Assessment of toxicological profiles of the municipal wastewater effluents using chemical analyses and bioassays

The hazardous chemical contamination of untreated wastewater and secondary effluent from the wastewater treatment plant (WWTP) of the city of Zagreb, Croatia was comprehensively characterized using large-volume solid-phase extraction (SPE) and silica gel fractionation, followed by a detailed analysis of the resulting extracts by a combination of chemical and bioassay methods. Over 100 individual contaminants or closely related-contaminant groups were identified by high-resolution gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-QTOF). Ecotoxicity profiling of the investigated samples, including cytotoxicity, chronic toxicity and EROD activity; inhibition of the multixenobiotic resistance (MXR), genotoxicicity and estrogenic potential, revealed the most significant contribution of toxic compounds to be present in polar fractions. Wastewater treatment using conventional activated sludge process reduced the initial toxicity of raw wastewater to various extents, ranging from 28% for algal toxicity to 73.2% for an estrogenic activity. The most efficient toxicity removal was observed for the polar compounds.     

How three countries in the Americas are fortifying dietary salt reduction: A north and south perspective

A chronic disease/risk factor prevention framework with three policy environments - communications, physical and economic - was used to organize population level interventions that address the “over consumption of dietary salt”, a key risk factor for cardiovascular diseases. The framework was then used to map the population based strategies to reduce dietary salt consumption being applied in three countries in the Americas - Argentina, Canada and Chile - each with a history of multi-sector approaches to deal with the risk factors for chronic disease, offering a north versus south perspective.Results show that in all three countries policy instruments are concentrated in the communications environment, e.g. media and education campaigns and/or regulations for standardized information on the salt or sodium content of packaged food products. Notable gaps are the requirement for nutrient information on meals and food items prepared by food establishments and restrictions on advertising and marketing of foods to children. In the physical environment, referring to the sodium levels in commercially prepared foods and meals available on the market, voluntary reformulation of food products is underway at this time in the three countries. Argentina and Chile began with bread and have gradually added other food categories; Canada at the outset is addressing all food categories where products have added salt. Argentina alone is at this point actively approaching regulations to limit the salt content of food, preferring this over ongoing monitoring of voluntary targets. No government in the three counties has yet considered action in the economic environment, a complex area where the research on and initiatives to limit or disadvantage energy-dense food products to address obesity may also capture foods that are highly salted. In the meantime, with recent research estimating substantially higher gains in population health from government legislation to limit salt in foods compared to voluntary approaches, decision makers in countries, whether in the north or south, committed to reducing dietary salt can take Argentina's example to strengthen their interventions in the physical environment with regulatory instruments. This will sustain reformulations made to date, “level the playing field” industry-wide and broadly and equitably distribute the health benefits of low salt foods.     

Alterations in thymopoiesis in intact and peripubertally orchidectomized adult rats of different age.

In adult rats 3, 6 and 9 months post-orchidectomy performed at the age of 30 days the thymus weight, thymocyte yield and relative proportions of thymocyte subsets (delineated by expression of CD4/CD8 molecules and TCRalphabeta) were analyzed in order to elucidate a putative role of male gonadal hormones in the shaping of thymus size and intrathymic T cell maturation. In 4-month-old control rats the thymus size and cellularity returned to the corresponding levels in 1-month-old rats. These levels were sustained during the following 6 months. In spite of that, the distribution of the main thymocyte subsets in these rats was subjected to significant changes, probably due to an age-associated diminishing thymus ability to provide efficient T cell differentiation. The results added further weight to a potential feedback regulatory role of CD4+8- cells in thymopoiesis. Furthermore, they revealed that the orchidectomy-induced (i) enlargement of the thymus size and enrichment of the thymic lymphoid cell content are of a limited duration; and (ii) alterations in the relative proportion of thymocytes become quantitatively more pronounced with duration of the gonadal deprivation. Thus, the study also indicates that the age-associated changes in gonadal hormones may be, at least partly, responsible for the age-related reshaping of the T cell maturation sequence, and hence for remodeling T cell dependent immune functions.     

Soluble adhesion molecules in acute ischemic stroke

BACKGROUND: Inflammatory adhesion molecules play a key role in the development of ischemic lesions. Elevated plasma concentrations of soluble adhesion molecules are reported in stroke patients, but data are still controversial. Our aim was to explore the potential association of plasma levels of soluble (s) intercellular and vascular cellular adhesion molecules-1 (sICAM-1 and sVCAM-1), sE-selectin and sL-selectin with acute ischemic stroke. METHODS: At our university hospital in Zagreb, Croatia, we prospectively enrolled 67 subjects with acute ischemic stroke, as well as 76 consecutive healthy individuals as controls who were visiting the centre for reasons unrelated to stroke. Serum concentrations of the molecules of interest were determined by means of quantitative sandwich enzyme immunoassay. RESULTS: Mean levels of sICAM-1 (p < 0.001), sVCAM-1 (p < 0.034) and sE-selectin (p < 0.002) were higher in patients than in controls, whereas sL-selectin was lower in patients (p = 0.043). In patients, levels of soluble adhesion molecules were independent of age and sex except for sL-selectin, which was inversely correlated with age (r = -0.260, p = 0.034) and higher in women (p = 0.006) and diabetics (n = 14; p = 0.004). Serum levels did not differ significantly with respect to carotid atherosclerotic disease, smoking status, hypertension or hypercholesterolemia. As well as correlating with each other, concentrations of soluble adhesion molecules in patients correlated with traditional biochemical markers of inflammation: total leukocyte count, erythrocyte sedimentation rate (ESR) and C-reactive protein level. Concentrations of sICAM-1 and high-density lipoprotein-cholesterol and ESR were identified as significant independent predictors/indicators of acute ischemic stroke. CONCLUSIONS: Acute ischemic stroke is associated with elevated plasma levels of sICAM-1, sVCAM-1 and sE-selectin, independent of age, sex and other recognized risk factors for stroke. Decreased levels of sL-selectin are associated with acute stroke. The observed changes in serum concentrations of adhesion molecules indicate inflammatory process occurring during acute cerebral ischemia.     

Cell cycle analysis of the CD133+ and CD133- cells isolated from umbilical cord blood

BACKGROUND: Umbilical cord blood cells (stem/progenitor cells) exhibit high proliferative capacities leading to a large expansion of cells in appropriate cell culture conditions. The aim of this study was to evaluate by flow cytometry the cycling status of CD133+ and CD133- cells depending on various culture conditions, such as sera, stem cell factor (SCF), interleukin 3 (IL-3) and interleukin 6 (IL-6). METHODS: An immunomagnetic system was used for cell separation. CD133+ and CD133- cells were seeded in Iscove's Modified Dulbecco's Medium (IMDM) with different serum concentrations and were stimulated with SCF (100 ng/ml), IL-3 (50 ng/ml) and IL-6 (50 ng/ml). RESULTS: Our experiments demonstrated that immediately after separation, 96.75+/-0.58% of CD133+ cells and 97.04+/-1.76% of CD133- cells were in G0/G1-phase, while 2.02+/-0.38% and 0.88+/-0.52% were in the S-phase, respectively. Our data documented that CD133+ cells are more active than CD133- cells after the first week of cultivation (p<0.01). Statistically significant difference was found for CD133+ cells vs. CD133- cells after second week of cultivation in G0/G1- and S-phases under all tested conditions. A combination of 12.5% FCS+12.5% HS yielded the highest cell expansion for CD133+ cells; this was concomitant with highest percentage of S-phase and G2M-phase. Our data show that the medium with 25% HS was the best for cell expansion and cycling of the CD133- cells for the first week, followed by the 12.5% FCS+12.5% HS. After 2 weeks of cultivation, obviously 12.5% HS and 12.5% FCS+12.5% HS exhibited similar S-phase amounts in CD133- cells. A decrease of HS concentrations seemed to stimulate CD133- cells' S-phase after the second week. CONCLUSIONS: Our data indicate that the source and the concentration of the serum used for cultivation have an impact on both cell populations: CD133+ cells are most comfortable with a combination of FCS and HS; CD133- cells prefer media-containing HS. Cell cycle status may be an important factor for defining cultivation strategies for stem cell expansion.     

Selection of Klebsiella pneumoniae mutants with high-level cefotaxime resistance during growth in serum containing therapeutic concentrations of cefotaxime

BACKGROUND: In a previous investigation of the genetic characterization of extended-spectrum beta-lactamases (ESBLs) in Klebsiella pneumoniae from Zagreb, Croatia, 20 strains were found to produce SHV-2 beta-lactamase. Those strains displayed varying degrees of beta-lactam resistance and a wide range of beta-lactamase activity. We concluded that more resistant isolates were hyperproducers of SHV-2 beta-lactamase. METHODS: In this investigation, we tried to develop hyperproducing variants from 8 low-level SHV-2 beta-lactamase-producing Klebsiella strains by subculturing them in serum containing therapeutic concentrations of cefotaxime (CTX). RESULTS: In most cases, there was a moderate increase in CTX resistance (twofold to threefold), except in one strain which displayed a 16-fold increase in the minimum inhibitory concentration (MIC) of CTX after incubation in the serum. That strain showed a marked increase in enzyme activity as well. The strains with a moderate increase in CTX MIC did not produce more enzyme after exposure to the serum, except for one strain which had a threefold rise in beta-lactamase activity after exposure to serum. CONCLUSIONS: In this investigation, it was established that the mutants with high-level CTX resistance developed very quickly in the biological fluids containing therapeutic concentrations of CTX. It is reasonable to expect that a similar process occurs in patients infected with an ESBL-producing K. pneumoniae strain during antibiotic treatment. Since most of the high-level CTX-resistant mutants did not have a marked rise in beta-lactamase activity after exposure to serum, it is possible that the elevated resistance was due to some other mechanism, such as reduced penicillin-binding protein affinity, changes in outer membrane proteins or efflux by multidrug efflux pumps.     

Sleep and hippocampus: Do we search for the right things?

In addition to its established function in brain restoration, energy saving, circadian homeostasis, thermoregulation, and ontogenetic brain development, sleep is involved in replay and restructuring of memory representations that may lead to memory consolidation. The degree of availability of these memory-related functions in various species, and in disparate environmental and behavioral situations is widely debated. Generally it seems that species which can afford to sleep deeply show an involvement of sleep in learning and memory, both, hippocampus-dependent and hippocampus-independent. Inconsistencies in the sleep literature concerning the importance of certain sleep states for learning of various tasks and the involvement of different types of memory do not disprove that sleep plays a role in memory consolidation. In this review, we attempt to reconcile some of the seemingly antagonistic theories of sleep function in a succinct and unbiased manner and develop an eclectic view of its role in learning and memory.     

Optimal nutritional status assessment parameters in gastroenterological patients on hospital admission

BACKGROUND/AIM: There are no recommendations for the optimal nutritional status assessment parameters (NSAPs) in the current literature. The aim of this study was to define the optimal NSAPs for nutritional status assessing in gastroenterological patients on hospital admission. METHODS: Nutritional status of 612 gastroenterological patients was evaluated at the admission using 6 NSAPs: unintentional weight loss (WL), body mass index (BMI), triceps skinfold thickness (TSF), mid-upper arm muscle circumference (MAMC), serum albumin concentration (ALB), and lymphocyte counts (LYM). According to their nutritive status, the patients were classified as well nourished (normally nourished and obese), moderately undernourished and severely undernourished. Based on the similarities and differences in the assessment results, obtained according to each of 6 parameters, the optimal nutritional assessment parameters were defined, separately for the well-nourished/undernourished patients and for moderately/severely undernourished patients. RESULTS: The incidence of malnutrition was in the range 5.9-29.7%. The results based on MAMC, ALB, and LYM were similar (25.2-29.7%; p > 0.05), while the results based on WL, BMI, and TSF differed significantly (5.9-19.9%; p = 0.001-0.015). The assessment based on BMI was the most severe criterion, while the assessment according to MAMC was the mildest criterion in selection of malnourished patients. The assessment according to MAMC was the mildest criterion for the selection of severe malnourished patients (severely malnouorished patients accounted for 70.1%), while BMI and LYM were the most severe criteria (severely malnouorished patients accounted for 22.2% and 27.3%, respectively). The results based on BMI and LYM were similar (Wilcoxon test; p > 0.05). CONCLUSION: Combining BMI with MAMC is sufficient for the assessment of the nutritional status of gastroenterological patients on admission, as well as for differentiation between severely malnourished and moderately malnourished patients.