Genome-Wide DNA Methylation in Policemen Working in Cities Differing by Major Sources of Air Pollution

DNA methylation is the most studied epigenetic mechanism that regulates gene expression, and it can serve as a useful biomarker of prior environmental exposure and future health outcomes. This study focused on DNA methylation profiles in a human cohort, comprising 125 nonsmoking city policemen (sampled twice), living and working in three localities (Prague, Ostrava and Ceske Budejovice) of the Czech Republic, who spent the majority of their working time outdoors. The main characterization of the localities, differing by major sources of air pollution, was defined by the stationary air pollution monitoring of PM2.5, B[a]P and NO₂. DNA methylation was analyzed by a genome-wide microarray method. No season-specific DNA methylation pattern was discovered; however, we identified 13,643 differentially methylated CpG loci (DML) for a comparison between the Prague and Ostrava groups. The most significant DML was cg10123377 (log₂FC = −1.92, p = 8.30 × 10⁻⁴) and loci annotated to RPTOR (total 20 CpG loci). We also found two hypomethylated loci annotated to the DNA repair gene XRCC5. Groups of DML annotated to the same gene were linked to diabetes mellitus (KCNQ1), respiratory diseases (PTPRN2), the dopaminergic system of the brain and neurodegenerative diseases (NR4A2). The most significant possibly affected pathway was Axon guidance, with 86 potentially deregulated genes near DML. The cluster of gene sets that could be affected by DNA methylation in the Ostrava groups mainly includes the neuronal functions and biological processes of cell junctions and adhesion assembly. The study demonstrates that the differences in the type of air pollution between localities can affect a unique change in DNA methylation profiles across the human genome.     

Age-Related DNA Methylation in Normal Kidney Tissue Identifies Epigenetic Cancer Risk Susceptibility Loci in the ANKRD34B and ZIC1 Genes

Both age-dependent and age-independent alteration of DNA methylation in human tissues are functionally associated with the development of many malignant and non-malignant human diseases. TCGA-KIRC data were biometrically analyzed to identify new loci with age-dependent DNA methylation that may contribute to tumor risk in normal kidney tissue. ANKRD34B and ZIC1 were evaluated as candidate genes by pyrosequencing of 539 tissues, including 239 normal autopsy, 157 histopathologically tumor-adjacent normal, and 143 paired tumor kidney samples. All candidate CpG loci demonstrated a strong correlation between relative methylation levels and age (R = 0.70–0.88, p < 2 × 10⁻¹⁶) and seven out of 10 loci were capable of predicting chronological age in normal kidney tissues, explaining 84% of the variance (R = 0.92). Moreover, significantly increased age-independent methylation was found for 9 out of 10 CpG loci in tumor-adjacent tissues, compared to normal autopsy tissues (p = 0.001–0.028). Comparing tumor and paired tumor-adjacent tissues revealed two patient clusters showing hypermethylation, one cluster without significant changes in methylation, and a smaller cluster demonstrating hypomethylation in the tumors (p < 1 × 10⁻¹⁰). Taken together, our results show the presence of additional methylation risk factors besides age for renal cancer in normal kidney tissue. Concurrent tumor-specific hypermethylation suggests a subset of these loci are candidates for epigenetic renal cancer susceptibility.     

Implication of N-Methyl-d-Aspartate Receptor in Homocysteine-Induced Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a leading cause of vision loss. Elevated homocysteine (Hcy) (Hyperhomocysteinemia) (HHcy) has been reported in AMD. We previously reported that HHcy induces AMD-like features. This study suggests that N-Methyl-d-aspartate receptor (NMDAR) activation in the retinal pigment epithelium (RPE) is a mechanism for HHcy-induced AMD. Serum Hcy and cystathionine-β-synthase (CBS) were assessed by ELISA. The involvement of NMDAR in Hcy-induced AMD features was evaluated (1) in vitro using ARPE-19 cells, primary RPE isolated from HHcy mice (CBS), and mouse choroidal endothelial cells (MCEC); (2) in vivo using wild-type mice and mice deficient in RPE NMDAR (NMDAR^R−/−) with/without Hcy injection. Isolectin-B4, Ki67, HIF-1α, VEGF, NMDAR1, and albumin were assessed by immunofluorescence (IF), Western blot (WB), Optical coherence tomography (OCT), and fluorescein angiography (FA) to evaluate retinal structure, fluorescein leakage, and choroidal neovascularization (CNV). A neovascular AMD patient’s serum showed a significant increase in Hcy and a decrease in CBS. Hcy significantly increased HIF-1α, VEGF, and NMDAR in RPE cells, and Ki67 in MCEC. Hcy-injected WT mice showed disrupted retina and CNV. Knocking down RPE NMDAR improved retinal structure and CNV. Our findings underscore the role of RPE NMDAR in Hcy-induced AMD features; thus, NMDAR inhibition could serve as a promising therapeutic target for AMD.     

KIT Expression Is Regulated by DNA Methylation in Uveal Melanoma Tumors

Uveal melanoma (UM) is an ocular tumor with a dismal prognosis. Despite the availability of precise molecular and cytogenetic techniques, clinicopathologic features with limited accuracy are widely used to predict metastatic potential. In 51 UM tissues, we assessed a correlation between the expression of nine proteins evaluated by immunohistochemistry (IHC) (Melan-A, S100, HMB45, Cyclin D1, Ki-67, p53, KIT, BCL2, and AIFM1) and the presence of UM-specific chromosomal rearrangements measured by multiplex ligation-dependent probe amplification (MLPA), to find IHC markers with increased prognostic information. Furthermore, mRNA expression and DNA methylation values were extracted from the whole-genome data, achieved by analyzing 22 fresh frozen UM tissues. KIT positivity was associated with monosomy 3, increasing the risk of poor prognosis more than 17-fold (95% CI 1.53–198.69, p = 0.021). A strong negative correlation was identified between mRNA expression and DNA methylation values for 12 of 20 analyzed positions, five located in regulatory regions of the KIT gene (r = −0.658, p = 0.001; r = −0.662, p = 0.001; r = −0.816; p < 0.001; r = −0.689, p = 0.001; r = −0.809, p < 0.001, respectively). DNA methylation β values were also inversely associated with KIT protein expression (p = 0.001; p = 0.001; p = 0.015; p = 0.025; p = 0.002). Our findings, showing epigenetic deregulation of KIT expression, may contribute to understanding the past failure to therapeutically target KIT in UM.     

Energy Metabolites as Biomarkers in Ischemic and Dilated Cardiomyopathy

With more than 25 million people affected, heart failure (HF) is a global threat. As energy production pathways are known to play a pivotal role in HF, we sought here to identify key metabolic changes in ischemic- and non-ischemic HF by using a multi-OMICS approach. Serum metabolites and mRNAseq and epigenetic DNA methylation profiles were analyzed from blood and left ventricular heart biopsy specimens of the same individuals. In total we collected serum from n = 82 patients with Dilated Cardiomyopathy (DCM) and n = 51 controls in the screening stage. We identified several metabolites involved in glycolysis and citric acid cycle to be elevated up to 5.7-fold in DCM (p = 1.7 × 10⁻⁶). Interestingly, cardiac mRNA and epigenetic changes of genes encoding rate-limiting enzymes of these pathways could also be found and validated in our second stage of metabolite assessment in n = 52 DCM, n = 39 ischemic HF and n = 57 controls. In conclusion, we identified a new set of metabolomic biomarkers for HF. We were able to identify underlying biological cascades that potentially represent suitable intervention targets.     

BDNF rs6265 Polymorphism and Its Methylation in Patients with Stroke Undergoing Rehabilitation

Brain-Derived Neurotrophic Factor (BDNF) and its rs6265 single nucleotide polymorphism (SNP) play an important role in post-stroke recovery. We investigated the correlation between BDNF rs6265 SNP and recovery outcome, measured by the modified Barthel index, in 49 patients with stroke hospitalized in our rehabilitation center at baseline (T0) and after 30 sessions of rehabilitation treatment (T1); moreover, we analyzed the methylation level of the CpG site created or abolished into BDNF rs6265 SNP. In total, 11 patients (22.4%) were heterozygous GA, and 32 (65.3%) and 6 (12.2%) patients were homozygous GG and AA, respectively. The univariate analysis showed a significant relationship between the BDNF rs6265 SNP and the modified Barthel index cut-off (χ²(1, N = 48) = 3.86, p = 0.049), considering patients divided for carrying (A+) or not carrying (A−) the A allele. A higher percentage of A− patients obtained a favorable outcome, as showed by the logistic regression model corrected by age and time since the stroke onset, compared with the A+ patients (OR: 5.59). At baseline (T0), the percentage of BDNF methylation was significantly different between GG (44.6 ± 1.1%), GA (39.5 ± 2.8%) and AA (28.5 ± 1.7%) alleles (p < 0.001). After rehabilitation (T1), only patients A− showed a significant increase in methylation percentages (mean change = 1.3, CI: 0.4–2.2, p = 0.007). This preliminary study deserves more investigation to confirm if BDNF rs6265 SNP and its methylation could be used as a biological marker of recovery in patients with stroke undergoing rehabilitation treatment.     

Retinal Degeneration and Microglial Dynamics in Mature Progranulin-Deficient Mice

Progranulin (PGRN) is a secreted glycoprotein that regulates numerous cellular processes. The role of PGRN as a regulator of lysosomes has recently received attention. The purpose of this study was to characterize the retinal phenotype in mature PGRN knockout (Grn^−/−) mice. The a-wave amplitude of scotopic electroretinogram and outer nuclear thickness were significantly reduced at 6 months of age in Grn^−/− mice compared to wild-type (Grn^+/+) mice. In Grn^−/− mice, retinal microglial cells accumulated on the retinal pigment epithelium (RPE) apical layer, and the number of infiltrated microglia and white fundus lesions between 2 and 6 months of age showed a close affinity. In Grn^+/+ mice, PGRN was located in the retina, while the strongest PGRN signals were detected in the RPE-choroid. The different effects of PGRN deficiency on the expression of lysosomal proteins between the retina and RPE-choroid were demonstrated. Our data suggest that the subretinal translocation of microglia is a characteristic phenotype in the retina of mature PGRN knockout mice. The different effects of PGRN deficiency on the expression of lysosomal proteins between the retina and RPE-choroid might modulate microglial dynamics in PGRN knockout mice.     

Minocycline Counteracts Ectopic Calcification in a Murine Model of Pseudoxanthoma Elasticum: A Proof-of-Concept Study

Pseudoxanthoma elasticum (PXE) is an intractable Mendelian disease characterized by ectopic calcification in skin, eyes and blood vessels. Recently, increased activation of the DNA damage response (DDR) was shown to be involved in PXE pathogenesis, while the DDR/PARP1 inhibitor minocycline was found to attenuate aberrant mineralization in PXE cells and zebrafish. In this proof-of-concept study, we evaluated the anticalcifying properties of minocycline in Abcc6^−/− mice, an established mammalian PXE model. Abcc6^−/− mice received oral minocycline supplementation (40 mg/kg/day) from 12 to 36 weeks of age and were compared to untreated Abcc6^−/− and Abcc6^+/+ siblings. Ectopic calcification was evaluated using X-ray microtomography with three-dimensional reconstruction of calcium deposits in muzzle skin and Yasue’s calcium staining. Immunohistochemistry for the key DDR marker H2AX was also performed. Following minocycline treatment, ectopic calcification in Abcc6^−/− mice was significantly reduced (−43.4%, p < 0.0001) compared to untreated Abcc6^−/− littermates. H2AX immunostaining revealed activation of the DDR at sites of aberrant mineralization in untreated Abcc6^−/− animals. In conclusion, we validated the anticalcifying effect of minocycline in Abcc6^−/− mice for the first time. Considering its favorable safety profile in humans and low cost as a generic drug, minocycline may be a promising therapeutic compound for PXE patients.     

Conditional Loss of the Exocyst Component Exoc5 in Retinal Pigment Epithelium (RPE) Results in RPE Dysfunction,Photoreceptor Cell Degeneration,and Decreased Visual Function

To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5^−/− zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5^−/− mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.     

Dysfunctional cGMP Signaling Leads to Age-Related Retinal Vascular Alterations and Astrocyte Remodeling in Mice

The nitric oxide–guanylyl cyclase-1–cyclic guanylate monophosphate (NO–GC-1–cGMP) pathway is integral to the control of vascular tone and morphology. Mice lacking the alpha catalytic domain of guanylate cyclase (GC1^−/−) develop retinal ganglion cell (RGC) degeneration with age, with only modest fluctuations in intraocular pressure (IOP). Increasing the bioavailability of cGMP in GC1^−/− mice prevents neurodegeneration independently of IOP, suggesting alternative mechanisms of retinal neurodegeneration. In continuation to these studies, we explored the hypothesis that dysfunctional cGMP signaling leads to changes in the neurovascular unit that may contribute to RGC degeneration. We assessed retinal vasculature and astrocyte morphology in young and aged GC1^−/− and wild type mice. GC1^−/− mice exhibit increased peripheral retinal vessel dilation and shorter retinal vessel branching with increasing age compared to Wt mice. Astrocyte cell morphology is aberrant, and glial fibrillary acidic protein (GFAP) density is increased in young and aged GC1^−/− mice, with areas of dense astrocyte matting around blood vessels. Our results suggest that proper cGMP signaling is essential to retinal vessel morphology with increasing age. Vascular changed are preceded by alterations in astrocyte morphology which may together contribute to retinal neurodegeneration and loss of visual acuity observed in GC1^−/− mice.     

Identifying Genetic Biomarkers Predicting Response to Anti-Vascular Endothelial Growth Factor Injections in Diabetic Macular Edema

Intraocular anti-vascular endothelial growth factor (VEGF) therapies are the front-line treatment for diabetic macular edema (DME); however, treatment response varies widely. This study aimed to identify genetic determinants associated with anti-VEGF treatment response in DME. We performed a genome-wide association study on 220 Australian patients with DME treated with anti-VEGF therapy, genotyped on the Illumina Global Screening Array, and imputed to the Haplotype Reference Consortium panel. The primary outcome measures were changes in central macular thickness (CMT in microns) and best-corrected visual acuity (BCVA in ETDRS letters) after 12 months. Association between single nucleotide polymorphism (SNP) genotypes and DME outcomes were evaluated by linear regression, adjusting for the first three principal components, age, baseline CMT/BCVA, duration of diabetic retinopathy, and HbA1c. Two loci reached genome-wide significance (p < 5 × 10⁻⁸) for association with increased CMT: a single SNP on chromosome 6 near CASC15 (rs78466540, p = 1.16 × 10⁻⁹) and a locus on chromosome 12 near RP11-116D17.1 (top SNP rs11614480, p = 2.69 × 10⁻⁸). Four loci were significantly associated with reduction in BCVA: two loci on chromosome 11, downstream of NTM (top SNP rs148980760, p = 5.30 × 10⁻⁹) and intronic in RP11-744N12.3 (top SNP rs57801753, p = 1.71 × 10⁻⁸); one near PGAM1P1 on chromosome 5 (rs187876551, p = 1.52 × 10⁻⁸); and one near TBC1D32 on chromosome 6 (rs118074968, p = 4.94 × 10⁻⁸). In silico investigations of each locus identified multiple expression quantitative trait loci and potentially relevant candidate genes warranting further analysis. Thus, we identified multiple genetic loci predicting treatment outcomes for anti-VEGF therapies in DME. This work may potentially lead to managing DME using personalized treatment approaches.     

Collagen Family and Other Matrix Remodeling Proteins Identified by Bioinformatics Analysis as Hub Genes Involved in Gastric Cancer Progression and Prognosis

Gastric cancer has remained in the top five cancers for over ten years, both in terms of incidence and mortality due to the shortage of biomarkers for disease follow-up and effective therapies. Aiming to fill this gap, we performed a bioinformatics assessment on our data and two additional GEO microarray profiles, followed by a deep analysis of the 40 differentially expressed genes identified. PPI network analysis and MCODE plug-in pointed out nine upregulated hub genes coding for proteins from the collagen family (COL12A1, COL5A2, and COL10A1) or involved in the assembly (BGN) or degradation of collagens (CTHRC1), and also associated with cell adhesion (THBS2 and SPP1) and extracellular matrix degradation (FAP, SULF1). Those genes were highly upregulated at the mRNA and protein level, the increase being correlated with pathological T stages. The high expression of BGN (p = 8 × 10⁻¹²), THBS2 (p = 1.2 × 10⁻⁶), CTHRC1 (p = 1.1 × 10⁻⁴), SULF1 (p = 3.8 × 10⁻⁴), COL5A1 (p = 1.3 × 10⁻⁴), COL10A1 (p = 5.7 × 10⁻⁴), COL12A1 (p = 2 × 10⁻³) correlated with poor overall survival and an immune infiltrate based especially on immunosuppressive M2 macrophages (p-value range 4.82 × 10⁻⁷–1.63 × 10⁻¹³). Our results emphasize that these genes could be candidate biomarkers for GC progression and prognosis and new therapeutic targets.     

Genetic Susceptibility to Periodontal Disease in Down Syndrome: A Case-Control Study

Severe periodontitis is prevalent in Down syndrome (DS). This study aimed to identify genetic variations associated with periodontitis in individuals with DS. The study group was distributed into DS patients with periodontitis (n = 50) and DS patients with healthy periodontium (n = 36). All samples were genotyped with the “Axiom Spanish Biobank” array, which contains 757,836 markers. An association analysis at the individual marker level using logistic regression, as well as at the gene level applying the sequence kernel association test (SKAT) was performed. The most significant genes were included in a pathway analysis using the free DAVID software. C12orf74 (rs4315121, p = 9.85 × 10⁻⁵, OR = 8.84), LOC101930064 (rs4814890, p = 9.61 × 10⁻⁵, OR = 0.13), KBTBD12 (rs1549874, p = 8.27 × 10⁻⁵, OR = 0.08), PIWIL1 (rs11060842, p = 7.82 × 10⁻⁵, OR = 9.05) and C16orf82 (rs62030877, p = 8.92 × 10⁻⁵, OR = 0.14) showed a higher probability in the individual analysis. The analysis at the gene level highlighted PIWIL, MIR9-2, LHCGR, TPR and BCR. At the signaling pathway level, PI3K-Akt, long-term depression and FoxO achieved nominal significance (p = 1.3 × 10⁻², p = 5.1 × 10⁻³, p = 1.2 × 10⁻², respectively). In summary, various metabolic pathways are involved in the pathogenesis of periodontitis in DS, including PI3K-Akt, which regulates cell proliferation and inflammatory response.     

Molecular Cloning and Characterization of the Calcineurin Subunit A from Plutella xylostella

Calcineurin (or PP2B) has been reported to be involved in an array of physiological process in insects, and the calcineurin subunit A (CNA) plays a central role in calcineurin activity. We cloned the CNA gene from Plutella xylostella (PxCNA). This gene contains an ORF of 1488 bp that encodes a 495 amino acid protein, showing 98%, and 80% identities to the CNA of Bombyx mori, and humans respectively. The full-length of PxCNA and its catalytic domain (CNA_1–341, defined as PxCNα) were both expressed in Escherichia coli. Purified recombinant PxCNA displayed no phosphatase activity, whereas recombinant PxCNα showed high phosphatase activity with a Km of 4.6 mM and a kcat of 0.66 S⁻¹ against pNPP. It could be activated at different degrees by Mn²⁺, Ni²⁺, Mg²⁺, and Ca²⁺. The optimum reaction pH was about 7.5 and the optimum reaction temperature was around 45 °C. An in vitro inhibition assay showed that okadaic acid (OA) and cantharidin (CTD) competitively inhibited recombinant PxCNα activity with the IC₅₀ values of 8.95 μM and 77.64 μM, respectively. However, unlike previous reports, pyrethroid insecticides were unable to inhibit recombinant PxCNα, indicating that the P. xylostella calcineurin appears not to be sensitive to class II pyrethroid insecticides.     

Global Changes of 5-mC/5h-mC Ratio and Methylation of Adiponectin and Leptin Gene in Placenta Depending on Mode of Delivery

It was suggested that the epigenetic alterations of the placenta are associated with obesity, as well as the delivery mode. This study aimed to assess the effect of maternal outcome and delivery procedure on global placental DNA methylation status, as well as selected 5’-Cytosine-phosphate-Guanine-3’ (CpG) sites in ADIPOQ and LEP genes. Global DNA methylation profile in the placenta was assessed using the 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) ratio evaluated with the ELISA, followed by target gene methylation patterns at selected gene regions which were determined using methylation-specific qPCR in 70 placentas from healthy, pregnant women with single pregnancy. We found no statistically significant differences in 5-mC/5-hmC ratio between intrapartum cesarean sections (CS) and vaginal deliveries (p = 0.214), as well as between elective cesarean sections and vaginal deliveries (p = 0.221). In intrapartum cesarean sections, the ADIPOQ demethylation index was significantly higher (the average: 1.75) compared to elective cesarean section (the average: 1.23, p = 0.010) and vaginal deliveries (the average: 1.23, p = 0.011). The LEP demethylation index did not significantly differ among elective CS, intrapartum CS, and vaginal delivery groups. The demethylation index of ADIPOQ correlated negatively with LEP in the placenta in the vaginal delivery group (r = −0.456, p = 0.017), but not with the global methylation. The methylation of a singular locus might be different depending on the mode of delivery and uterine contractions. Further studies should be conducted with locus-specific analysis of the whole genome to detect the methylation index of specific genes involved in metabolism.     

Deep Transcranial Magnetic Stimulation Affects Gut Microbiota Composition in Obesity: Results of Randomized Clinical Trial

Growing evidence highlights the crucial role of gut microbiota in affecting different aspects of obesity. Considering the ability of deep transcranial magnetic stimulation (dTMS) to modulate the cortical excitability, the reward system, and, indirectly, the autonomic nervous system (ANS), we hypothesized a potential role of dTMS in affecting the brain-gut communication pathways, and the gut microbiota composition in obesity. In a hospital setting, 22 subjects with obesity (5 M, 17 F; 44.9 ± 2.2 years; BMI 37.5 ± 1.0 kg/m²) were randomized into three groups receiving 15 sessions (3 per week for 5 weeks) of high frequency (HF), low frequency (LF) dTMS, or sham stimulation. Fecal samples were collected at baseline and after 5 weeks of treatment. Total bacterial DNA was extracted from fecal samples using the QIAamp DNA Stool Mini Kit (Qiagen, Italy) and analyzed by a metagenomics approach (Ion Torrent Personal Genome Machine). After 5 weeks, a significant weight loss was found in HF (HF: −4.1 ± 0.8%, LF: −1.9 ± 0.8%, sham: −1.3 ± 0.6%, p = 0.042) compared to LF and sham groups, associated with a decrease in norepinephrine compared to baseline (HF: −61.5 ± 15.2%, p < 0.01; LF: −31.8 ± 17.1%, p < 0.05; sham: −35.8 ± 21.0%, p > 0.05). Furthermore, an increase in Faecalibacterium (+154.3% vs. baseline, p < 0.05) and Alistipes (+153.4% vs. baseline, p < 0.05) genera, and a significant decrease in Lactobacillus (−77.1% vs. baseline, p < 0.05) were found in HF. Faecalibacterium variations were not significant compared to baseline in the other two groups (LF: +106.6%, sham: +27.6%; p > 0.05) as well as Alistipes (LF: −54.9%, sham: −15.1%; p > 0.05) and Lactobacillus (LF: −26.0%, sham: +228.3%; p > 0.05) variations. Norepinephrine change significantly correlated with Bacteroides (r² = 0.734; p < 0.05), Eubacterium (r² = 0.734; p < 0.05), and Parasutterella (r² = 0.618; p < 0.05) abundance variations in HF. In conclusion, HF dTMS treatment revealed to be effective in modulating gut microbiota composition in subjects with obesity, reversing obesity-associated microbiota variations, and promoting bacterial species representative of healthy subjects with anti-inflammatory properties.     

Intronic Polymorphisms in the CDKN2B-AS1 Gene Are Strongly Associated with the Risk of Myocardial Infarction and Coronary Artery Disease in the Saudi Population

Recent genome-wide association studies identified single nucleotide polymorphisms (SNPs) on the chromosome 9p21.3 conferring the risk for CAD (coronary artery disease) in individuals of Caucasian ancestry. We performed a genetic association study to investigate the effect of 12 candidate SNPs within 9p21.3 locus on the risk of CAD in the Saudi population of the Eastern Province of Saudi Arabia. A total of 250 Saudi CAD patients who had experienced an myocardial infarction (MI) and 252 Saudi age-matched healthy controls were genotyped using TaqMan assay. Controls with evidenced lack of CAD provided 90% of statistical power at the type I error rate of 0.05. Five percent of the results were rechecked for quality control using Sanger sequencing, the results of which concurred with the TaqMan genotyping results. Association analysis of 12 SNPs indicated a significant difference in the genotype distribution for four SNPs between cases and controls (rs564398 p = 0.0315, χ² = 4.6, odds ratio (OD) = 1.5; rs4977574 p = 0.0336, χ² = 4.5, OD = 1.4; rs2891168 p = 1.85 × 10 − 10, χ² = 40.6, OD = 2.1 and rs1333042 p = 5.14 × 10 − 9, χ² = 34.1, OD = 2.2). The study identified three protective haplotypes (TAAG p = 1.00 × 10 − 4; AGTA p = 0.022 and GGGCC p = 0.0175) and a risk haplotype (TGGA p = 2.86 × 10 − 10) for the development of CAD. This study is in line with others that indicated that the SNPs located in the intronic region of the CDKN2B-AS1 gene are associated with CAD.     

Acetylsalicylic Acid Reduces Passive Aortic Wall Stiffness and Cardiovascular Remodelling in a Mouse Model of Advanced Atherosclerosis

Acetylsalicylic acid (ASA) is widely used in secondary prevention of cardiovascular (CV) disease, mainly because of its antithrombotic effects. Here, we investigated whether ASA can prevent the progression of vessel wall remodelling, atherosclerosis, and CV complications in apolipoprotein E deficient (ApoE^−/−) mice, a model of stable atherosclerosis, and in ApoE^−/− mice with a mutation in the fibrillin-1 gene (Fbn1^C1039G+/−), which is a model of elastic fibre fragmentation, accompanied by exacerbated unstable atherosclerosis. Female ApoE^−/− and ApoE^−/−Fbn1^C1039G+/− mice were fed a Western diet (WD). At 10 weeks of WD, the mice were randomly divided into four groups, receiving either ASA 5 mg/kg/day in the drinking water (ApoE^−/− (n = 14), ApoE^−/−Fbn1^C1039G+/− (n = 19)) or plain drinking water (ApoE^−/− (n = 15), ApoE^−/−Fbn1^C1039G+/− (n = 21)) for 15 weeks. ApoE^−/−Fbn1^C1039G+/− mice showed an increased neutrophil–lymphocyte ratio (NLR) compared to ApoE^−/− mice, and this effect was normalised by ASA. In the proximal ascending aorta wall, ASA-treated ApoE^−/−Fbn1^C1039G+/− mice showed less p-SMAD2/3 positive nuclei, a lower collagen percentage and an increased elastin/collagen ratio, consistent with the values measured in ApoE^−/− mice. ASA did not affect plaque progression, incidence of myocardial infarction and survival of ApoE^−/−Fbn1^C1039G+/− mice, but systolic blood pressure, cardiac fibrosis and hypertrophy were reduced. In conclusion, ASA normalises the NLR, passive wall stiffness and cardiac remodelling in ApoE^−/−Fbn1^C1039G+/− mice to levels observed in ApoE^−/− mice, indicating additional therapeutic benefits of ASA beyond its classical use.