Exploring Metabolic and Gut Microbiome Responses to Paraquat Administration in Male Wistar Rats: Implications for Oxidative Stress.

In this study, we examined the metabolic and gut microbiome responses to paraquat (PQ) in male Wistar rats, focusing on oxidative stress effects. Rats received a single intraperitoneal injection of PQ at 15 and 30 mg/kg, and various oxidative stress parameters (i.e., MDA, SOD, ROS, 8-isoprostanes) were assessed after three days. To explore the omic profile, GC-qTOF and UHPLC-qTOF were performed to assess the plasma metabolome; 1H-NMR was used to assess the urine metabolome; and shotgun metagenomics sequencing was performed to study the gut microbiome. Our results revealed reductions in body weight and tissue changes, particularly in the liver, were observed, suggesting a systemic effect of PQ. Elevated lipid peroxidation and reactive oxygen species levels in the liver and plasma indicated the induction of oxidative stress. Metabolic profiling revealed changes in the tricarboxylic acid cycle, accumulation of ketone body, and altered levels of key metabolites, such as 3-hydroxybutyric acid and serine, suggesting intricate links between energy metabolism and redox reactions. Plasma metabolomic analysis revealed alterations in mitochondrial metabolism, nicotinamide metabolism, and tryptophan degradation. The gut microbiome showed shifts, with higher PQ doses influencing microbial populations (e.g., Escherichia coli and Akkermansia muciniphila) and metagenomic functions (pyruvate metabolism, fermentation, nucleotide and amino acid biosynthesis). Overall, this study provides comprehensive insights into the complex interplay between PQ exposure, metabolic responses, and gut microbiome dynamics. These findings enhance our understanding of the mechanisms behind oxidative stress-induced metabolic alterations and underscore the connections between xenobiotic exposure, gut microbiota, and host metabolism.

Gut Microbiome and Small RNA Integrative-Omic Perspective of Meconium and Milk-FED Infant Stool Samples.

The human gut microbiome plays an important role in health, and its initial development is conditioned by many factors, such as feeding. It has also been claimed that this colonization is guided by bacterial populations, the dynamic virome, and transkingdom interactions between host and microbial cells, partially mediated by epigenetic signaling. In this article, we characterized the bacteriome, virome, and smallRNome and their interaction in the meconium and stool samples from infants. Bacterial and viral DNA and RNA were extracted from the meconium and stool samples of 2- to 4-month-old milk-fed infants. The bacteriome, DNA and RNA virome, and smallRNome were assessed using 16S rRNA V4 sequencing, viral enrichment sequencing, and small RNA sequencing protocols, respectively. Data pathway analysis and integration were performed using the R package mixOmics. Our findings showed that the bacteriome differed among the three groups, while the virome and smallRNome presented significant differences, mainly between the meconium and stool of milk-fed infants. The gut environment is rapidly acquired after birth, and it is highly adaptable due to the interaction of environmental factors. Additionally, transkingdom interactions between viruses and bacteria can influence host and smallRNome profiles. However, virome characterization has several protocol limitations that must be considered.

DDR1 and Its Ligand, Collagen IV, Are Involved in In Vitro Oligodendrocyte Maturation.

Discoidin domain receptor 1 (DDR1) is a tyrosine kinase receptor expressed in epithelial cells from different tissues in which collagen binding activates pleiotropic functions. In the brain, DDR1 is mainly expressed in oligodendrocytes (OLs), the function of which is unclear. Whether collagen can activate DDR1 in OLs has not been studied. Here, we assessed the expression of DDR1 during in vitro OL differentiation, including collagen IV incubation, and the capability of collagen IV to induce DDR1 phosphorylation. Experiments were performed using two in vitro models of OL differentiation: OLs derived from adult rat neural stem cells (NSCs) and the HOG16 human oligodendroglial cell line. Immunocytofluorescence, western blotting, and ELISA were performed to analyze these questions. The differentiation of OLs from NSCs was addressed using oligodendrocyte transcription factor 2 (Olig2) and myelin basic protein (MBP). In HOG16 OLs, collagen IV induced DDR1 phosphorylation through slow and sustained kinetics. In NSC-derived OLs, DDR1 was found in a high proportion of differentiating cells (MBP+/Olig2+), but its protein expression was decreased in later stages. The addition of collagen IV did not change the number of DDR1+/MBP+ cells but did accelerate OL branching. Here, we provide the first demonstration that collagen IV mediates the phosphorylation of DDR1 in HOG16 cells and that the in vitro co-expression of DDR1 and MBP is associated with accelerated branching during the differentiation of primary OLs.

A mixture of four dietary fibres ameliorates adiposity and improves metabolic profile and intestinal health in cafeteria-fed obese rats: an integrative multi-omics approach.

The aim of this study was to assess the effects of a mixture of four dietary fibers on obese rats. Four groups of male Wistar rats were fed with either standard chow (STD) or cafeteria diet (CAF) and were orally supplemented with either fibre mixture (2 g kg-1 of body weight) (STD+F or CAF+F groups) or vehicle (STD+VH or CAF+VH groups). We studied a wide number of biometric, biochemical, transcriptomic, metagenomic and metabolomic variables and applied an integrative multivariate approach based on multiple factor analysis and Pearson's correlation analysis. A significant reduction in body weight, adiposity, HbA1c and HDL-cholesterol serum levels, and colon MPO activity was observed, whereas cecal weight and small intestine length:weight ratio were significantly increased in F-treated groups compared to control animals. CAF+F rats displayed a significant enhancement in energy expenditure, fat oxidation and fresh stool weight, and a significant reduction in adiponectin and LPS serum levels, compared to control group. Animals in STD+F group showed reduced serum LDL-cholesterol levels and a significant reduction in total cholesterol levels in the liver compared to STF+VH group. The intervention effect was reflected at the metabolomic (i.e., production of short-chain fatty acids, phenolic acids, and amino acids), metagenomic (i.e., modulation of Ruminococcus and Lactobacillus genus) and transcriptomic (i.e., expression of tight junctions and proteolysis) levels. Altogether, our integrative multi-omics approach highlights the potential of supplementation with a mixture of fibers to ameliorate the impairments triggered by obesity in terms of adiposity, metabolic profile, and intestinal health.

The Catalan Surveillance Network of SARS-CoV-2 in Sewage: design, implementation, and performance.

Wastewater-based epidemiology has shown to be an efficient tool to track the circulation of SARS-CoV-2 in communities assisted by wastewater treatment plants (WWTPs). The challenge comes when this approach is employed to help Health authorities in their decision-making. Here, we describe the roadmap for the design and deployment of SARSAIGUA, the Catalan Surveillance Network of SARS-CoV-2 in Sewage. The network monitors, weekly or biweekly, 56 WWTPs evenly distributed across the territory and serving 6 M inhabitants (80% of the Catalan population). Each week, samples from 45 WWTPs are collected, analyzed, results reported to Health authorities, and finally published within less than 72 h in an online dashboard ( https://sarsaigua.icra.cat ). After 20 months of monitoring (July 20-March 22), the standardized viral load (gene copies/day) in all the WWTPs monitored fairly matched the cumulative number of COVID-19 cases along the successive pandemic waves, showing a good fit with the diagnosed cases in the served municipalities (Spearman Rho = 0.69). Here we describe the roadmap of the design and deployment of SARSAIGUA while providing several open-access tools for the management and visualization of the surveillance data.

Imbalances in TCA, Short Fatty Acids and One-Carbon Metabolisms as Important Features of Homeostatic Disruption Evidenced by a Multi-Omics Integrative Approach of LPS-Induced Chronic Inflammation in Male Wistar Rats.

Chronic inflammation is an important risk factor in a broad variety of physical and mental disorders leading to highly prevalent non-communicable diseases (NCDs). However, there is a need for a deeper understanding of this condition and its progression to the disease state. For this reason, it is important to define metabolic pathways and complementary biomarkers associated with homeostatic disruption in chronic inflammation. To achieve that, male Wistar rats were subjected to intraperitoneal and intermittent injections with saline solution or increasing lipopolysaccharide (LPS) concentrations (0.5, 5 and 7.5 mg/kg) thrice a week for 31 days. Biochemical and inflammatory parameters were measured at the end of the study. To assess the omics profile, GC-qTOF and UHPLC-qTOF were performed to evaluate plasma metabolome; 1H-NMR was used to evaluate urine metabolome; additionally, shotgun metagenomics sequencing was carried out to characterize the cecum microbiome. The chronicity of inflammation in the study was evaluated by the monitoring of monocyte chemoattractant protein-1 (MCP-1) during the different weeks of the experimental process. At the end of the study, together with the increased levels of MCP-1, levels of interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-α) and prostaglandin E2 (PGE2) along with 8-isoprostanes (an indicative of oxidative stress) were significantly increased (p-value < 0.05). The leading features implicated in the current model were tricarboxylic acid (TCA) cycle intermediates (i.e., alpha-ketoglutarate, aconitic acid, malic acid, fumaric acid and succinic acid); lipids such as specific cholesterol esters (ChoEs), lysophospholipids (LPCs) and phosphatidylcholines (PCs); and glycine, as well as N, N-dimethylglycine, which are related to one-carbon (1C) metabolism. These metabolites point towards mitochondrial metabolism through TCA cycle, ß-oxidation of fatty acids and 1C metabolism as interconnected pathways that could reveal the metabolic effects of chronic inflammation induced by LPS administration. These results provide deeper knowledge concerning the impact of chronic inflammation on the disruption of metabolic homeostasis.

Alterations in Metabolome and Microbiome Associated with an Early Stress Stage in Male Wistar Rats: A Multi-Omics Approach.

Stress disorders have dramatically increased in recent decades becoming the most prevalent psychiatric disorder in the United States and Europe. However, the diagnosis of stress disorders is currently based on symptom checklist and psychological questionnaires, thus making the identification of candidate biomarkers necessary to gain better insights into this pathology and its related metabolic alterations. Regarding the identification of potential biomarkers, omic profiling and metabolic footprint arise as promising approaches to recognize early biochemical changes in such disease and provide opportunities for the development of integrative candidate biomarkers. Here, we studied plasma and urine metabolites together with metagenomics in a 3 days Chronic Unpredictable Mild Stress (3d CUMS) animal approach that aims to focus on the early stress period of a well-established depression model. The multi-omics integration showed a profile composed by a signature of eight plasma metabolites, six urine metabolites and five microbes. Specifically, threonic acid, malic acid, alpha-ketoglutarate, succinic acid and cholesterol were proposed as key metabolites that could serve as key potential biomarkers in plasma metabolome of early stages of stress. Such findings targeted the threonic acid metabolism and the tricarboxylic acid (TCA) cycle as important pathways in early stress. Additionally, an increase in opportunistic microbes as virus of the Herpesvirales was observed in the microbiota as an effect of the primary stress stages. Our results provide an experimental biochemical characterization of the early stage of CUMS accompanied by a subsequent omic profiling and a metabolic footprinting that provide potential candidate biomarkers.

Discoidin domain receptor 1 gene variants are associated with decreased white matter fractional anisotropy and decreased processing speed in schizophrenia.

DDR1 has been linked to schizophrenia (SZ) and myelination. Here, we tested whether DDR1 variants in people at risk for SZ influence white matter (WM) structural variations and cognitive processing speed (PS). First, following a case-control design (Study 1), SZ patients (N = 1193) and controls (N = 1839) were genotyped for rs1264323 and rs2267641 at DDR1, and the frequencies were compared. We replicated the association between DDR1 and SZ (rs1264323, adjusted P = 0.015). Carriers of the rs1264323AA combined with the rs2267641AC or CC genotype are at risk to develop SZ compared to the other genotype combinations. Second, SZ patients (Study 2, N = 194) underwent an evaluation of PS using the Trail Making Test (TMT) and DDR1 genotyping. To compare PS between DDR1 genotype groups, we conducted an analysis of covariance (including rs1264323 as a covariate) and found that SZ patients with the rs2267641CC genotype had decreased PS compared to patients with the AA and AC genotypes. Third, 54 patients (Study 3) from Study 2 were selected based on rs1264323 genotype to undergo reevaluation, including a DTI-MRI brain scan. To test for associations between PS, WM microstructure and DDR1 genotype, we first localized those WM regions where fractional anisotropy (FA) was correlated with PS and tested whether FA showed differences between the rs1264323 genotypes. SZ patients with the rs1264323AA genotype showed decreased FA in WM regions associated with decreased PS. We conclude that DDR1 variants may confer a risk of SZ through WM microstructural alterations leading to cognitive dysfunction.

Genetic and clinical evidence of mitochondrial dysfunction in autism spectrum disorder and intellectual disability.

Clinical conditions commonly associated with mitochondrial disorders (CAMDs) are often present in autism spectrum disorders (ASD) and intellectual disability (ID). Therefore, the mitochondrial dysfunction hypothesis has been proposed as a transversal mechanism that may function in both disorders. Here, we investigated the presence of conditions associated with mitochondrial disorders and mitochondrial DNA (mtDNA) alterations in 122 subjects who presented ASD with ID (ASD group), 115 subjects who presented ID but not ASD (ID group) and 112 healthy controls (HC group). We assessed in the three study groups the presence of the clinical conditions through a questionnaire and the mtDNA content of two mitochondrial genes, MT-ND1 and MT-ND4, by qPCR. The mtDNA sequences of 98 ASD and 95 ID subjects were obtained by mtDNA-targeted next generation sequencing and analysed through the MToolBox pipeline to identify mtDNA mutations. Subjects with ASD and ID showed higher frequencies of constipation, edema, seizures, vision alterations, strabismus and sphincter incontinence than HCs subjects. ASD and ID subjects showed significantly lower mtDNA content than HCs in both MT-ND1 and MT-ND4 genes. In addition, we identified 49 putative pathogenic variants with a heteroplasmy level higher than 60%: 8 missense, 29 rRNA and 12 tRNA variants. A total of 28.6% of ASD and 30.5% of ID subjects carried at least one putative pathogenic mtDNA mutation. The high frequency of CAMDs, the low mtDNA content and the presence of putative pathogenic mtDNA mutations observed in both ASD and ID subjects are evidence of mitochondrial dysfunction in ASD and ID.

Mitochondrial DNA (mtDNA) variants in the European haplogroups HV, JT, and U do not have a major role in schizophrenia.

It has been reported that certain genetic factors involved in schizophrenia could be located in the mitochondrial DNA (mtDNA). Therefore, we hypothesized that mtDNA mutations and/or variants would be present in schizophrenia patients and may be related to schizophrenia characteristics and mitochondrial function. This study was performed in three steps: (1) identification of pathogenic mutations and variants in 14 schizophrenia patients with an apparent maternal inheritance of the disease by sequencing the entire mtDNA; (2) case-control association study of 23 variants identified in step 1 (16 missense, 3 rRNA, and 4 tRNA variants) in 495 patients and 615 controls, and (3) analyses of the associated variants according to the clinical, psychopathological, and neuropsychological characteristics and according to the oxidative and enzymatic activities of the mitochondrial respiratory chain. We did not identify pathogenic mtDNA mutations in the 14 sequenced patients. Two known variants were nominally associated with schizophrenia and were further studied. The MT-RNR2 1811A > G variant likely does not play a major role in schizophrenia, as it was not associated with clinical, psychopathological, or neuropsychological variables, and the MT-ATP6 9110T > C p.Ile195Thr variant did not result in differences in the oxidative and enzymatic functions of the mitochondrial respiratory chain. The patients with apparent maternal inheritance of schizophrenia did not exhibit any mutations in their mtDNA. The variants nominally associated with schizophrenia in the present study were not related either to phenotypic characteristics or to mitochondrial function. We did not find evidence pointing to a role for mtDNA sequence variation in schizophrenia.

Mitochondrial DNA (mtDNA) in brain samples from patients with major psychiatric disorders: gene expression profiles, mtDNA content and presence of the mtDNA common deletion.

Several lines of evidence support a mitochondrial dysfunction in major psychiatric disorders. The objective of this study was to determine whether mitochondrial DNA (mtDNA) expression or content are implicated in the mitochondrial dysfunction observed in schizophrenia (SCH), bipolar disorder (BD), and major depressive disorder (MDD). MtDNA gene expression and mtDNA content (including the MT-ND4 deletion) were measured by RT-qPCR and qPCR, respectively. Post-mortem brain tissue from 60 subjects, divided evenly into four diagnostic groups (SCH, BD, MDD, and control (C)), was analyzed. MT-ND1 gene expression was significantly increased in the BD group compared with the C group. MDD and SCH patients showed a similar pattern of mtDNA expression, which was different from that in BD patients. Similarly, a larger number of MDD and SCH patients tended to have the MT-ND4 gene deleted compared with BD and C subjects. However, no other significant differences were observed in mtDNA gene expression and mtDNA content. Notably, high variability was observed in the mtDNA gene expression and content in each diagnostic group. Previous studies and the present work provide evidence for a role of mtDNA in SCH, BD and MDD. However, further studies with larger patient and control groups as well as by analyzing distinct brain regions are needed to elucidate the role of mtDNA in major psychiatric disorders.

No evidence that major mtDNA European haplogroups confer risk to schizophrenia.

Previous studies suggest that genetic factors could be involved in mitochondrial dysfunction observed in schizophrenia (SZ), some of them claiming a role of mtDNA common variants (mtSNPs) and/or haplogroups (hgs) in developing this disorder. These studies, however, have mainly been undertaken on relatively small cohorts of patients and control individuals and most have not yet been replicated. To further analyze the role of mtSNPs in SZ risk, we have carried out the largest genotyping effort to date using two Spanish case-control samples comprising a total of 942 schizophrenic patients and 1,231 unrelated controls: 454 patients and 616 controls from Santiago de Compostela (Galicia) and 488 patients and 615 controls from Reus (Catalonia). A set of 25 mtSNPs representing main branches of the European mtDNA phylogeny were genotyped in the Galician cohort and a subset of 16 out of these 25 mtSNPs was genotyped in the Catalan cohort. These 16 common variants characterize the most common European branches of the mtDNA phylogeny. We did not observe any positive association of mtSNPs and hgs with SZ. We discuss several deficiencies of previous studies that might explain the false positive nature of previous findings, including the confounding effect of population sub-structure and deficient statistical methodologies. It is unlikely that mtSNPs defining the most common European mtDNA haplogroups are related to SZ.

RT-qPCR study on post-mortem brain samples from patients with major psychiatric disorders: reference genes and specimen characteristics.

BACKGROUND: Gene expression studies conducted in post-mortem human brain samples have the potential to identify relevant genes implicated in psychiatric disorders. Although reverse transcription quantitative real-time PCR (RT-qPCR) has emerged as the method of choice for specific gene expression studies, it requires the use of stable reference genes, and it is necessary to control for pre- and post-mortem factors to obtain reliable data. OBJECTIVE: The aim of this study was to identify suitable reference genes and specimen characteristics that can be taken into account when comparing mRNA expression data between post-mortem brain specimens from psychiatric patients and controls. METHOD: We used a selection of suitably matched occipital cortex specimens from subjects in each of the following groups: schizophrenia (N = 15), bipolar disorder (N = 13), major depressive disorder (N = 15), and control (N = 15). Quantitative and qualitative RNA analyses were performed prior to RT-qPCR and gene expression stability was evaluated with geNorm and NormFinder. RESULTS: We identified GAPDH, RPS17, RPL30, RPLP0, and TFRC as potential reference genes from a sample plate containing 32 candidates commonly used as reference genes. Further analyses of these 5 genes highlighted that 1) they are suitable reference genes for RT-qPCR studies in these post-mortem brain samples from psychiatric patients, and 2) the RNA quality index is highly correlated with gene expression values (r = -0.681, p < 0.0001). CONCLUSIONS: In addition to controlling for pre- and post-mortem factors and selecting stable reference genes for normalization, sample sets should be matched with regard to RNA quality.

Association study between polymorphims in GAS6-TAM genes and carotid atherosclerosis.

Carotid atherosclerosis (CA) is one of the most common causes of stroke, and recent studies suggest that pathways initiated by the interaction of the plasma vitamin K-dependent protein GAS6 with the tyrosine kinase receptors TYRO3, AXL and MERTK (TAM) may have a relevant role in atherogenesis. Furthermore, our previous studies indicated an association between GAS6 and stroke. The aim of this study was to analyse the genetic association between SNPs and haplotypes in GAS6-TAM genes and CA. We performed a case-control study with 233 CA patients confirmed by nuclear magnetic resonance angiography and 202 patients who suffered from cardioembolic (non atherogenic) stroke. For all included subjects information on established risk factors was available. Genotyping of 16 selected tagSNPs was performed by real-time PCR, using either FRET or TaqMan probes. Adjusted logistic regression (LR) analyses indicated that rs2289743 in TYRO3 and rs869016 in MERTK were associated to CA, decreasing its risk (OR [95%CI]=0.39 [0.16-0.94] and OR [95%CI]=0.31 [0.14-0.69], respectively). Linkage disequilibrium results were consistent with the haplotype blocks described in HapMap and adjusted LR analyses revealed that the haplotype ACAA in MERTK , containing the minor allele of the associated SNP, was also associated to CA. No association was observed with GAS6 and AXL variants, which suggests that CA is not the mechanism underlying the reported association between GAS6 and stroke. The association between TYRO3 and MERTK variants and carotid atherosclerosis found in this study reinforces a physiological role of the GAS6-TAM pathway in atherogenesis.