Metagenomic Investigation of the Short-Term Temporal and Spatial Dynamics of the Bacterial Microbiome and the Resistome Downstream of a Wastewater Treatment Plant in the Iskar River in Bulgaria.

Waste Water Treatment Plants (WWTP) aim to reduce contamination in effluent water; however, studies indicate antimicrobial resistance genes (ARGs) persist post-treatment, potentially leading to their spread from human populated areas into the environment. This study evaluated the impact of a large WWTP serving 125,000 people on the Iskar River in Bulgaria, by characterizing the spatial and short-term temporal dynamics in bacterial community dynamics and resistance profiles of the surface water. Pairs of samples were collected biweekly on four dates from two different locations, one about 800 m after the WWTP effluents and the other 10 km downstream. Taxonomic classification revealed the dominance of Pseudomonodota and Bacteriodota, notably the genera Flavobacterium, Aquirufa, Acidovorax, Polynucleobacter, and Limnohabitans. The taxonomic structure corresponded with both lentic and lotic freshwater habitats, with Flavobacterium exhibiting a significant decrease over the study period. Principal Coordinate Analysis revealed statistically significant differences in bacterial community composition between samples collected on different dates. Differential abundance analysis identified notable enrichment of Polynucleobacter and Limnohabitans. There were shifts within the enriched or depleted bacterial taxa between early and late sampling dates. High relative abundance of the genes erm(B), erm(F), mph(E), msr(E) (macrolides); tet(C), tet(O), tet(W), tet(Q) and tet(X) (tetracyclines); sul1 and sul2 (sulphonamides); and cfxA3, cfxA6 (beta-lactams) were detected, with trends of increased presence in the latest sampling dates and in the location closer to the WWTP. Of note, genes conferring resistance to carbapenems blaOXA-58 and blaIMP-33-like were identified. Co-occurrence analysis of ARGs and mobile genetic elements on putative plasmids showed few instances, and the estimated human health risk score (0.19) according to MetaCompare2.0 was low. In total, 29 metagenome-assembled genomes were recovered, with only a few harbouring ARGs. This study enhances our understanding of freshwater microbial community dynamics and antibiotic resistance profiles, highlighting the need for continued ARGs monitoring.

Prevalence of Irritable Bowel Syndrome, Functional Dyspepsia and their Overlap in Bulgaria: a Population-Based Study.

AIMS: We aimed to evaluate the prevalence of irritable bowel syndrome (IBS), functional dyspepsia (FD), and their overlap syndrome (OS) in the Bulgarian population and to assess the risk factors associated with these disorders. METHODS: We sent an internet-based survey to Bulgarian adults. The survey collected data on socio- demographic, behavioral and lifestyle characteristics, and diagnostic questions following the Rome IV criteria to assess IBS, FD and their overlap occurrence. RESULTS: Data was collected from 1,896 individuals (mean age = 35.5 years, 18-65, SD=11.7), 73.1% females. The prevalence of IBS was 20% (14% were with predominant constipation, 32% with predominant diarrhea, 52% had IBS with mixed bowel habits, and 2% unclassified IBS). Gender (p=0.005), age (p<0.001), marital status (p=0.009), occupation (p=0.001), alcohol consumption (p=0.013), sexual problems (p<0.001), FD (p<0.001), and milk intolerance (p<0.001) were significantly associated with IBS. Females (p=0.032; OR: 1.50), patients with FD (p<0.001; OR: 104.98), sexual problems (p= 0.001; ОR: 1.55 ), and milk intolerance (p<0.001; OR: 2.22) are at a higher risk of having IBS. The prevalence of FD was 12.7% (39% had postprandial distress syndrome, 33% epigastric pain syndrome, and 28% had the overlapping variant). Patients with IBS (p<0.001; OR: 127.88) and milk intolerance (p<0.001) were significantly associated with FD prevalence. The prevalence of OS was 11.7%. Gender (p=0.013), milk intolerance (p<0.001, OR: 1.65), urinary (p=0.035) and sexual problems (p<0.001, ОR: 1.80) were associated with OS prevalence. CONCLUSION: This is the first study to estimate the prevalence of IBS, FD, and their OS and assess the behavioral and demographic risk factors associated with these disorders in the Bulgarian population. Our results are valuable in filling in the epidemiological data gap regarding IBS, FD, and OS in Eastern Europe.

The evaluation of anthropogenic impact on the ecological stability of landscape.

The model area is the northern surrounding of the water reservoir Zemplinska Irava in the east of Slovakia. Selection of the examined territory and the time horizons was not random. The aim was to capture the intensity level of anthropogenic impact on the values of the coefficient of ecological stability after the construction of water reservoir, Zempifnska Irava. The contribution evaluates ecological stability of landscape in the years 1956 and 2009 by GIS technology, using two methods. The first method determines the rate of ecological stability of landscape on the basis of the significance of land cover classes in the regular network of squares (the real size of the square is 0.5 square km). The second method determines the ecological stability of landscape secondary on the basis of the man influence on the landscape. A comparison of two methods has been made, as well as interpretation of the output data (e.g., monitoring the impact of marginal land cover classes with the minimal surfaces in the grid of square at the fluctuation of the index of ecological stability, respectively, it considers the possibilities to streamline the research results using homogeneous spatial units) and it also allows to track the changes in the ecological stability of the landscape in chronological development.

Quercetin improves postischemic recovery of heart function in doxorubicin-treated rats and prevents doxorubicin-induced matrix metalloproteinase-2 activation and apoptosis induction.

Quercetin (QCT) is flavonoid that possesses various biological functions including anti-oxidative and radical-scavenging activities. Moreover, QCT exerts some preventive actions in treatment of cardiovascular diseases. The aim of present study was to explore effects of prolonged administration of QCT on changes induced by repeated application of doxorubicin (DOX) in rat hearts. We focused on the ultrastructure of myocardium, matrix metalloproteinases (MMPs), biometric parameters, and apoptosis induction. Our aim was also to examine effects of QCT on ischemic tolerance in hearts exposed to chronic effects of DOX, and to determine possible mechanisms underlying effects of QCT. Our results showed that QCT prevented several negative chronic effects of DOX: (I) reversed DOX-induced blood pressure increase; (II) mediated improvement of deleterious effects of DOX on ultrastructure of left ventricle; (III) prevented DOX-induced effects on tissue MMP-2 activation; and (iv) reversed effects of DOX on apoptosis induction and superoxide dismutase inhibition. Moreover, we showed that rat hearts exposed to effects of QCT were more resistant to ischemia/reperfusion injury. Effects of QCT on modulation of ischemic tolerance were linked to Akt kinase activation and connexin-43 up-regulation. Taken together, these results demonstrate that prolonged treatment with QCT prevented negative chronic effects of DOX on blood pressure, cellular damage, MMP-2 activation, and apoptosis induction. Moreover, QCT influenced myocardial responses to acute ischemic stress. These facts bring new insights into mechanisms of QCT action on rat hearts exposed to the chronic effects of DOX.

Activity profiles of soccer players during the 2010 world cup.

The main objective of this study was to analyse the distance covered and the activity profile that players presented at the FIFA World Cup in 2010. Complementarily, the distance covered by each team within the same competition was analysed. For the purposes of this study 443 players were analysed, of which 35 were goalkeepers, 84 were external defenders, 77 were central defenders, 182 were midfielders, and 65 were forwards. Afterwards, a thorough analysis was performed on 16 teams that reached the group stage, 8 teams that achieved the round of 16, 4 teams that reached the quarter-finals, and 4 teams that qualified for the semi-finals and finals. A comparison of the mean distance covered per minute among the playing positions showed statistically significant differences (F(4,438) = 559.283; p < 0.001; 2 = 0.836; Power = 1.00). A comparison of the activity time among tactical positions also resulted in statistically significant differences, specifically, low activity (F(4,183.371) = 1476.844; p < 0.001; 2 = 0.742; Power = 1.00), medium activity (F(4,183.370) = 1408.106; p < 0.001; 2 = 0.731; Power = 1.00), and high activity (F(4,182.861) = 1152.508; p < 0.001; 2 = 0.703; Power = 1.00). Comparing the mean distance covered by teams, differences that are not statistically significant were observed (F(3,9.651) = 4.337; p < 0.035; 2 = 0.206; Power = 0.541). In conclusion, the tactical positions of the players and their specific tasks influence the activity profile and physical demands during a match.

Chronic cardiotoxicity of doxorubicin involves activation of myocardial and circulating matrix metalloproteinases in rats.

AIM: To investigate the role of matrix metalloproteinases (MMPs) in the responses of rats to a prolonged doxorubicin (DOX) treatment. METHODS: Male Wistar rats were used. DOX was administered by intraperitoneal injections of seven doses (cumulative dose was 15 mg/kg). Control animals were treated with saline. Tissue or plasma samples were collected at four and eight weeks after the application of the last dose. Protein levels were determined by immunoblot assay, and MMP activities were measured by gelatin zymography. Superoxide content was analyzed using a lucigenin chemiluminescence assay and superoxide dismutase (SOD) activities with a SOD assay kit. Qualitative structural alterations of the heart were characterized by transmission electron microscopy. RESULTS: Systolic blood pressure was higher in DOX-treated rats as compared with the control rats at 8 weeks after treatment. In contrast, there were no differences in the heart rate between the control and DOX-treated rats. DOX treatment caused marked heterogeneous subcellular alterations of cardiomyocytes and structural disorganizations of the cardiac extracellular space. The effects of DOX were linked to a stimulation of plasma MMP-2 and MMP-9 activities that had already increased by 4 weeks after the end of the treatment. In the left ventricle, however, DOX only led to increased MMP-2 activation at 8 weeks after the end of treatment. These changes in tissue MMP-2 were connected with stimulation of Akt kinase activation, inhibition of SOD, an increase in superoxide levels, induction of iNOS protein expression and caspase-3 activation. CONCLUSION: Our results show that MMPs are involved in the chronic cardiotoxicity of DOX in rats. The data also suggest that reactive oxygen species (superoxide), NO production (iNOS) and the Akt kinase pathway can modulate MMP-2 activities in rat hearts influenced by DOX.

Activation of Akt kinase accompanies increased cardiac resistance to ischemia/reperfusion in rats after short-term feeding with lard-based high-fat diet and increased sucrose intake.

High-fat or high-carbohydrate food consumption contributes to changes in myocardial tolerance to ischemia. However, with respect to experimental models, most studies used diets with very high doses of cholesterol, saturated fatty acids, or fructose. In our study, we fed rats a high-fat diet based on lard in combination with administration of a sweet beverage (30% sucrose solution) (high-fat sucrose diet [HFS]). This diet was used to simulate the unhealthy dietary habit typical for developed countries. We hypothesized that the application of HFS diet for 48 days might initiate progression of pathologic changes in the heart associated with myocardial remodeling and activation of adaptive mechanisms. We investigated the influence of HFS diet on cardiac function and vulnerability to ischemia-reperfusion (I/R) injury in Langendorff-perfused rat hearts subjected to 30-minute global ischemia and 120-minute reperfusion as well as on Akt kinase and matrix metalloproteinases. We found lower food consumption in HFS group compared with controls, but a significant increase in visceral fat mass and concentrations of triacylglycerol, low-density lipoprotein, and very low-density lipoprotein cholesterol. Baseline heart functional parameters and their postischemic recovery were not affected by HFS diet. On the other hand, hearts of HFS group were more resistant to lethal I/R injury manifested by significantly smaller infarct size. In addition, there was lower content of collagen I and III in the left ventricle associated with Akt kinase activation and matrix metalloproteinase 9 up-regulation. In conclusion, feeding rats with HFS diet resulted in heart remodeling associated with activation of some adaptive mechanisms, which can contribute to modulation of myocardial resistance to I/R injury.

Influence of ischemia/reperfusion and modulation of PI3K/Akt kinase pathway on matrix metalloproteinase-2 in rat hearts.

Matrix metalloproteinases (MMPs) are enzymes that play an important role in degradation and remodeling of extracellular matrix and MMP-2 has been also shown as a primary mediator of the acute mechanical dysfunction of the heart immediately after ischemia/reperfusion (I/R). The aims of the study were to investigate the influence of I/R on MMP-2 and to study the effects of wortmannin on modulation of MMP-2 activities after cycle of short I/R procedures (ischemic preconditioning, IP). Wortmannin is a specific inhibitor of PI3K/Akt kinase pathway activation of which was found to play a role in infarct size limiting mechanisms in the rat heart. In the study isolated Langendorff-perfused rat hearts subjected to protocols of prolonged (test) I/R and/or IP were used. Wortmannin was infused before and during the reperfusion phase of IP. The levels and activation of proteins were determined by immunoblot assay. The MMP-2 activities were measured by zymography. We found that ischemia induced time-dependent activation of tissue pro-MMP-2. Strong activation occurred after 15 min ischemia, during prolonged ischemia and following reperfusion the activities of this form of MMP-2 declined. The specific activities of both 72 and 63 kDa forms of MMP-2 were increased in perfusates collected during reperfusion after 30 min ischemia and these activities peaked in the first minute of reperfusion. Cycle of short ischemia and reperfusion that led to increased cardiac tolerance against prolonged I/R reduced 72 kDa MMP-2 activities and induced also an activation of Akt kinase. The application of wortmannin was connected with inhibition of IP-mediated Akt kinase activation. Moreover, the actions of wortmannin were linked with modulation of MMP-2 activities. Our results suggest that MMP-2 may be involved in the responses of rat hearts to ischemia and point to possible relationship between Akt kinase and modulation of MMP-2 activities in rat hearts.