin wt. Strikingly, under these conditions, vimentin C328H expression blunts electrophile-elicited stress fiber formation, apparently acting upstream of RhoA. Analysis of additional vimentin C328 mutants shows that electrophile-sensitive and assembly-defective vimentin variants permit induction of stress fibers by reactive species, whereas electrophile-resistant filamentous vimentin structures prevent it. Together, our results suggest that vimentin acts as a break for actin stress fibers formation, which would be released by C328-aided disruption, thus allowing full actin remodeling in response to oxidants and electrophiles. These observations postulate C328 as a "sensor" transducing structurally diverse modifications into fine-tuned vimentin network rearrangements, and a gatekeeper for certain electrophiles in the interplay with actin.

The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase.

BACKGROUND: Dynamic chromatin remodeling is associated with changes in the epigenetic pattern of histone acetylations and methylations required for processes based on dynamic chromatin remodeling and implicated in different nuclear functions. These histone epigenetic modifications need to be coordinated, a role that may be mediated by chromatin kinases such as VRK1, which phosphorylates histones H3 and H2A. METHODS: The effect of VRK1 depletion and VRK1 inhibitor, VRK-IN-1, on the acetylation and methylation of histone H3 in K4, K9 and K27 was determined under different conditions, arrested or proliferating cells, in A549 lung adenocarcinoma and U2OS osteosarcoma cells. RESULTS: Chromatin organization is determined by the phosphorylation pattern of histones mediated by different types of enzymes. We have studied how the VRK1 chromatin kinase can alter the epigenetic posttranslational modifications of histones by using siRNA, a specific inhibitor of this kinase (VRK-IN-1), and of histone acetyl and methyl transferases, as well as histone deacetylase and demethylase. Loss of VRK1 implicated a switch in the state of H3K9 posttranslational modifications. VRK1 depletion/inhibition causes a loss of H3K9 acetylation and facilitates its methylation. This effect is similar to that of the KAT inhibitor C646, and to KDM inhibitors as iadademstat (ORY-1001) or JMJD2 inhibitor. Alternatively, HDAC inhibitors (selisistat, panobinostat, vorinostat) and KMT inhibitors (tazemetostat, chaetocin) have the opposite effect of VRK1 depletion or inhibition, and cause increase of H3K9ac and a decrease of H3K9me3. VRK1 stably interacts with members of these four enzyme families. However, VRK1 can only play a role on these epigenetic modifications by indirect mechanisms in which these epigenetic enzymes are likely targets to be regulated and coordinated by VRK1. CONCLUSIONS: The chromatin kinase VRK1 regulates the epigenetic patterns of histone H3 acetylation and methylation in lysines 4, 9 and 27. VRK1 is a master regulator of chromatin organization associated with its specific functions, such as transcription or DNA repair.

Evaluation of hygiene practices and microbiological status of ready-to-eat vegetable salads in Spanish school canteens.

BACKGROUND: This study was conducted in eight Spanish school canteens during the period 2008-2009. Food handlers' practices, kitchen equipment, hygiene/sanitation conditions and handling practices were evaluated using checklists. In parallel, the microbiological quality and safety of ready-to-eat (RTE) vegetable salads were assessed. In addition, food contact surfaces and environmental air quality of different areas were analysed. The study determined the relationship between the microbiological quality of RTE foods and food handling practices, together with the degree of contamination of working surfaces and environmental contamination of processing and distribution areas. RESULTS: Some deficiencies were found regarding the use and change of gloves, hand-washing and cleanliness of working surfaces. The microbial levels detected in the foods examined indicated the absence of pathogens in the samples analysed. Surface counts were higher on cutting boards and faucets, showing insufficient cleanliness procedures. CONCLUSION: This study constitutes a descriptive analysis of the hygiene/sanitation conditions implemented in food service systems in eight Spanish school canteens. The results should help risk managers to better define control measures to be adopted in order to prevent foodborne infections.

The VRK1 chromatin kinase regulates the acetyltransferase activity of Tip60/KAT5 by sequential phosphorylations in response to DNA damage.

The regulation of histone epigenetic modifications mediates the adaptation of chromatin to different biological processes. DNA damage causes a local relaxation of chromatin associated to histone H4 acetylation in K16, mediated by Tip60/KAT5. In this work, we have studied the role that the VRK1 chromatin kinase plays on the activation of Tip60 during this process. In the DNA damage response induced by doxorubicin, VRK1 directly phosphorylates Tip60. However, the phosphorylated Tip60 residues and their functional roles are unknown. In DDR, we have identified these two Tip60 phosphorylated residues and the cooperation of the participating kinases. The T158 phosphorylation, mediated by VRK1, is early and transient, preceding that of S199, which is more sustained in time, and mediated by DNA-PK. The role of each phosphorylated residues was determined by using phosphomimetic and phosphonull mutants and their combination. T158 phosphorylation protects Tip60 from ubiquitin-mediated degradation, promotes its recruitment to chromatin from the nucleoplasm, and is necessary for its full trans-acetylase activity. The phosphorylation in S199 by DNA-PK directly facilitates Tip60 autoacetylation, but it is not enough for trans-acetylation of two of its targets, histone H4 and ATM, which requires a double phosphorylation of Tip60 in T158 and S199. DNA-PK inhibitors block the phosphorylation of S199. We propose a model in which the cooperation between VRK1 and DNA-PK mediates the sequential phosphorylation of Tip60/KAT5, and contributes to the recruitment of this protein to initiate the sequential remodeling of chromatin in DDR. Both proteins are candidates for novel synthetic lethality strategies in cancer treatment.

Cellulose Nanofibers from Olive Tree Pruning as Food Packaging Additive of a Biodegradable Film.

A biodegradable packaging film containing cellulose nanofibers from olive tree pruning, a by-product of olives production, was obtained using a solvent casting method. Nanocellulose was added to polyvinyl alcohol (PVA) to enhance the technological properties of the composite film as food packaging material. Nanocellulose was obtained from unbleached and bleached pulp through a mechanical and TEMPO pretreatment. Crystalline and chemical structure, surface microstructure, UV and gas barrier, optical, mechanical and antioxidant properties, as well as thermal stability were evaluated. Regarding optical properties, the UV barrier was increased from 6% for the pure PVA film to 50% and 24% for unbleached and bleached nanocellulose, respectively. The antioxidant capacity increased significantly in unbleached mechanical nanocellulose-films (5.3%) compared to pure PVA film (1.7%). In terms of mechanical properties, the tensile strength of the 5% unbleached mechanical nanocellulose films was significantly improved compared to the pure PVA film. Similarly, the 5% nanocellulose films had increased the thermal stability and improved barrier properties, reducing water vapor permeability by 38-59% and presenting an oxygen barrier comparable to aluminum layer and plastic films. Our results support the use of the developed films as a green alternative material for food packaging.

VRK1 Depletion Facilitates the Synthetic Lethality of Temozolomide and Olaparib in Glioblastoma Cells.

BACKGROUND: Glioblastomas treated with temozolomide frequently develop resistance to pharmacological treatments. Therefore, there is a need to find alternative drug targets to reduce treatment resistance based on tumor dependencies. A possibility is to target simultaneously two proteins from different DNA-damage repair pathways to facilitate tumor cell death. Therefore, we tested whether targeting the human chromatin kinase VRK1 by RNA interference can identify this protein as a novel molecular target to reduce the dependence on temozolomide in combination with olaparib, based on synthetic lethality. MATERIALS AND METHODS: Depletion of VRK1, an enzyme that regulates chromatin dynamic reorganization and facilitates resistance to DNA damage, was performed in glioblastoma cells treated with temozolomide, an alkylating agent used for GBM treatment; and olaparib, an inhibitor of PARP-1, used as sensitizer. Two genetically different human glioblastoma cell lines, LN-18 and LN-229, were used for these experiments. The effect on the DNA-damage response was followed by determination of sequential steps in this process: H4K16ac, γH2AX, H4K20me2, and 53BP1. RESULTS: The combination of temozolomide and olaparib increased DNA damage detected by labeling free DNA ends, and chromatin relaxation detected by H4K16ac. The combination of both drugs, at lower doses, resulted in an increase in the DNA damage response detected by the formation of γH2AX and 53BP1 foci. VRK1 depletion did not prevent the generation of DNA damage in TUNEL assays, but significantly impaired the DNA damage response induced by temozolomide and olaparib, and mediated by γH2AX, H4K20me2, and 53BP1. The combination of these drugs in VRK1 depleted cells resulted in an increase of glioblastoma cell death detected by annexin V and the processing of PARP-1 and caspase-3. CONCLUSION: Depletion of the chromatin kinase VRK1 promotes tumor cell death at lower doses of a combination of temozolomide and olaparib treatments, and can be a novel alternative target for therapies based on synthetic lethality.

Valorisation of Olea europaea L. Olive Leaves through the Evaluation of Their Extracts: Antioxidant and Antimicrobial Activity.

Olea europaea L. leaves constitute a source of bioactive compounds with recognized benefits for both human health and technological purposes. In the present work, different extracts from olive leaves were obtained by the application of two extraction methods, Soxhlet and microwave-assisted extraction (MAE), and six solvents (distilled water, ethanolic and glycerol mixtures solvents). MAE was applied under 40, 60 and 80 °C for 3, 6.5 and 10 min. The effect of the extraction method, solvent and treatment factors (the latter in MAE) on the total phenol content (TPC), the antioxidant activity (AA) and the phenolic profile of the extracts were all evaluated. The extracts showed high values of TPC (up to 76.1 mg GAE/g DW) and AA (up to 78 mg TE/g DW), with oleuropein being the most predominant compound in all extracts. The Soxhlet extraction method exhibited better yields in TPC than in MAE, although both methods presented comparable AA values. The water MAE extract presented the strongest antimicrobial activity against five foodborne pathogens, with minimum inhibitory concentration (MIC) values ranging from 2.5 to 60 mg/mL. MAE water extract is proposed to be exploited in the food and nutraceutical industry in the frame of a sustainable economy.

Lysine Methyltransferase Inhibitors Impair H4K20me2 and 53BP1 Foci in Response to DNA Damage in Sarcomas, a Synthetic Lethality Strategy.

BACKGROUND: Chromatin is dynamically remodeled to adapt to all DNA-related processes, including DNA damage responses (DDR). This adaptation requires DNA and histone epigenetic modifications, which are mediated by several types of enzymes; among them are lysine methyltransferases (KMTs). METHODS: KMT inhibitors, chaetocin and tazemetostat (TZM), were used to study their role in the DDR induced by ionizing radiation or doxorubicin in two human sarcoma cells lines. The effect of these KMT inhibitors was tested by the analysis of chromatin epigenetic modifications, H4K16ac and H4K20me2. DDR was monitored by the formation of γH2AX, MDC1, NBS1 and 53BP1 foci, and the induction of apoptosis. RESULTS: Chaetocin and tazemetostat treatments caused a significant increase of H4K16 acetylation, associated with chromatin relaxation, and increased DNA damage, detected by the labeling of free DNA-ends. These inhibitors significantly reduced H4K20 dimethylation levels in response to DNA damage and impaired the recruitment of 53BP1, but not of MDC1 and NBS1, at DNA damaged sites. This modification of epigenetic marks prevents DNA repair by the NHEJ pathway and leads to cell death. CONCLUSION: KMT inhibitors could function as sensitizers to DNA damage-based therapies and be used in novel synthetic lethality strategies for sarcoma treatment.