Effects of Liquid Fructose Supplementation and Chronic Unpredictable Stress on Uterine Contractile Activity in Nonpregnant Rats.

Increased fructose consumption and chronic stress, the major characteristics of modern lifestyle, impact human health; however, the consequences of their combination on the uterus remain understudied. In this study, we investigated contractile activity, morphology, and intracellular activity of antioxidant enzymes in uteri from virgin Wistar rats subjected to liquid fructose supplementation and/or unpredictable stress over 9 weeks. Contractile activity and uterine response to oxytocin or adrenaline were examined ex vivo using isolated bath chambers. Fructose supplementation, irrespective of stress, affected uterine morphology by increasing endometrium while decreasing myometrium volume density, attenuated uterine response to increasing doses of oxytocin, and increased glutathione peroxidase activity. Stress, irrespective of fructose, attenuated dose-dependent adrenaline-induced uterine relaxation. Stress, when applied solely, decreased mitochondrial superoxide dismutase activity. In the combined treatment, irregular estrous cycles and both reduced response to oxytocin and to adrenaline (as a consequence of fructose consumption and exposure to stress), along with fructose-related alteration of uterine morphology, were detected. In conclusion, fructose and stress affect uterine contractile activity, irrespective of each other, by inducing completely distinct responses in isolated uteri. In the combined treatment, the effects of both factors were evident, suggesting that the combination exerts more detrimental effects on the uterus than each factor individually.

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.

Exosomal Prostate-Specific Membrane Antigen (PSMA) and Caveolin-1 as Potential Biomarkers of Prostate Cancer-Evidence from Serbian Population.

Prostate-specific membrane antigen (PSMA) and caveolin-1 are membrane proteins that are overexpressed in prostate cancer (PCa) and are involved in tumor growth and increase in aggressiveness. The aim of the present study is therefore to evaluate PSMA and caveolin-1 proteins from plasma exosomes as effective liquid biopsy biomarkers for PCa. This study included 39 patients with PCa and 33 with benign prostatic hyperplasia (BPH). The shape and size of the exosomes were confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis. Immunogold analysis showed that PSMA is localized to the membrane of exosomes isolated from the plasma of both groups of participants. The relative protein levels of PSMA and caveolin-1 in the plasma exosomes of PCa and BPH patients were determined by Western blot analysis. The relative level of the analyzed plasma exosomal proteins was compared between PCa and BPH patients and the relevance of the exosomal PSMA and caveoin-1 level to the clinicopathological parameters in PCa was investigated. The analysis performed showed an enrichment of exosomal PSMA in the plasma of PCa patients compared to the exosomes of men with BPH. The level of exosomal caveolin-1 in plasma was significantly higher in PCa patients with high PSA levels, clinical-stage T3 or T4 and in the group of PCa patients with aggressive PCa compared to favorable clinicopathological features or tumor aggressiveness. Plasma exosomes may serve as a suitable object for the identification of potential biomarkers for the early diagnosis and prognosis of PCa as well as carriers of therapeutic agents in precision medicine of PCa treatment.

Crocus sativus tepals extract suppresses subcutaneous adipose tissue hypertrophy and improves systemic insulin sensitivity in mice on high-fat diet.

Obesity is a pressing problem worldwide for which standard therapeutic strategies have limited effectiveness. The use of natural products seems to be a promising approach to alleviate obesity and its associated complications. The tepals of Crocus sativus (Cr) plant, usually wasted in saffron production, are an unexplored source of bioactive compounds. Our aim was to elucidate the mechanisms of Cr tepals extract in obesity by investigating its effects on adipocyte differentiation, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) hypertrophy, and lipid metabolism in an animal model of diet-induced obesity. To this end, mouse 3T3-F442A preadipocytes were treated with Cr tepals extract and the expression of adipocyte differentiation genes was determined. Caloric intake, body mass, triglycerides, systemic insulin sensitivity, histology, insulin signaling, and lipid metabolism in VAT and SAT were analyzed in mice fed a 60% fat diet for 14 weeks and treated orally with Cr tepals extract during the last 5 weeks of the diet. We demonstrated for the first time that Cr tepals extract inhibits adipocyte differentiation in vitro. The animal model confirmed that oral treatment with Cr tepals extract results in weight loss, improved systemic insulin sensitivity, lower triglycerides, and improved lipid peroxidation. The suppressive effect of Cr tepals extract on adipocyte hypertrophy and inflammation was observed only in SAT, which, together with preserved SAT insulin signaling, most likely contributed to improved systemic insulin sensitivity. Our results suggest the functionality of SAT as a possible target for the treatment of obesity and its complications.

Effects of early life overnutrition and hyperandrogenism on spatial learning and memory in a rat model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex disorder characterized by endocrine and metabolic abnormalities such as obesity and insulin resistance. PCOS is also associated with psychiatric disorders and cognitive impairment. The animal model of PCOS was induced by treating rats with 5α-dihydrotestosterone (5α-DHT) and additionally modified to induce adiposity by litter size reduction (LSR). Spatial learning and memory were assessed using the Barnes Maze test, and striatal markers of synaptic plasticity were analyzed. Striatal insulin signaling was estimated by the levels of insulin receptor substrate 1 (IRS1), its inhibitory phosphorylation at Ser307, and glycogen synthase kinase-3α/ß (GSK3α/ß) activity. Both LSR and DHT treatment significantly decreased striatal protein levels of IRS1, followed by increased GSK3α/ß activity in small litters. Results of the behavioral study showed that LSR had a negative effect on learning rate and memory retention, whereas DHT treatment did not induce impairment in memory formation. While protein levels of synaptophysin, GAP43, and postsynaptic density protein 95 (PSD-95) were not altered by the treatments, DHT treatment induced an increase in phosphorylation of PSD-95 at Ser295 in both normal and small litters. This study revealed that LSR and DHT treatment suppressed insulin signaling by downregulating IRS1 in the striatum. However, DHT treatment did not have an adverse effect on learning and memory, probably due to compensatory elevation in pPSD-95-Ser295, which had a positive effect on synaptic strength. This implies that hyperandrogenemia in this setting does not represent a threat to spatial learning and memory, opposite to the effect of overnutrition-related adiposity.

AMPK Activation as a Protective Mechanism to Restrain Oxidative Stress in the Insulin-Resistant State in Skeletal Muscle of Rat Model of PCOS Subjected to Postnatal Overfeeding.

Polycystic ovary syndrome (PCOS) is a common endocrinopathy in women of reproductive age, often associated with obesity and insulin resistance. Childhood obesity is an important predisposing factor for the development of PCOS later in life. Being particularly interested in the interplay between prepubertal obesity and hyperandrogenemia, we investigated the effects of early postnatal overfeeding, accomplished by reducing litter size during the period of suckling, on energy sensing and insulin signaling pathways in the gastrocnemius muscle of a rat model of PCOS-induced by 5α-dihydrotestosterone (DHT). The combination of overfeeding and DHT treatment caused hyperinsulinemia and decreased systemic insulin sensitivity. Early postnatal overfeeding induced defects at critical nodes of the insulin signaling pathway in skeletal muscle, which was associated with reduced glucose uptake in the presence of hyperandrogenemia. In this setting, under a combination of overfeeding and DHT treatment, skeletal muscle switched to mitochondrial ß-oxidation of fatty acids, resulting in oxidative stress and inflammation that stimulated AMP-activated protein kinase (AMPK) activity and its downstream targets involved in mitochondrial biogenesis and antioxidant protection. Overall, a combination of overfeeding and hyperandrogenemia resulted in a prooxidative and insulin-resistant state in skeletal muscle. This was accompanied by the activation of AMPK, which could represent a potential therapeutic target in insulin-resistant PCOS patients.

Oxidative Stress Linking Obesity and Cancer: Is Obesity a 'Radical Trigger' to Cancer?

Obesity is on the rise worldwide, and consequently, obesity-related non-communicable diseases are as well. Nutritional overload induces metabolic adaptations in an attempt to restore the disturbed balance, and the byproducts of the mechanisms at hand include an increased generation of reactive species. Obesity-related oxidative stress causes damage to vulnerable systems and ultimately contributes to neoplastic transformation. Dysfunctional obese adipose tissue releases cytokines and induces changes in the cell microenvironment, promoting cell survival and progression of the transformed cancer cells. Other than the increased risk of cancer development, obese cancer patients experience higher mortality rates and reduced therapy efficiency as well. The fact that obesity is considered the second leading preventable cause of cancer prioritizes the research on the mechanisms connecting obesity to cancerogenesis and finding the solutions to break the link. Oxidative stress is integral at different stages of cancer development and advancement in obese patients. Hypocaloric, balanced nutrition, and structured physical activity are some tools for relieving this burden. However, the sensitivity of simultaneously treating cancer and obesity poses a challenge. Further research on the obesity-cancer liaison would offer new perspectives on prevention programs and treatment development.

Fructose diet ameliorate effects of macrophage migration inhibitory factor deficiency on prefrontal cortex inflammation, neural plasticity, and behavior in male mice.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that represents a link between diet-induced inflammation and insulin resistance. Our aim was to examine whether fructose diet affects inflammation and insulin signaling in the prefrontal cortex (PFC) of Mif knockout mice (MIF-KO), and their possible link to neural plasticity and behavior. We analyzed nuclear factor κB (NF-κB) and glucocorticoid signaling, expression of F4/80, IL-1ß, TNF-α, TLR-4, MyD88, arginase 1 (Arg-1), mannose receptor (Mrc-1), and leukemia inhibitory factor (Lif) to assess inflammation in the PFC of C57/BL6J and MIF-KO mice consuming 20% fructose solution for 9 weeks. Insulin receptor (IR), IRS-1 serine phosphorylations (307 and 1101) and activity of PKCα, Akt, GSK-3ß and AMPKα were used to analyze insulin signaling. Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1) mRNA levels, together with synapthophysin and PSD-95 protein level and calcium calmodulin-dependent kinase 2 (CaMKII) activity, were used as plasticity markers. Behavior was examined in elevated plus maze, light dark box and novel object recognition test. The results showed concomitant increase of Tnf-α, Tlr-4, MyD88 and M2 microglia markers (Arg-1, Mrc-1, Lif) in the PFC of MIF-KO, paralleled with unchanged glucocorticoid and insulin signaling. Increase of BDNF and IGF-1 was paralleled with increased CaMKII activity, decreased PSD-95 protein level, anxiogenic behavior, and impaired memory in MIF-KO mice. Fructose feeding restored these parameters in the PFC to the control level and mitigated behavioral changes, suggesting that ameliorating effects of fructose on neuroinflammation and behavior depend on the presence of MIF.

Identification and functional interpretation of miRNAs affected by rare CNVs in CAKUT.

Rare copy number variants (CNVs) are among the most common genomic disorders underlying CAKUT. miRNAs located in rare CNVs represent well-founded functional variants for human CAKUT research. The study aimed to identify and functionally interpret miRNAs most frequently affected by rare CNVs in CAKUT and to estimate the overall burden of rare CNVs on miRNA genes in CAKUT. The additional aim of this study was to experimentally confirm the effect of a rare CNV in CAKUT on candidate miRNA's expression and the subsequent change in mRNA levels of selected target genes. A database of CAKUT-associated rare CNV regions, created by literature mining, was used for mapping of the miRNA precursors. miRNAs and miRNA families, most frequently affected by rare CAKUT-associated CNVs, have been subjected to bioinformatic analysis. CNV burden analysis was performed to identify chromosomes with over/underrepresentation of miRNA genes in rare CNVs associated with CAKUT. A functional study was performed on HEK293 MIR484+/- KO and HEK293 WT cell lines, followed by the analysis of relative miRNA and mRNA target gene levels. 80% of CAKUT patients with underlying rare CNV had at least one miRNA gene overlapping the identified CNV. Network analysis of the most frequently affected miRNAs has revealed the dominant regulation of the two miRNAs, hsa-miR-484 and hsa-miR-185-5p. Additionally, miR-548 family members have shown substantial enrichment in rare CNVs in CAKUT. An over/underrepresentation of miRNA genes in rare CNVs associated with CAKUT was observed in multiple chromosomes, such as chr16, chr20, and chr21. A significant 0.37 fold downregulation of hsa-miR-484, followed by a notable upregulation of MDM2 and APAF1 and downregulation of NOTCH3 was detected in HEK293 MIR484+/- KO compared to HEK293 WT cell lines, supporting the study hypothesis. miRNA genes are frequently affected by rare CNVs in CAKUT patients. Understanding the potential of CNV-affected miRNAs to participate in CAKUT as genetic drivers represent a crucial implication for the development of novel therapeutic approaches.

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.

AMPK Activation Is Important for the Preservation of Insulin Sensitivity in Visceral, but Not in Subcutaneous Adipose Tissue of Postnatally Overfed Rat Model of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a well-known reproductive syndrome usually associated with obesity, insulin resistance, and hyperinsulinemia. Although the first signs of PCOS begin early in adolescence, it is underexplored whether peripubertal obesity predisposes women to PCOS metabolic disturbances. To highlight that, we examined the impact of postnatal overfeeding-induced obesity, achieved by litter size reduction during the suckling period, on metabolic disturbances associated with visceral and subcutaneous adipose tissue (VAT and SAT) function in the 5α-dihydrotestosterone (5α-DHT)-induced animal model of PCOS. We analyzed markers of insulin signaling, lipid metabolism, and energy sensing in the VAT and SAT. Our results showed that postnatally overfed DHT-treated Wistar rats had increased VAT mass with hypertrophic adipocytes, together with hyperinsulinemia and increased HOMA index. In the VAT of these animals, insulin signaling remained unchanged while lipogenic markers decreased, which was accompanied by increased AMPK activation. In the SAT of the same animals, markers of lipogenesis and lipolysis increased, while the activity of AMPK decreased. Taken together, obtained results showed that postnatal overfeeding predisposes development of PCOS systemic insulin resistance, most likely as a result of worsened metabolic function of SAT, while VAT preserved its tissue insulin sensitivity through increased activity of AMPK.

Effects of Fructose and Stress on Rat Renal Copper Metabolism and Antioxidant Enzymes Function.

The effects of a fructose-rich diet and chronic stress on copper metabolism in the kidneys are still understudied. We investigated whether fructose and/or chronic unpredictable stress modulate copper metabolism in a way that affects redox homeostasis, thus contributing to progression of metabolic disturbances in the kidney. We determined protein level of copper transporters, chaperones, and cuproenzymes including cytochrome c oxidase, as well as antioxidant enzymes function in the kidneys of male Wistar rats subjected to 20% liquid fructose supplementation and/or chronic stress. Liquid fructose supplementation increased level of copper chaperone of superoxide dismutase and decreased metallothionein level, while rendering the level of copper importer and copper chaperones involved in copper delivery to mitochondria and trans Golgi network unaffected. Stress had no effect on renal copper metabolism. The activity and expression of renal antioxidant enzymes remained unaltered in all experimental groups. In conclusion, fructose, independently of stress, decreased renal copper level, and modulated renal copper metabolism as to preserve vital cellular function including mitochondrial energy production and antioxidative defense, at the expense of intracellular copper storage.

Combination of chronic stress with fructose diet increases AMP-activated protein kinase phosphorylation and affects agouti-related protein and proopiomelanocortin expression in the hypothalamus of male Wistar rats.

Appetite regulation in the hypothalamus is dependent on hormonal signals from the periphery, such as insulin and leptin, and can be modulated by both sugar-rich diet and stress. Our aim was to explore the effects of 9-week feeding with 20% fructose solution combined with 4-week chronic unpredictable stress, on appetite-regulating neuropeptides and modulatory role of leptin and insulin signalling in the hypothalamus of male Wistar rats. Energy intake, body mass and adiposity, as well as circulatory leptin and insulin concentrations were assessed. Hypothalamic insulin signalling was analysed at the level of glucose transporters, as well as at the protein level and phosphorylation of insulin receptor, insulin receptor supstrate-1, Akt and ERK. Phosphorylation of AMP-activated protein kinase (AMPK), level of protein tyrosine phosphatase 1B (PTP1B) and expression of leptin receptor (ObRb) and suppressor of cytokine signalling 3 (SOCS3) were also analysed, together with the expression of orexigenic agouti-related protein (AgRP) and anorexigenic proopiomelanocortin (POMC) neuropeptides. The results revealed that stress decreased body mass and adiposity, blood leptin level and expression of ObRb, SOCS3 and POMC, while combination with fructose diet led to marked increase of AgRP, associated with AMPK phosphorylation despite increased plasma insulin. Reduced Akt, enhanced ERK activity and elevated PTP1B were also observed in the hypothalamus of these animals. In conclusion, our results showed that joint effects of fructose diet and stress are more deleterious than the separate ones, since inappropriate appetite control in the hypothalamus may provide a setting for the disturbed energy homeostasis in the long run.

Obesity-related prepartal insulin resistance in dairy cows is associated with increased lipin 1 and decreased FATP 1 expression in skeletal muscle.

A number of alterations have been identified in lipid metabolism within adipose tissue and liver in obesity. Less is known about the capacity of skeletal muscle for the metabolism of fatty acids in obesity-related insulin resistance, though it is evident that dry cow muscles may contain increased triglyceride content. The current study was therefore undertaken to evaluate the skeletal muscle expression of proteins of the fatty acid metabolism in dry cows with different body condition scores (BCS). Sixteen Holstein-Friesian close-up cows were divided into 2 equal groups based on their BCS as optimal (3.25 ≤ BCS ≤ 3.5) and high (4.0 ≤ BCS ≤ 4.25). Blood samples collection and skeletal muscle biopsies were carried out at day 10 before calving. Blood serum was assayed for concentration of resistin using a bovine specific ELISA. Protein expression of insulin receptor beta subunit (IRß), glucose transporter 4 (GLUT4), fatty acid translocase (FAT/CD36), fatty acid transporter 1 (FATP1), carnitine palmitoyltransferase 1 (CPT1), AMP-acitvated protein kinase (AMPK) and lipin 1 were analyzed in semitendinosus muscle by immunoblot. Resistin differed non-significantly between high-BCS and optimal-BCS cows. Insulin-resistant lipid metabolism in obese cows was paralleled with increased skeletal muscle expression of lipin 1 and GLUT4, and decreased expression of IRß and FATP1. These data suggest that in obesity-related insulin resistance, metabolic capacity in dry cow skeletal muscles appears to be organized towards the synthesis of signaling intermediates rather than fatty acids oxidation and that altered fatty acid uptake does not contribute to this disposition.

Late-Onset Calorie Restriction Improves Lipid Metabolism and Aggravates Inflammation in the Liver of Old Wistar Rats.

Aging is a progressive process that could disturb metabolic homeostasis in the liver via ectopic lipid accumulation, oxidative stress, and deterioration of inflammatory response. Although calorie restriction (CR) is recognized as beneficial for life span and health span prolongation, it is still unclear how late-onset CR, characterized by late beginning and short duration, affects age-related processes. The aim of this study was to examine how late-onset CR-induced metabolic adjustments impact lipid status and inflammation in the liver of old rats. The experiments were conducted on aging male Wistar rats fed ad libitum (AL) or exposed to late-onset CR (60% of AL daily intake) from 21st to 24th month. The results showed that late-onset CR reduces body weight, visceral adipose tissue and liver mass, and triglyceride levels when compared to old animals on AL diet. The ameliorating effects of CR on lipid metabolism include increased activity of AMP-activated protein kinase, suppressed de novo fatty acid synthesis, stimulated ß-oxidation, decreased lipotoxicity, and limited triglyceride synthesis and packaging in the liver. Restricted diet regime, however, does not improve expression of antioxidant enzymes, although it leads to progression of age-related inflammation in the liver, partially through lower corticosterone concentration and decreased activation of glucocorticoid receptor. In conclusion, late-onset CR is able to restore age-related imbalance of lipid metabolism in the liver, but has a negative impact on hepatic inflammatory status, implying that the type of diet for older individuals must be balanced and chosen carefully with appropriate duration and start point.

Wastewater-based epidemiology in countries with poor wastewater treatment - Epidemiological indicator function of SARS-CoV-2 RNA in surface waters.

Wastewater-based epidemiology (WBE) surveillance of COVID-19 and other future outbreaks is a challenge for developing countries as most households are not connected to a sewerage system. In December 2019, SARS-CoV-2 RNA was detected in the Danube River at a site severely affected by wastewaters from Belgrade. Rivers are much more complex systems than wastewater systems, and efforts are needed to address all the factors influencing the adoption of WBE as an alternative to targeting raw wastewater. Our objective was to provide a more detailed insight into the potential of SARS-CoV-2 surveillance in Serbian surface waters for epidemiological purposes. Water samples were collected at 12 sites along the Sava and Danube rivers in Belgrade during the fourth COVID-19 wave in Serbia that started in late February 2021. RNA was concentrated using Amicon Ultra-15 centrifugal filters and quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. Microbiological (faecal indicator bacteria and human and animal genetic faecal source tracking markers), epidemiological, physicochemical and hydromorphological parameters were analysed in parallel. From 44 samples, SARS-CoV-2 RNA was detected in 31, but only at 4 concentrations above the level of quantification (ranging from 8.47 × 103 to 2.07 × 104 gc/L). The results indicated that surveillance of SARS-CoV-2 RNA in surface waters as ultimate recipients could be used as an epidemiological early-warning tool in countries lacking wastewater treatment and proper sewerage infrastructure. The performance of the applied approach, including advanced sampling site characterization to trace and identify sites with significant raw sewage influence from human populations, could be further improved by adaptation of the methodology for processing higher volumes of samples and enrichment factors, which should provide the quantitative instead of qualitative data needed for WBE.

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.

Variants Tagging LGALS-3 Haplotype Block in Association with First Myocardial Infarction and Plasma Galectin-3 Six Months after the Acute Event.

Galectin-3 is encoded by LGALS-3, located in a unique haplotype block in Caucasians. According to the Tagger server, rs4040064, rs11628437, and rs7159490 cover 82% (r2 > 0.8) of the genetic variance of this HapBlock. Our aims were to examine the association of their haplotypes with first myocardial infarction (MI), changes in left ventricular echocardiographic parameters over time, and impact on plasma galectin-3 and LGALS-3 mRNA in peripheral blood mononuclear cells, both 6 months post-MI. The study group consisted of 546 MI patients and 323 controls. Gene expression was assessed in 92 patients and plasma galectin-3 in 189 patients. Rs4040064, rs11628437, rs7159490, and LGALS-3 mRNA expression were detected using TaqMan® technology. Plasma galectin-3 concentrations were determined by the ELISA method. We found that the TGC haplotype could have a protective effect against MI (adjusted OR 0.19 [0.05-0.72], p = 0.015) and that the GAC haplotype had significantly higher galectin-3 concentrations (48.3 [37.3-59.4] ng/mL vs. 18.9 [14.5-23.4] ng/mL, p < 0.0001), both in males and compared to the referent haplotype GGC. Higher plasma Gal-3 was also associated with higher NYHA class and systolic dysfunction. Our results suggest that variants tagging LGALS-3 HapBlock could reflect plasma Gal-3 levels 6 months post-MI and may have a potential protective effect against MI in men. Further replication, validation, and functional studies are needed.

Fructose Induces Visceral Adipose Tissue Inflammation and Insulin Resistance Even Without Development of Obesity in Adult Female but Not in Male Rats.

Introduction: Obesity and related metabolic disturbances are frequently related to modern lifestyle and are characterized by excessive fructose intake. Visceral adipose tissue (VAT) inflammation has a central role in the development of insulin resistance, type 2 diabetes (T2D), and metabolic syndrome. Since sex-related differences in susceptibility and progression of metabolic disorders are not yet fully understood, our aim was to examine inflammation and insulin signaling in VAT of fructose-fed female and male adult rats. Methods: We analyzed effects of 9-week 10% fructose-enriched diet on energy intake, VAT mass and histology, and systemic insulin sensitivity. VAT insulin signaling and markers of VAT inflammation, and antioxidative defense status were also evaluated. Results: The fructose diet had no effect on VAT mass and systemic insulin signaling in the female and male rats, while it raised plasma uric acid, increased PPARγ level in the VAT, and initiated the development of a distinctive population of small adipocytes in the females. Also, adipose tissue insulin resistance, evidenced by increased PTP1B and insulin receptor substrate 1 (IRS1) inhibitory phosphorylation and decreased Akt activity, was detected. In addition, fructose stimulated the nuclear accumulation of NFκB, increased expression of proinflammatory cytokines (IL-1ß, IL-6, and TNFα), and protein level of macrophage marker F4/80, superoxide dismutase 1, and glutathione reductase. In contrast to the females, the fructose diet had no effect on plasma uric acid and VAT inflammation in the male rats, but less prominent alterations in VAT insulin signaling were observed. Conclusion: Even though dietary fructose did not elicit changes in energy intake and led to obesity in the females, it initiated the proliferation of small-sized adipocytes capable of storing fats further. In contrast to the males, this state of VAT was accompanied with enhanced inflammation, which most likely contributed to the development of insulin resistance. The observed distinction could possibly originate from sex-related differences in uric acid metabolism. Our results suggest that VAT inflammation could precede obesity and start even before the measurable increase in VAT mass, making it a silent risk factor for the development of T2D. Our results emphasize that adipose tissue dysfunction, rather than its simple enlargement, could significantly contribute to the onset and development of obesity and related metabolic disorders.

Fructose-Rich Diet Attenuates Stress-Induced Metabolic Disturbances in the Liver of Adult Female Rats.

BACKGROUND: Both fructose consumption and chronic stress contribute to the development of metabolic disorders. The consequences of such combination are not fully understood. OBJECTIVE: We investigated whether fructose supplementation and chronic stress synergistically disturb hepatic lipid and glucose metabolism. The role of energy sensing, redox, and inflammatory status during development of metabolic disturbances was investigated. METHODS: Female Wistar rats, aged 2.5 mo, were divided into 4 experimental groups: control (C) fed a standard diet (commercial food and drinking water); fructose (F) fed the same food and 10% fructose solution; stress (S) fed the standard diet and subjected to chronic unpredictable stress and, stress + fructose (SF) combining conditions F and S as above. Stress included daily stressors: cold water forced swimming, physical restraint, cold room, wet bedding, rocking, switching, or tilting cages. After 9 wk, hepatic enzymes and transcription factors involved in gluconeogenesis, lipogenesis, fatty acid oxidation, antioxidative defence, energy sensing, and cytokines were assessed by qPCR, Western blotting, and spectrophotometry and analyzed by 2-factor ANOVA. RESULTS: Fructose increased AMP-activated protein kinase (AMPK) phosphorylation (40%; P < 0.05) and the ratio of inhibitory phosphorylation to total acetyl-CoA carboxylase (46%; P < 0.01), and decreased sterol regulatory element binding protein 1c nuclear translocation by 30% (P < 0.05) in F and SF compared with C rats. Increased phosPck (phoenolpyruvate carboxykinase) (85%) and G6pase(glucose-6-phosphatase) (55%) was observed in S rats (P < 0.05). A 40% decrease in Apob (apolipoprotein B-100) and an increase in hepatic lipids (P < 0.05), together with a double increase in TNF-α (P < 0.001), were observed in S rats, but without liver histopathological changes. These stress effects on lipid accumulation and TNF-α were abolished in SF rats (P < 0.05). CONCLUSIONS: Fructose does not enhance stress effects on hepatic lipid and glucose metabolism but attenuates its effects on hepatic lipid accumulation and inflammation, suggesting that, in female rats, AMPK activation prevails over stress-induced effects.