Presence of Zonula Occludens Toxin-Coding Genes among Vibrio parahaemolyticus Isolates of Clinical and Environmental Origin.

In recent studies, emphasis has been placed on the zonula occludens toxin (Zot) from the non-toxigenic Vibrio parahaemolyticus strain PMC53.7 as an agent inducing alterations in the actin cytoskeleton of infected Caco-2 cells and which appears as a relevant virulence factor. Universal zot primers were designed by the alignment of different types of zot gene and identification of conserved sequences to investigate the presence in diverse environmental and clinical V. parahaemolyticus isolates, in co-occurrence with virulence factors, such a hemolysins and secretion systems. The study screened a total of 390 isolates from environmental sources from Chile and Italy and 95 Chilean clinical isolates. The results revealed that around 37.2% of Chilean environmental strains and 25.9% of Italian strains, and 24.2% of clinical isolates carried the zot gene. The Zot-C2 cluster was present in 71.4% of Chilean environmental strains but absent in clinical isolates, while the Zot-C4 cluster was identified in 28.6% of environmental and 100% of clinical isolates. Understanding the role of zot in V. parahaemolyticus virulence is crucial, especially considering the risk associated with consuming diverse isolates from bivalves and the co-occurrence with virulence factors such as TDH, TRH or T3SS2.

Unveiling antimicrobial resistance in Chilean fertilized soils: a One Health perspective on environmental AMR surveillance.

Antimicrobial resistance (AMR) poses a significant threat to humans and animals as well as the environment. Within agricultural settings, the utilization of antimicrobial agents in animal husbandry can lead to the emergence of antimicrobial resistance. In Chile, the widespread use of animal-derived organic amendments, including manure and compost, requires an examination of the potential emergence of AMR resulting from their application. The aim of this research was to identify and compare AMR genes found in fertilized soils and manure in Los Andes city, Chile. Soil samples were collected from an agricultural field, comprising unamended soils, amended soils, and manure used for crop fertilization. The selected genes (n = 28) included genes associated with resistance to beta-lactams, tetracyclines, sulfonamides, polymyxins, macrolides, quinolones, aminoglycosides, as well as mobile genetic elements and multidrug resistance genes. Twenty genes were successfully identified in the samples. Tetracycline resistance genes displayed the highest prevalence, followed by MGE and sulfonamides, while quinolone resistance genes were comparatively less abundant. Notably, blaOXA, sulA, tetO, tetW, tetM, aac (6) ib., and intI1, exhibited higher frequencies in unamended soils, indicating their potential persistence within the soil microbiome and contribution to the perpetuation of AMR over time. Given the complex nature of AMR, it is crucial to adopt an integrated surveillance framework that embraces the One Health approach, involving multiple sectors, to effectively address this challenge. This study represents the first investigation of antimicrobial resistance genes in agricultural soils in Chile, shedding light on the presence and dynamics of AMR in this context.

Effectiveness of anthocyanins rich foods on cardiometabolic factors in individuals with metabolic syndrome: a systematic review and meta-analysis.

PURPOSE: The aim of this systematic review with meta-analysis was to determine whether anthocyanin-rich foods are effective to improve cardiometabolic markers in individuals with metabolic syndrome (MetS), compared with placebo or control interventions. METHODS: We searched MEDLINE, CENTRAL, Embase, LILACS, CINAHL, and Web of Science from their inception up to March 2022. We include clinical trials (randomized clinical trials, controlled clinical trials, and cross-over trials) with anthocyanin-rich foods versus placebo or control intervention that assessment cardiometabolic factors. RESULTS: We found 14 clinical trials that met the eligibility criteria, and we included 10 studies for the quantitative synthesis. For anthocyanin-rich foods versus control interventions, the mean difference (MD) for low-density lipoprotein (LDL) was - 7.98 mg/dL (CI = - 15.20 to - 0.77, GRADE: Very low). For homeostatic model assessment for insulin resistance (HOMA-IR), the MD was 0.04 (CI = 0.08 to 0.16, GRADE: Moderate). The MD for interleukin 6 was 0.00 pg/mL (CI = - 0.01 to 0.00, GRADE: Low). For tumor necrosis factor alpha (TNF-α), the standardized mean difference (SMD) was - 0.52 pg/mL (CI = 0.85 to 0.19 GRADE: Very low) when compared with the control interventions. The certainty of the evidence for the other outcomes it is very low. CONCLUSION: Our findings suggest that anthocyanin-rich foods could improve certain cardiometabolic markers (e.g., TC, TG, LDL, and TNF-α) among individuals with MetS (with very low quality evidence according to GRADE), compared with placebo or other control interventions. PROSPERO REGISTRATION NUMBER: CRD42020187287.

The High Risk of Bivalve Farming in Coastal Areas With Heavy Metal Pollution and Antibiotic-Resistant Bacteria: A Chilean Perspective.

Anthropogenic pollution has a huge impact on the water quality of marine ecosystems. Heavy metals and antibiotics are anthropogenic stressors that have a major effect on the health of the marine organisms. Although heavy metals are also associate with volcanic eruptions, wind erosion or evaporation, most of them come from industrial and urban waste. Such contamination, coupled to the use and subsequent misuse of antimicrobials in aquatic environments, is an important stress factor capable of affecting the marine communities in the ecosystem. Bivalves are important ecological components of the oceanic environments and can bioaccumulate pollutants during their feeding through water filtration, acting as environmental sentinels. However, heavy metals and antibiotics pollution can affect several of their physiologic and immunological processes, including their microbiome. In fact, heavy metals and antibiotics have the potential to select resistance genes in bacteria, including those that are part of the microbiota of bivalves, such as Vibrio spp. Worryingly, antibiotic-resistant phenotypes have been shown to be more tolerant to heavy metals, and vice versa, which probably occurs through co- and cross-resistance pathways. In this regard, a crucial role of heavy metal resistance genes in the spread of mobile element-mediated antibiotic resistance has been suggested. Thus, it might be expected that antibiotic resistance of Vibrio spp. associated with bivalves would be higher in contaminated environments. In this review, we focused on co-occurrence of heavy metal and antibiotic resistance in Vibrio spp. In addition, we explore the Chilean situation with respect to the contaminants described above, focusing on the main bivalves-producing region for human consumption, considering bivalves as potential vehicles of antibiotic resistance genes to humans through the ingestion of contaminated seafood.

Effectiveness of multicomponent treatment in patients with fibromyalgia: protocol for a systematic review and meta-analysis.

BACKGROUND: The purpose of this protocol is to provide a new systematic review with meta-analysis using the current methodology to compare the effectiveness of multicomponent treatment versus other interventions for patients with fibromyalgia. METHODS: This protocol conforms to the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) and the recommendations of the Cochrane Collaboration Handbook. An electronic search will be conducted in MEDLINE, EMBASE, Web of Science, Cochrane CENTRAL, LILACS, CINAHL, and PEDro, from inception until April 2022. There will be no language restrictions. The Cochrane Collaboration tool for assessing the risk of bias (RoB2) will be used. The Grading of Recommendations, Assessment, Development and Evaluation (GRADE) scale will be used to evaluate the strength of the evidence. The Hartung-Knapp-Sidik-Jonkman random effects or Mantel-Haenszel fixed effects methods will be used, depending on the heterogeneity, to compute a pooled estimate of the mean difference (MD) or standardized mean difference (SMD) and respective 95% confidence intervals for clinical outcomes. DISCUSSION: This systematic review will synthesize evidence on the effectiveness of multicomponent treatment in patients with fibromyalgia and could add important evidence in the treatment of FM to improve clinical practice and decision-making/actions in this field. This new systematic review will try to show the effects of multicomponent treatment by type (endurance, resistance, stretching, or mind-body exercises [pilates or taichi]) and intensity (light, moderate, moderate-to-vigorous, vigorous) of exercise in patients with FM. The results will be disseminated by publication in a peer-reviewed journal. Ethics approval will not be needed because the data used for this systematic review will be obtained from individual trials and there will be no concerns about privacy. However, if we identify ethical issues during the development of the systematic review, these findings will be reported in the discussion of the study. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020142082.

Effectiveness of aloe vera in patients with type 2 Diabetes Mellitus and pre-diabetes: An overview of systematic reviews.

BACKGROUND AND AIMS: The effects of aloe vera are inconsistent and unclear. The aim of this study is to analyze the effects of aloe vera in metabolic profiles. METHODS: An electronic search of systematic reviews (SRs) was performed in seven databases up to June 2021. RESULTS: Four SRs met the eligibility criteria. In T2DM, SMD for FBG = -5.61 (p < 0.001). For HbA1c, MD = -0.95 (p = 0.02). In pre-diabetes, SMD for FBG = -1.41 (p = 0.02). For HbA1c, MD = -0.31 (p = 0.02). For TG, MD = -4.99 (p = 0.000). CONCLUSION: There exist a moderate to high quality of evidence in favor of the effects of aloe vera in patients with T2DM and pre-diabetes.

Tomato Cultivars With Variable Tolerances to Water Deficit Differentially Modulate the Composition and Interaction Patterns of Their Rhizosphere Microbial Communities.

Since drought is the leading environmental factor limiting crop productivity, and plants have a significant impact in defining the assembly of plant-specific microbial communities associated with roots, we aimed to determine the effect of thoroughly selected water deficit tolerant and susceptible Solanum lycopersicum cultivars on their rhizosphere microbiome and compared their response with plant-free soil microbial communities. We identified a total of 4,248 bacterial and 276 fungal different operational taxonomic units (OTUs) in soils by massive sequencing. We observed that tomato cultivars significantly affected the alpha and beta diversity of their bacterial rhizosphere communities but not their fungal communities compared with bulk soils (BSs), showing a plant effect exclusively on the bacterial soil community. Also, an increase in alpha diversity in response to water deficit of both bacteria and fungi was observed in the susceptible rhizosphere (SRz) but not in the tolerant rhizosphere (TRz) cultivar, implying a buffering effect of the tolerant cultivar on its rhizosphere microbial communities. Even though water deficit did not affect the microbial diversity of the tolerant cultivar, the interaction network analysis revealed that the TRz microbiota displayed the smallest and least complex soil network in response to water deficit with the least number of connected components, nodes, and edges. This reduction of the TRz network also correlated with a more efficient community, reflected in increased cooperation within kingdoms. Furthermore, we identified some specific bacteria and fungi in the TRz in response to water deficit, which, given that they belong to taxa with known beneficial characteristics for plants, could be contributing to the tolerant phenotype, highlighting the metabolic bidirectionality of the holobiont system. Future assays involving characterization of root exudates and exchange of rhizospheres between drought-tolerant and susceptible cultivars could determine the effect of specific metabolites on the microbiome community and may elucidate their functional contribution to the tolerance of plants to water deficit.

Intestinal Transcriptome Analysis Reveals Enrichment of Genes Associated with Immune and Lipid Mechanisms, Favoring Soybean Meal Tolerance in High-Growth Zebrafish (Danio Rerio).

The molecular mechanisms underlying fish tolerance to soybean meal (SBM) remain unclear. Identifying these mechanisms would be beneficial, as this trait favors growth. Two fish replicates from 19 experimental families were fed fishmeal-(100FM) or SBM-based diets supplemented with saponin (50SBM + 2SPN) from juvenile to adult stages. Individuals were selected from families with a genotype-by-environment interaction higher (HG-50SBM + 2SPN, 170 ± 18 mg) or lower (LG-50SBM + 2SPN, 76 ± 10 mg) weight gain on 50SBM + 2SPN for intestinal transcriptomic analysis. A histological evaluation confirmed middle intestinal inflammation in the LG- vs. HG-50SBM + 2SPN group. Enrichment analysis of 665 differentially expressed genes (DEGs) identified pathways associated with immunity and lipid metabolism. Genes linked to intestinal immunity were downregulated in HG fish (mpx, cxcr3.2, cftr, irg1l, itln2, sgk1, nup61l, il22), likely dampening inflammatory responses. Conversely, genes involved in retinol signaling were upregulated (rbp4, stra6, nr2f5), potentially favoring growth by suppressing insulin responses. Genes associated with lipid metabolism were upregulated, including key components of the SREBP (mbtps1, elov5l, elov6l) and cholesterol catabolism (cyp46a1), as well as the downregulation of cyp7a1. These results strongly suggest that transcriptomic changes in lipid metabolism mediate SBM tolerance. Genotypic variations in DEGs may become biomarkers for improving early selection of fish tolerant to SMB or others plant-based diets.

Analysis of the Zonula occludens Toxin Found in the Genome of the Chilean Non-toxigenic Vibrio parahaemolyticus Strain PMC53.7.

Vibrio parahaemolyticus non-toxigenic strains are responsible for about 10% of acute gastroenteritis associated with this species, suggesting they harbor unique virulence factors. Zonula occludens toxin (Zot), firstly described in Vibrio cholerae, is a secreted toxin that increases intestinal permeability. Recently, we identified Zot-encoding genes in the genomes of highly cytotoxic Chilean V. parahaemolyticus strains, including the non-toxigenic clinical strain PMC53.7. To gain insights into a possible role of Zot in V. parahaemolyticus, we analyzed whether it could be responsible for cytotoxicity. However, we observed a barely positive correlation between Caco-2 cell membrane damage and Zot mRNA expression during PMC53.7 infection and non-cytotoxicity induction in response to purified PMC53.7-Zot. Unusually, we observed a particular actin disturbance on cells infected with PMC53.7. Based on this observation, we decided to compare the sequence of PMC53.7-Zot with Zot of human pathogenic species such as V. cholerae, Campylobacter concisus, Neisseria meningitidis, and other V. parahaemolyticus strains, using computational tools. The PMC53.7-Zot was compared with other toxins and identified as an endotoxin with conserved motifs in the N-terminus and a variable C-terminal region and without FCIGRL peptide. Notably, the C-terminal diversity among Zots meant that not all of them could be identified as toxins. Structurally, PMC53.7-Zot was modeled as a transmembrane protein. Our results suggested that it has partial 3D structure similarity with V. cholerae-Zot. Probably, the PMC53.7-Zot would affect the actin cytoskeletal, but, in the absence of FCIGRL, the mechanisms of actions must be elucidated.

Melatonin triggers metabolic and gene expression changes leading to improved quality traits of two sweet cherry cultivars during cold storage.

Sweet cherry is a valuable non-climacteric fruit with elevated phytonutrients, whose fruit quality attributes are prone to rapid deterioration after harvest, especially peel damage and water loss of stem. Here the metabolic and transcriptional response of exogenous melatonin was assessed in two commercial cultivars of sweet cherry (Santina and Royal Rainier) during cold storage. Gene expression profiling revealed that cuticle composition and water movement may underlie the effect of melatonin in delaying weight loss. An effect of melatonin on total soluble solids and lower respiration rate was observed in both cultivars. Melatonin induces overexpression of genes related to anthocyanin biosynthesis, which correlates with increased anthocyanin levels and changes in skin color (Chroma). Our results indicate that along with modulating antioxidant metabolism, melatonin improves fruit quality traits by triggering a range of metabolic and gene expression changes, which ultimately contribute to extend sweet cherry postharvest storability.

Skin Multi-Omics-Based Interactome Analysis: Integrating the Tissue and Mucus Exuded Layer for a Comprehensive Understanding of the Teleost Mucosa Functionality as Model of Study.

From a general structural perspective, a mucosal tissue is constituted by two main matrices: the tissue and the secreted mucus. Jointly, they fulfill a wide range of functions including the protection of the epithelial layer. In this study, we simultaneously analyzed the epithelial tissue and the secreted mucus response using a holistic interactome-based multi-omics approach. The effect of the gilthead sea bream (Sparus aurata) skin mucosa to a dietary inclusion of spray-dried porcine plasma (SDPP) was evaluated. The epithelial skin microarrays-based transcriptome data showed 194 differentially expressed genes, meanwhile the exuded mucus proteome analysis 35 differentially synthesized proteins. Separately, the skin transcripteractome revealed an expression profile that favored biological mechanisms associated to gene expression, biogenesis, vesicle function, protein transport and localization to the membrane. Mucus proteome showed an enhanced protective role with putatively higher antioxidant and antimicrobial properties. The integrated skin mucosa multi-interactome analysis evidenced the interrelationship and synergy between the metabolism and the exuded mucus functions improving specifically the tissue development, innate defenses, and environment recognition. Histologically, the skin increased in thickness and in number of mucous cells. A positive impact on animal performance, growth and feed efficiency was also registered. Collectively, the results suggest an intimate crosstalk between skin tissue and its exuded mucus in response to the nutritional stimulus (SDPP supplementation) that favors the stimulation of cell protein turnover and the activation of the exudation machinery in the skin mucosa. Thus, the multi-omics-based interactome analysis provides a comprehensive understanding of the biological context of response that takes place in a mucosal tissue. In perspective, this strategy is applicable for evaluating the effect of any experimental variable on any mucosal tissue functionality, including the benefits this assessment may provide on the study of the mammalian mucosa.

Effectiveness of black tea versus placebo in subjects with hypercholesterolemia: A PRISMA systematic review and meta-analysis.

AIM: To determine if the black tea is more effective in serum lipid profile that placebo in subjects with hypercholesterolemia. DESIGN: Systematic review with meta-analysis of randomized clinical trials (RCTs). DATA SOURCES: The databases Medline, Central, Embase, Lilacs, Cinahl, SPORTDiscus, and Web of Science were searched from inception up to January 2019. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: RCTs that compared black tea versus placebo, that included serum lipid profile outcomes in subjects older than 18 years of age with hypercholesterolemia. RESULTS: Seven RCTs met the eligibility criteria, and for the quantitative synthesis, six studies were included. Mean difference for total cholesterol was 1.67 mg/dl 95% CI = -5.47 to 8.80 (p = 0.65), mean difference 0.28 mg/dl, 95% CI = -3.89 to 4.45 (p = 0.90) for triglycerides, mean difference 3.21 mg/dl, 95% CI = -11.02 to 4.60 (p = 0.42) for low density lipoprotein-cholesterol, mean difference 0.38 mg/dl, 95% CI = -1.12 to 1.87 (p = 0.62) for high density lipoprotein-cholesterol. CONCLUSION: In the short term, no significant differences were found in lipid serum profile comparing black tea consumption with placebo.

Conservation of Small Regulatory RNAs in Vibrio parahaemolyticus: Possible role of RNA-OUT Encoded by the Pathogenicity Island (VPaI-7) of Pandemic Strains.

Small regulatory RNAs (sRNAs) are molecules that play an important role in the regulation of gene expression. sRNAs in bacteria can affect important processes, such as metabolism and virulence. Previous studies showed a significant role of sRNAs in the Vibrio species, but knowledge about Vibrio parahaemolyticus is limited. Here, we examined the conservation of sRNAs between V. parahaemolyticus and other human Vibrio species, in addition to investigating the conservation between V. parahaemolyticus strains differing in pandemic origin. Our results showed that only 7% of sRNAs were conserved between V. parahaemolyticus and other species, but 88% of sRNAs were highly conserved within species. Nonetheless, two sRNAs coding to RNA-OUT, a component of the Tn10/IS10 system, were exclusively present in pandemic strains. Subsequent analysis showed that both RNA-OUT were located in pathogenicity island-7 and would interact with transposase VPA1379, according to the model of pairing of IS10-encoded antisense RNAs. According to the location of RNA-OUT/VPA1379, we also investigated if they were expressed during infection. We observed that the transcriptional level of VPA1379 was significantly increased, while RNA-OUT was decreased at three hours post-infection. We suggest that IS10 transcription increases in pandemic strains during infection, probably to favor IS10 transposition and improve their fitness when they are facing adverse conditions.

Comparative Transcriptome Profiling in a Segregating Peach Population with Contrasting Juiciness Phenotypes.

Cold storage of fruit is one of the methods most commonly employed to extend the postharvest lifespan of peaches ( Prunus persica (L.) Batsch). However, fruit quality in this species is affected negatively by mealiness, a physiological disorder triggered by chilling injury after long periods of exposure to low temperatures during storage and manifested mainly as a lack of juiciness, which ultimately modifies the organoleptic properties of peach fruit. The aim of this study was to identify molecular components and metabolic processes underlying mealiness in susceptible and nonsusceptible segregants. Transcriptome and qRT-PCR profiling were applied to individuals with contrasting juiciness phenotypes in a segregating F2 population. Our results suggest that mealiness is a multiscale phenomenon, because juicy and mealy fruit display distinctive reprogramming processes affecting translational machinery and lipid, sugar, and oxidative metabolism. The candidate genes identified may be useful tools for further crop improvement.

Accessory Toxins of Vibrio Pathogens and Their Role in Epithelial Disruption During Infection.

Gastrointestinal episodes associated with Vibrio species have been rising worldwide in the last few years. Consequently, it is important to comprehend how occurs the production of diarrhea, to establish new preventive and therapeutic measures. Besides the classical CT and TCP toxins, Zot, RTX, and Ace among others have been deeply studied in V. cholerae. However, in other Vibrio species of clinical interest, where some of these toxins have been reported, there is practically no information. Zot activates a cascade of signals inside of the cell that increase the permeability of epithelial barrier, while RTX causes depolymerization of the actin cytoskeleton and Ace increases the permeability of intestinal cell monolayers. The goal of this study is to acquire information about the distribution of these toxins in human pathogenic Vibrios and to review the progress in the study of their role in the intestinal epithelium during infection.

Exploring the Genomic Traits of Non-toxigenic Vibrio parahaemolyticus Strains Isolated in Southern Chile.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. As reported in other countries, after the rise and fall of the pandemic strain in Chile, other post-pandemic strains have been associated with clinical cases, including strains lacking the major toxins TDH and TRH. Since the presence or absence of tdh and trh genes has been used for diagnostic purposes and as a proxy of the virulence of V. parahaemolyticus isolates, the understanding of virulence in V. parahaemolyticus strains lacking toxins is essential to detect these strains present in water and marine products to avoid possible food-borne infection. In this study, we characterized the genome of four environmental and two clinical non-toxigenic strains (tdh-, trh-, and T3SS2-). Using whole-genome sequencing, phylogenetic, and comparative genome analysis, we identified the core and pan-genome of V. parahaemolyticus of strains of southern Chile. The phylogenetic tree based on the core genome showed low genetic diversity but the analysis of the pan-genome revealed that all strains harbored genomic islands carrying diverse virulence and fitness factors or prophage-like elements that encode toxins like Zot and RTX. Interestingly, the three strains carrying Zot-like toxin have a different sequence, although the alignment showed some conserved areas with the zot sequence found in V. cholerae. In addition, we identified an unexpected diversity in the genetic architecture of the T3SS1 gene cluster and the presence of the T3SS2 gene cluster in a non-pandemic environmental strain. Our study sheds light on the diversity of V. parahaemolyticus strains from the southern Pacific which increases our current knowledge regarding the global diversity of this organism.

Administration of high doses of copper to capuchin monkeys does not cause liver damage but induces transcriptional activation of hepatic proliferative responses.

Liver cells respond to copper loading upregulating protective mechanisms. However, to date, except for liver content, there are no good indicators that identify individuals with excess liver copper. We hypothesized that administering high doses of copper to young (5.5 mg Cu · kg⁻¹ . d⁻¹) and adult (7.5 mg Cu · kg⁻¹ . d⁻¹) capuchin monkeys would induce detectable liver damage. Study groups included adult monkeys (2 females, 2 males) 3-3.5 y old at enrollment treated with copper for 36 mo (ACu); age-matched controls (1 female, 3 males) that did not receive additional copper (AC); young monkeys (2 female, 2 males) treated from birth with copper for 36 mo (YCu); and young age-matched controls (2 female, 2 males) that did not receive additional copper (YC). We periodically assessed clinical, blood biochemical, and liver histological indicators and at 36 mo the hepatic mRNA abundance of MT2a, APP, DMT1, CTR1, HGF, TGFß, and NFκΒ only in adult monkeys. After 36 mo, the liver copper concentration was 4-5 times greater in treated monkeys relative to controls. All monkeys remained healthy with normal routine serum biochemical indices and there was no evidence of liver tissue damage. Relative mRNA abundance of HGF, TGFß and NFκB was significantly greater in ACu than in AC monkeys. In conclusion, capuchin monkeys exposed to copper at doses up to 50 times the current upper level enhanced expression of genes related to inflammation and injury without clinical, blood biochemical, or histological evidence of liver damage.

Alteration of gene expression profile in Niemann-Pick type C mice correlates with tissue damage and oxidative stress.

BACKGROUND: Niemann-Pick type C disease (NPC) is a neurovisceral lipid storage disorder mainly characterized by unesterified cholesterol accumulation in lysosomal/late endosomal compartments, although there is also an important storage for several other kind of lipids. The main tissues affected by the disease are the liver and the cerebellum. Oxidative stress has been described in various NPC cells and tissues, such as liver and cerebellum. Although considerable alterations occur in the liver, the pathological mechanisms involved in hepatocyte damage and death have not been clearly defined. Here, we assessed hepatic tissue integrity, biochemical and oxidative stress parameters of wild-type control (Npc1(+/+); WT) and homozygous-mutant (Npc1(-/-); NPC) mice. In addition, the mRNA abundance of genes encoding proteins associated with oxidative stress, copper metabolism, fibrosis, inflammation and cholesterol metabolism were analyzed in livers and cerebella of WT and NPC mice. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed various oxidative stress parameters in the liver and hepatic and cerebellum gene expression in 7-week-old NPC1-deficient mice compared with control animals. We found signs of inflammation and fibrosis in NPC livers upon histological examination. These signs were correlated with increased levels of carbonylated proteins, diminished total glutathione content and significantly increased total copper levels in liver tissue. Finally, we analyzed liver and cerebellum gene expression patterns by qPCR and microarray assays. We found a correlation between fibrotic tissue and differential expression of hepatic as well as cerebellar genes associated with oxidative stress, fibrosis and inflammation in NPC mice. CONCLUSIONS/SIGNIFICANCE: In NPC mice, liver disease is characterized by an increase in fibrosis and in markers associated with oxidative stress. NPC is also correlated with altered gene expression, mainly of genes involved in oxidative stress and fibrosis. These findings correlate with similar parameters in cerebellum, as has been previously reported in the NPC mice model.

Genome-wide identification of new Wnt/beta-catenin target genes in the human genome using CART method.

BACKGROUND: The importance of in silico predictions for understanding cellular processes is now widely accepted, and a variety of algorithms useful for studying different biological features have been designed. In particular, the prediction of cis regulatory modules in non-coding human genome regions represents a major challenge for understanding gene regulation in several diseases. Recently, studies of the Wnt signaling pathway revealed a connection with neurodegenerative diseases such as Alzheimer's. In this article, we construct a classification tool that uses the transcription factor binding site motifs composition of some gene promoters to identify new Wnt/beta-catenin pathway target genes potentially involved in brain diseases. RESULTS: In this study, we propose 89 new Wnt/beta-catenin pathway target genes predicted in silico by using a method based on multiple Classification and Regression Tree (CART) analysis. We used as decision variables the presence of transcription factor binding site motifs in the upstream region of each gene. This prediction was validated by RT-qPCR in a sample of 9 genes. As expected, LEF1, a member of the T-cell factor/lymphoid enhancer-binding factor family (TCF/LEF1), was relevant for the classification algorithm and, remarkably, other factors related directly or indirectly to the inflammatory response and amyloidogenic processes also appeared to be relevant for the classification. Among the 89 new Wnt/beta-catenin pathway targets, we found a group expressed in brain tissue that could be involved in diverse responses to neurodegenerative diseases, like Alzheimer's disease (AD). These genes represent new candidates to protect cells against amyloid beta toxicity, in agreement with the proposed neuroprotective role of the Wnt signaling pathway. CONCLUSIONS: Our multiple CART strategy proved to be an effective tool to identify new Wnt/beta-catenin pathway targets based on the study of their regulatory regions in the human genome. In particular, several of these genes represent a new group of transcriptional dependent targets of the canonical Wnt pathway. The functions of these genes indicate that they are involved in pathophysiology related to Alzheimer's disease or other brain disorders.

Calcium/calmodulin-dependent protein kinase type IV is a target gene of the Wnt/beta-catenin signaling pathway.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a key role in the regulation of calcium-dependent gene expression. The expression of CaMKIV and the activation of CREB regulated genes are involved in memory and neuronal survival. We report here that: (a) a bioinformatic analysis of 15,476 promoters of the human genome predicted several Wnt target genes, being CaMKIV a very interesting candidate; (b) CaMKIV promoter contains TCF/LEF transcription motifs similar to those present in Wnt target genes; (c) biochemical studies indicate that lithium and the canonical ligand Wnt-3a induce CaMKIV mRNA and protein expression levels in rat hippocampal neurons as well as CaMKIV promoter activity; (d) treatment of hippocampal neurons with Wnt-3a increases the binding of beta-catenin to the CaMKIV promoter: (e) In vivo activation of the Wnt signaling improve spatial memory impairment and restores the expression of CaMKIV in a mice double transgenic model for Alzheimer's disease which shows decreased levels of the kinase. We conclude that CaMKIV is regulated by the Wnt signaling pathway and that its expression could play a role in the neuroprotective function of the Wnt signaling against the Alzheimer's amyloid peptide.