Targeted RNAseq Revealed the Gene Expression Signature of Ferroptosis-Related Processes Associated with Disease Severity in Patients with Multiple Sclerosis.

Detrimental molecular processes in multiple sclerosis (MS) lead to the cellular accumulation of lipid peroxidation products and iron in the CNS, which represents the main driving force for ferroptosis. Ferroptosis is an iron-dependent form of regulated cell death, with proposed roles in neurodegeneration, oligodendrocyte loss and neuroinflammation in the pathogenesis of MS. Ferroptosis-related gene expression signature and molecular markers, which could reflect MS severity and progression, are currently understudied in humans. To tackle these challenges, we have applied a curated approach to create and experimentally analyze a comprehensive panel of ferroptosis-related genes covering a wide range of biological processes associated with ferroptosis. We performed the first ferroptosis-related targeted RNAseq on PBMCs from highly distinctive MS phenotype groups: mild relapsing-remitting (RR) (n = 24) and severe secondary progressive (SP) (n = 24), along with protein detection of GPX4 and products of lipid peroxidation (MDA and 4-HNE). Out of 138 genes, 26 were differentially expressed genes (DEGs), indicating changes in both pro- and anti-ferroptotic genes, representing a molecular signature associated with MS severity. The top three DEGs, as non-core ferroptosis genes, CDKN1A, MAP1B and EGLN2, were replicated by qPCR to validate findings in independent patient groups (16 RR and 16 SP MS). Co-expression and interactions of DEGs were presented as additional valuable assets for deeper understanding of molecular mechanisms and key targets related to MS severity. Our study integrates a wide genetic signature and biochemical markers related to ferroptosis in easily obtainable PBMCs of MS patients with clinical data and disease severity, thus providing novel molecular markers which can complement disease-related changes in the brain and undergo further research as potential therapeutic targets.

Essential trace element levels in multiple sclerosis: Bridging demographic and clinical gaps, assessing the need for supplementation.

BACKGROUND: Multiple sclerosis (MS) is a chronic demyelinating disorder intricately linked to perturbations in trace element levels. While previous studies have explored circulating trace elements in a limited sample, understanding the impact of demographic and clinical variables on the elemental profile within a larger cohort remains elusive. METHODS: This study aimed to evaluate essential trace elements (Cr, Mn, Co, Cu, Zn, and Se) in the sera of 215 MS patients compared to a meticulously matched control group of 100 individuals with similar gender and age. Our main objective was to identify potential variations in elemental profiles based on demographic and clinical parameters among MS patients, elucidating the prospective relevance of supplementing specific essential trace elements. RESULTS: Data indicated a significant decrease in serum levels of Mn, Co, Zn, and Se, and an increase in Cr in MS patients compared to controls. These trace elements not only discriminated between MS patients and controls but also exhibited distinctive capabilities among demographic subgroups. Gender, smoking habits, and age strata (20-40 years and 41-60 years) revealed discernible variations in elemental profiles between MS patients and their control counterparts. Se demonstrated the singular ability to stratify cases of extreme MS severity, mild relapsing-remitting MS (RRMS) and highly severe secondary progressive MS (SPMS). In contrast, Co significantly differentiated RRMS from primary progressive MS (PPMS), while Cu significantly differentiated SPMS from PPMS. Additionally, Cu showed a negative correlation with MSSS, while Mn and Zn showed a positive correlation with EDSS. CONCLUSION: These findings underscore a substantive deficiency in Mn, Co, Zn, and Se in the MS cohort, supporting targeted supplementation with these trace elements. This study provides a comprehensive understanding of the intricate relationship between essential trace elements and MS, paving the way for further research into personalized nutritional interventions for this complex neurological disorder.

Association of MMP1 and MMP3 haplotypes with myocardial infarction and echocardiographic parameters of the left ventricle.

BACKGROUND: Myocardial infarction (MI) leads to ischemia and afterward to left ventricular (LV) remodeling. Matrix metalloproteinase-1 (MMP1) and -3 (MMP3) belong to the family of endopeptidases and together they can dissolve most of the components of the extracellular matrix. MMP1 and MMP3 variants have been investigated solely in association with ischemic heart disease and LV dysfunction, but not in haplotype. The aims of this study were to investigate the association of haplotypes inferred from MMP1 rs1799750 (-1607 1G/2G; NC_000011.9:g.102670497del) and MMP3 rs35068180 (-1612 5A/6A; NC_000011.9:g.102715952dup) with MI and their effect on the change in echocardiographic parameters of LV structure and function in patients within 6 months after MI. METHODS: The study included 325 patients with the first MI and 283 healthy controls. Gene variants were detected by PCR-RFLP method. Parameters of LV structure and function were assessed by conventional 2D echocardiography, 3-5 days and 6 months after the first MI, on a subgroup of 160 patients. Haplotype analysis was performed with Thesias software. RESULTS: Haplotypes 2G-5A and 1G-6A were significantly and independently associated with MI compared with the reference haplotype 2G-6A (adjusted, p = 0.009 and p = 0.026, respectively). After Bonferroni correction for multiple testing, MMP1 and MMP3 haplotypes lost their association with the change in LV long diameter and stroke volume within 6 months after MI. CONCLUSION: MMP1 and MMP3 haplotypes are strongly associated with MI. Further studies are needed to validate this result and to examine their association with echocardiographic parameters of LV structure and function after MI.

Tag Variants of LGALS-3 Containing Haplotype Block in Advanced Carotid Atherosclerosis.

OBJECTIVES: Galectin-3 affects a variety of biological processes. It is encoded by LGALS-3, located in unique haplotype block in Caucasians. Most of the studies regarding the gal-3 role in atherosclerosis are focused exclusively on protein/mRNA levels. Genetic analyses of LGALS-3 are scarce. We sought to thoroughly examine the genetic background of gal-3 and to analyze tag variants that cover more than 80% variability of the LGALS-3 containing hap-block in association with carotid plaque presence (CPP). According to Tagger server, rs4040064 G/T, rs11628437 G/A and rs7159490 C/T cover 82% (r2 > 0.8) of the genetic variance of this hap-block. Our aims were to investigate possible association of rs4040064, rs11628437 and rs7159490 haplotypes with CPP in patients with advanced carotid atherosclerosis (CA) and to analyze their possible effect on LGALS-3 mRNA expression in carotid plaques. MATERIALS AND METHODS: Study group consisted of 468 patients and 296 controls. Rs4040064, rs11628437, rs7159490 and LGALS-3 mRNA expression were detected by TaqMan® technology. RESULTS: We have found that haplotype TAC was associated with the cerebrovascular insult (CVI) occurrence (OR = 1.68, 95% CI = 1.09-2.58, p = 0.02), compared to the referent haplotype. OR was adjusted for hypertension, age and BMI. TAC also showed higher, but not statistically significant, LGALS-3 expression in carotid plaques. CONCLUSIONS: Our results suggest that rs4040064, rs11628437 and rs7159490 bear no association with CPP, neither they affect LGALS-3 mRNA in carotid plaques. However, we showed a significant association of haplotype TAC with the CVI occurrence in CA patients from Serbia. Replication and validation of our results are required.

Association of PHACTR1 intronic variants with the first myocardial infarction and their effect on PHACTR1 mRNA expression in PBMCs.

BACKGROUND: Myocardial infarction (MI) and underlining atherosclerosis are the main causes of death worldwide. Phosphatase and actin regulator 1 (PHACTR1) variants have been associated with early onset MI, coronary artery disease and carotid dissection. PHACTR1 mRNA expression has been detected in tissues and cells related to atherosclerosis. Nonetheless, the true effect of PHACTR1 on vascular diseases is still unknown. Our aim was to examine the association of PHACTR1 intronic variants, rs9349379, rs2026458 and rs2876300, with MI and multi-vessel disease (MVD) and to assess their effect on PHACTR1 and EDN1 mRNA expression in PBMCs of patients six months after MI. METHODS: The study enrolled 537 patients with the first MI and 310 controls. Gene expression was assessed in 74 patients six months after MI and 37 healthy controls. Rs9349379, rs2026458, rs2876300 and relative mRNA expressions were detected by TaqMan® technology. RESULTS: The significant association between PHACTR1 variants and MI was not found, either individually or in haplotype. A higher frequency of rs2876300G-allele in MVD was rendered not significant after Bonferroni correction. PHACTR1 mRNA was significantly increased in PBMCs of patients six months after MI compared to controls (p = 0.02). Patients that carry ACG haplotype have increased PHACTR1 mRNA expression in PBMCs (p = 0.04). There was no effect of PHACTR1 variants on EDN1 mRNA expression. CONCLUSION: Our findings suggest that PHACTR1 intronic variants may have a role in severity and progression of coronary atherosclerosis. Future research is needed to clarify the mechanism underlying the role of PHACTR1 in coronary atherosclerosis and MI.

The Effects of Aronia melanocarpa Juice Consumption on the mRNA Expression Profile in Peripheral Blood Mononuclear Cells in Subjects at Cardiovascular Risk.

Foods and food products that contain polyphenols are proposed to modulate risk of cardiovascular disease. The aim of this three-arm, crossover, randomized, double-blind, placebo-controlled intervention study was to examine the impact of Aronia melanocarpa juice (AMJ), high-polyphenol (AMJ treatment, 1.17 g/100 mL polyphenols) and low-polyphenol (dAMJ treatment, 0.29 g/100 mL polyphenols) dose, on the transcriptome in peripheral blood mononuclear cells (PBMC) of 19 subjects at cardiovascular risk. Transcriptome data were obtained by microarray. Bioinformatic functional annotation analysis was performed on both the whole transcriptome datasets and the differentially expressed genes (DEGs). Expression of selected DEGs was validated by RT-qPCR. Administration of AMJ and dAMJ treatments during the two consecutive four-week treatment periods had additive effects on PBMC transcriptome profiles, with the most pronounced and specific effect noticed for AMJ in the last treatment period (TP3) of the trial. Between the high-dose and low-dose treatments in TP3, there was a multitude of overlapping DEGs and DEG-enriched biological processes and pathways, which primarily included immunomodulation and regulation of cell proliferation/death. Increased expression of TNF, IL1B, IL8, RGS1, OSM, and DUSP2 in TP3 was confirmed by RT-qPCR. The results suggest the immunomodulatory effects of prolonged habitual consumption of polyphenol-rich aronia juice in individuals at cardiovascular risk.

PHACTR1 haplotypes are associated with carotid plaque presence and affect PHACTR1 mRNA expression in carotid plaque tissue.

BACKGROUND: Carotid plaque is a hallmark of advanced carotid atherosclerosis and there is evidence of phosphatase and actin regulator 1 (PHACTR1) involvement in the processes that lead to atherosclerosis. PHACTR1 intronic variants have been associated with coronary artery disease and carotid dissection. Up to date the PHACTR1 haplotypes were not investigated in association with carotid plaque presence (CPP). So, the aims of this study were to investigate possible association of PHACTR1 haplotypes inferred from the intronic variants rs9349379, rs2026458 and rs2876300 with CPP in patients with advanced carotid atherosclerosis and to analyze their possible effect on PHACTR1 relative mRNA expression in carotid plaque tissue specimens. METHODS: The study group consisted of 501 patients with evidence of carotid plaque presence admitted for carotid endarterectomy and 310 healthy controls. PHACTR1 rs9349379, rs2026458, rs2876300 and relative mRNA expression were detected by TaqMan® technology. RESULTS: We have found significant and independent association of haplotype ACA with the CPP, compared to the referent haplotype GTA (adjusted OR = 1.54 95% CI = 1.07-2.21, p = 0.02). The OR was adjusted for gender, age, BMI, hypertension and total cholesterol. The relative expression of PHACTR1 mRNA in carotid plaque tissue proved to be significantly higher in carriers of the ACG haplotype compared to the referent haplotype GTA (p = 0.03). CONCLUSION: Our results suggest that PHACTR1 haplotypes inferred from the variants rs9349379, rs2026458 and rs2876300 affect PHACTR1 mRNA and bear the risk for CPP in patients with advanced carotid atherosclerosis. Further replication and validation studies are inevitable.

Polymorphisms in Toll-like receptors 2 and 4 genes and their expression in chronic suppurative otitis media.

OBJECTIVE: Toll-like receptors (TLRs) have a prominent role in inducing innate immune response. It has been suggested that regulation of TLRs is involved in the pathogenesis of chronic otitis media. TLR 2 and TLR 4 polymorphisms were connected with susceptibility to acute otitis and chronic otitis with effusion. The objective of this study was to establish expression of TLR 2 and 4 on middle ear mucosa in different types of chronic suppurative otitis media (CSOM), and the influence of gene polymorphisms TLR 2 Arg753Gln and TLR 4 Thr399Ile and Asp299Gly to susceptibility to CSOM. MATERIAL AND METHODS: Middle ear mucosa and full blood samples were obtained from 85 patients with chronic suppurative otitis media with and without cholesteatoma. Control group for mucosal TLR expression consisted of 71 samples of middle ear mucosa taken from patients with otosclerosis, and control group for DNA polymorphism consisted of 100 full blood samples in healthy subjects. DNA polymorphism detection was done with restriction fragment length polymorphism in RT PCR. Expression of TLR 2 and 4 was determined with immunohistochemical staining. RESULTS: TLR 2 and TLR 4 expression on the middle ear mucosa was not influenced by age of the patients with chronic otitis media. Incidence of TLR 2 Arg753Gln polymorphism was significantly higher in patients with chronic otitis media, compared to control group. Significant association between TLR 2 Arg753Gln polymorphism and different types of mucosal changes in patients with chronic otitis media was established. TLR 2 and 4 expression on experimental group mucosa was significantly different compared to control group, where there was no expression (p=0.000). Strong dependence of TLR 2 and TLR 4 expression on middle ear mucosa with different mucosal changes and immunohistochemical activity after staining was detected. CONCLUSION: Certain polymorphisms in TLR genes could be indicative for susceptibility to chronic otitis media. Expression of TLR 2 and 4 on middle ear mucosa was more dependable on different types of mucosal changes and type of CSOM than on bacteria found in the specimens. This can indicate that the type of mucosal changes are closely correlated with TLRs activity in middle ear.