Anti-herpesvirus bovine type 5 activities of extracts obtained from Plocamium brasiliense

Bovine herpesvirus type 5 (BoHV-5) is an important etiologic agent of meningoencephalitis in cattle and has been frequently identified in outbreaks of neurological disease in bovine in the southern hemisphere including Brazil. This study aimed to evaluate the cytotoxic effect and the antiviral properties of extracts obtained from Plocamium brasiliense (Greville) Howe and Taylor in BoHV-5 RJ42/01 replication. The cytotoxic effects were measured in Madin-Darbin bovine kidney cells (MDBK) and cytotoxic concentration (CC₅₀) values have been determined for acyclovir (ACV) (223 μg?±?2.0), ethyl acetate extract from P. brasiliense (2,109 μg?±?10), hexane extract from P. brasiliense (7.181 μg?±?5), dichloromethane extract from P. brasiliense (2.356 μg?±?6.5), and hydroalcoholic extract from P. brasiliense (1.408 μg?±?5.8). As a first approach to characterize the action of these extracts on BoHV-5 RJ42/01, a virucidal assay activity was performed. A virus suspension containing 1?×?10⁵ plaque-forming units (PFU) of the BoHV-5 RJ42/01 was mixed with 600 μg of extract and acyclovir and kept at room temperature (24 °C) for 3 h. Meanwhile, a control of untreated infected virus was performed in the same conditions. Then, treated virus suspension and untreated control were diluted, and percentage of inhibition of infectivity was determined by plaque assay: ethyl acetate extract (99 %), hexane extract (90 %), dichloromethane extract (99 %), and hydroalcoholic extract (27 %). Acyclovir had a slight virucidal activity on viral particle. The inhibition of attachment was performed in MDBK cells inoculated with 100 PFU of BoHV-5 RJ42/01 in the presence or absence of various concentrations of extracts (0.3, 0.9, and 1.5 μg mL^?1). Acyclovir was also assayed at 2.8 and 11.25 μg mL^?1. The inhibition of adsorption was also tested in MDBK cells treated with the same concentrations of the extracts before virus inoculation. Results: hexane extracts inhibited virus attachment in pre-treated cell 0.9 μg (55 %) and 1.5 μg (71 %) and untreated MDBK cell only with 1.5 μg (63 %). Ethyl acetate extract on cell pre-treated with 0.3 μg (67 %), 0.9 μg (81 %), and 1.5 μg (91 %). Ethyl acetate extract on pre-treated cell 0.3 μg (67 %), 0.9 μg (81 %), and 1.5 (91 %) but discrete inhibition on cell untreated. Dichloromethane extract and acyclovir slightly inhibited virus binding on MDBK cell.     

Changes of Concentrations of Elements in Deciduous Teeth with Age

The aim of this study was to describe the issue of changes in the concentration of some elements (Mn, Fe, Mg, Cu, K, Cr, Pb, Cd, and Ca) in the mineral composition of deciduous teeth and to answer the question whether there was a tendency to change the metal concentration, depending on children's age. Physiological replacement of deciduous teeth is preceded with the remodeling process occurring in the mineral structure. The elemental composition of deciduous teeth tissue during their natural loss is the result of these transformations. The subject of the study was deciduous teeth taken from the children (n?=?45, aged 5–14 years), residing in southern Poland, obtained as a result of noninvasive physiological replacement. The concentration of metals in samples was determined over the atomic absorption spectroscopy method. The results of the research showed a statistically significant decrease in the concentration of the analyzed elements in deciduous teeth of older children compared to younger children. The total concentration of all metals studied against the Ca content, expressed as the value of the constant cationic equilibrium, did not change in a statistically significant manner, depending on the age of the children (p?>?0.05).     

Urinary zearalenone measured with ELISA as a biomarker of zearalenone exposure in pigs

The suitability to assess zearalenone (ZEA) exposure in pigs of a commercial ELISA kit for ZEA analysis in urine was tested. A daily dose of 0, 5, 10, 20 and 40 μg synthetic ZEA per kilogram BW was administered via the feed to four gilts per dose group, and after 3 and after 7 days of ZEA intake, urine samples were assayed with the ELISA which has a relative cross-reactivity of 42 % with α-zearalenol. The concentration of urinary ZEA equivalents (ZEA plus 42 % of α-zearalenol present) did not differ between day 4 and day 8 (P?=?0.50) within each dose group. The urinary ZEA equivalent/creatinine ratio was tightly correlated with ZEA intake (r?=?0.95). The urinary ZEA equivalent/creatinine values at 0 and 40 μg/kg BW were distinctly different from those of the intermediate dose levels, whereas there was some overlapping of the individual values at the dose levels 5, 10 and 20 μg/kg BW. The urinary ZEA equivalent/creatinine ratio can be used as a biomarker for ZEA exposure in pigs provided that urine samples of several animals receiving the same diet are assayed, either separately or after pooling.     

Sterigmatocystin and aflatoxin B<Subscript>1</Subscript> contamination of corn,soybean meal,and formula feed in Japan

Sterigmatocystin (STC) and aflatoxin B₁ (AFB₁) were analyzed in 246 corn samples, 126 soybean meal samples, and 861 formula feed samples from the Japanese market between April 2010 and March 2015. The detection rate, the highest concentration, and the mean concentration of STC were respectively 14%, 6.4 μg/kg, and 1.2 μg/kg for corn; 14%, 1.1 μg/kg, and 0.63 μg/kg for soybean meal; and 43%, 9.1 μg/kg, and 0.97 μg/kg for formula feed. The detection rate, the highest concentration, and the mean concentration of AFB₁ were respectively 46%, 24 μg/kg, and 3.9 μg/kg for corn; 30%, 6.7 μg/kg, and 1.1 μg/kg for soybean meal; and 47%, 20 μg/kg, and 1.6 μg/kg for formula feed. A weak negative correlation between the STC and AFB₁ concentrations was observed: there was a high concentration of AFB₁ in samples that contained a lower concentration of STC and vice versa. Spearman’s rank correlation coefficient showed a weak negative correlation of ? 0.30 (p < 0.001, n = 128) for corn and ? 0.23 (p < 0.001, n = 575) for formula feed. In conclusion, no correlation was observed between the mean concentrations of STC contamination in formula feed (0.97 μg/kg) and in corn (1.2 μg/kg) and the blending rate (approximately 50%). The rate of STC contamination in the formula feed (43%) was higher than that in corn (14%). Therefore, it is likely that ingredients other than corn contribute to the contamination of formula feed with STC. In this study, regarding STC, problematic samples were not found.     

Supragingival Microbial Profiles of Permanent and Deciduous Teeth in Children with Mixed Dentition

Objectives

The present study was designed to investigate the microbial profiles of teeth in different locations in mixed-dentition-stage children, and to compare the microbiomes of permanent and deciduous teeth in the same healthy oral cavity.

Methods

Supragingival plaque samples of teeth in various locations—the first permanent molars, deciduous molars, deciduous canines and incisors and permanent incisors—were collected from 20 healthy mixed-dentition-stage children with 10–12 permanent teeth erupted. Plaque DNA was extracted, and the V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified and subjected to sequencing.

Results

On average, 18,051 high-quality sequences per sample were generated. Permanent tooth sites tended to host more diverse bacterial communities than those of deciduous tooth sites. A total of 12 phyla, 21 classes, 38 orders, 66 families, 74 genera were detected ultimately. Five predominant phyla (Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria and Actinobacteria) were highly variable among sites. Of 26 genera with a mean relative abundance of >0.1%, 16 showed significant differences in relative abundance among the groups. More than 20% of the total operational taxonomical units were detected only in permanent or deciduous teeth. The variation in the microbial community composition was due mainly to permanent teeth being enriched in Actinomyces and deciduous teeth in Treponema. The core microbiome of supragingival plaque in mixed dentition comprised 19 genera with complex correlationships.

Conclusion

Our results suggest differences in microbial diversity and composition between permanent and deciduous teeth sites in mixed dentition. Moreover, the core microbiome of these sites was determined. These findings enhance our understanding of the development of the native oral microbiota with age.     

Antibacterial activities of nemonoxacin against clinical isolates of Staphylococcus aureus: an in vitro comparison with three fluoroquinolones

In comparison with ciprofloxacin, levofloxacin and moxifloxacin, antimicrobial activity of nemonoxacin against ciprofloxacin-susceptible/-resistant methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) was determined with the availability to select resistant mutants evaluated. Minimum inhibitory concentrations and mutant prevention concentrations of quinolones were determined by agar dilution method, that concentrated bacterial cells were spread onto Mueller–Hinton agar plates containing antibacterials at different concentrations. Selection index (SI) was calculated. Minimum inhibitory concentration and mutant prevention concentration of nemonoxacin were 0.063 and 0.25 μg/mL for ciprofloxacin-susceptible MSSA and those were 0.5 and 4.0 μg/mL for ciprofloxacin-resistant MSSA, lower than observations of three fluoroquinolones distinctly. SI of nemonoxacin and moxifloxacin were similar, with narrower mutant selective window than levofloxacin and ciprofloxacin. Minimum inhibitory concentration and mutant prevention concentration of nemonoxacin were 0.25 and 2.0 μg/mL for ciprofloxacin-susceptible MRSA, which were 0.5 and 16.0 μg/mL for ciprofloxacin-resistant MRSA. Values were lower than those determined from fluoroquinolones. Nemonoxacin presents good antimicrobial activity against clinical isolates of S. aureus, especially for ciprofloxacin-resistant strains. But stepwise mutant accumulation of ciprofloxacin-resistant MRSA can be hardly inhibited by nemonoxacin with pharmacokinetic parameters considered.     

A comparative study of PKH67, DiI,and BrdU labeling techniques for tracing rat mesenchymal stem cells

Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 μM BrdU and 10 μM BrdU. The cells were then cultured for 6 d or passaged (1–3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3?±?1.6% PKH67+ and 98.4?±?1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2?±?4.6% 10 μM BrdU+ and 77.6?±?4.6% 5 μM BrdU+), which remained high for 6 d. Viability of labeled cells was 91?±?3.8% PKH67+, 90?±?1.5% DiI+, 91?±?0.8% 5 μM BrdU+, and 76.9?±?0.9% 10 μM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.     

Catesbeianin-1, a novel antimicrobial peptide isolated from the skin of <Emphasis Type="Italic">Lithobates catesbeianus</Emphasis> (American bullfrog)

Objectives

To identify and characterize a novel antimicrobial peptide, catesbeianin-1.

Results

Catesbeianin-1 is 25 amino acids long and is α-helical, cationic and amphipathic. It had antimicrobial activity against Gram-positive and Gram-negative bacteria. It was resistant against trypsin and pepsin. Catesbeianin-1 exhibited moderate hemolytic activity (approx 8%) at 100 μg/ml, and its HC₅₀ (50% hemolytic concentration) was 300 μg/ml. Its cytotoxicity was approx 10–20% at 100 μg/ml, and its CC₅₀ (50% cytotoxic concentration) was >100 μg/ml. The LD₅₀ of catesbeianin-1 in mice was 80 mg/kg. At 3.1 µg/ml, catesbeianin-1 significantly inhibited the growth of methicillin-resistant Staphylococcus aureus.

Conclusions

A new antimicrobial peptide from the skin of Lithobates catesbeianus (American bullfrog) may represent a template for the development of novel antimicrobial agents.

     

Correlation between lactoferrin and beneficial microbiota in breast milk and infant’s feces

Lactoferrin (LF) is a natural component of human milk with antimicrobial, immunostimulatory and immunomodulatory properties. Several in vitro studies suggest that LF could promote an environment in the gut of neonates that favors colonization with beneficial bacteria. However, clinical studies on the correlation between the concentration of LF in breast milk and feces of infants and the gut microbiota in infants are lacking. In our study we analyzed the content of LF and the microbiota of breast milk and feces of infants of 48 mother–infant pairs (34 full-term and 14 pre-term infants) at birth and 30 days after delivery. In the term group, a significant decrease of mean LF concentration between colostrum (7.0 ± 5.1 mg/ml) and mature milk (2.3 ± 0.4 mg/ml) was observed. In pre-term group, breast milk LF levels were similar to those observed in full-term group. Fecal LF concentration of healthy infants was extremely high both in term and pre-term infants, higher than the amount reported in healthy children and adults. In term infants mean fecal LF levels significantly increased from birth (994 ± 1,828 μg/ml) to 1 month of age (3,052 ± 4,323 μg/ml). The amount of LF in the feces of 30 day-old term infants was significantly associated with maternal mature milk LF concentration (p = 0.030) confirming that breast milk represents the main source of LF found in the gut of infants. A linear positive correlation between colostrum and mature milk LF concentration was observed (p = 0.008) indicating that milk LF levels reflect individual characteristics. In pre-term infants higher mean concentrations of fecal LF at birth (1,631 ± 2,206 μg/ml) and 30 days after delivery (7,633 ± 9,960 μg/ml) were observed in comparison to full-term infants. The amount of fecal bifidobacteria and lactobacilli resulted associated with the concentration of fecal LF 3 days after delivery (p = 0.017 and p = 0.026, respectively). These results suggest that high levels of fecal LF in neonates, particularly in the first days of life, could represent an important factor in the initiation, development and/or composition of the neonatal gut microbiota. Since early host–microbe interaction is a crucial component of healthy immune and metabolic programming, high levels of fecal LF in neonates may beneficially contribute to the immunologic maturation and well-being of the newborn, especially in pre-term infants.     

Candidacidal mechanism of the arenicin-3-derived peptide NZ17074 from Arenicola marina

The candidacidal mechanisms of NZ17074, which is a variant of arenicin-3 from Arenicola marina, against human pathogenic fungus Candida albicans are reported in this work. The minimum inhibitory concentration (MIC) of NZ17074 toward C. albicans was 4 μg/ml, and this peptide exerted marked candidacidal activity in an energy-dependent and salt-sensitive manner. The flow cytometric analysis using propidium iodide (PI) showed that the plasma membrane of cells treated with NZ17074 was perturbed and that the cells were arrested in the G2/M phase. The dihydrorhodamine-123 (DHR-123) staining showed that the reactive oxygen species (ROS) production of C. albicans increased after exposure to NZ17074. Typical cellular disruption events, such as mitochondrial degradation, nuclear fragmentation, nuclear membrane disruption, and chromatin condensation, were further revealed through rhodamine 123 (RH123) staining, 4′,6-diamidino-2-phenylindole (DAPI) staining, and transmission electron microscopy. In addition, the intracellular localization of this peptide was concentration dependent: it was located in the membrane at low concentrations (4 to 8 μg/ml) and penetrated into the cytoplasm at high concentrations (16 to 32 μg/ml). Our results suggested that NZ17074 exerts its candidacidal effects by disrupting the cell membrane, inducing apoptosis, and interrupting the cell cycle. These findings showed the potential of NZ17074 as a new candidacidal peptide, in addition to its antibacterial activities.     

Evaluation of an enzyme immunoassay for the detection of the mycotoxin tenuazonic acid in sorghum grains and sorghum-based infant food

An enzyme-linked immunosorbent assay (ELISA) for the Alternaria mycotoxin tenuazonic acid (TeA) was evaluated by comparative analysis of naturally contaminated sorghum grains and sorghum-based infant food, using a stable isotope dilution LC-MS assay (SIDA; limit of detection (LOD) 1.0 μg/kg) as the reference method. LODs of the ELISA were 30 μg/kg in sorghum grains and 220 μg/kg in sorghum-based infant cereals. With SIDA, 100% of the samples (n = 28) had been positive for TeA in a concentration range of 6–584 μg/kg (mean 113 μg/kg). The ELISA consistently detected TeA in all naturally contaminated samples at cut-off levels of 30–60 μg/kg (sorghum) and 200–300 μg/kg (infant cereals), as based on corresponding to SIDA values. Although the ELISA was much less sensitive than the SIDA method, it may be useful as a screening method for sorghum and sorghum-based infant foods and can be employed to identify samples containing elevated concentrations of TeA in food, well below the proposed level of concern (500 μg/kg).     

The Antiepileptic Effect of Ghrelin During Different Phases of the Estrous Cycle in PTZ-Induced Seizures in Rat

Catamenial epilepsy is a special form of epilepsy in women whom seizure aggravation is arranged with menstrual cycle that may affect up to 70 % of epileptic women. Antiepileptic effect of Ghrelin hormone has been proved recently. Due to effects of Ghrelin on GABA and LH concentration and periodic variation in the level of estrogen (E₂) and progesterone (P₄) during menstrual cycle, it seems that antiepileptic effect of Ghrelin can be different during various phases of estrous cycle. So this study was conducted to survey antiepileptic effect of Ghrelin during various phases of estrous cycle in rats. 72 adult female Wistar rats in three groups (control, 40 and 80 μg/kg of Ghrelin), each with four subgroups (proestrus, estrus, metestrus and diestrus) were used (n = 6). Then, intracerebroventricular (ICV) injection of Ghrelin (40 and 80 μg/kg) followed by intraperitoneal injection of 80 μg/kg pentylenetetrazole in control and treatment groups were done. Initiation time of myoclonic seizures (ITMS), initiation time of tonic–clonic seizures (ITTS), seizures duration and mortality rate were monitored and recorded for 30 min. Results showed that, ICV injection of Ghrelin significantly increased ITMS and ITTS during luteal phase than follicular phase compared to control group (P < 0.05). Also, seizure duration significantly decreased after ICV injection of Ghrelin during luteal phase and follicular phase compared to control group (P < 0.05). Furthermore, there was no mortality after ICV injection of Ghrelin (40 and 80 μg/kg) during luteal and follicular phases compared to control group (P < 0.05). These results suggest that Ghrelin has antiepileptic effects which are more prominent during luteal phase than follicular phase.     

Synthesis of thiazolidine-2,4-dione derivatives: anticancer,antimicrobial and DNA cleavage studies

In the search of efficient anticancer agents, here, new 5-(4-alkylbenzyledene)thiazolidine-2,4-dione derivatives (5a–g) have been successfully synthesized and characterized and are evaluated for anticancer and antimicrobial activities using DNA cleavage studies. In vitro studies on anticancer activity of compound 5d (NSC: 768619/1) was done against the full panel of 60 human tumor cell lines. The five-level dose activity results revealed that, the compound 5d was active against all the cell lines, it has shown potential activity against leukemia SR (GI₅₀: 2.04 μM), non-small cell lung cancer NCI-H522 (GI₅₀: 1.36 μM), colon cancer COLO 205 (GI₅₀: 1.64 μM), CNS cancer SF-539 (GI₅₀: 1.87 μM), melanoma SK-MEL-2 (GI₅₀: 1.64 μM), ovarian cancer OVCAR-3 (GI₅₀: 1.87 μM), renal cancer RXF 393 (GI₅₀: 1.15 μM), prostate cancer PC-3 (GI₅₀: 1.90 μM), and breast cancer MDA-MB-468(GI₅₀: 1.11 μM). DNA cleavage studies revealed that at 50 μg/mL concentration, partial DNA digestion was observed and when the concentration is increasing to threefold (150 μg/mL), complete linear DNA digestion and partial supercoiled DNA digestion was observed. Further antimicrobial studies indicate that all the synthesized compounds except compound 5a possess prominent activity against all the screened microbial species. This study throws a ray of light in the field of anticancer drugs.     

Distinctive Genetic Activity Pattern of the Human Dental Pulp between Deciduous and Permanent Teeth

Human deciduous and permanent teeth exhibit different developmental processes, morphologies, histological characteristics and life cycles. In addition, their pulp tissues react differently to external stimuli, such as the pulp sensitivity test, dental trauma and pulp therapy materials. These suggest differences in gene expression and regulation, and in this study we compared gene-expression profiles of the human dental pulp from deciduous and permanent teeth. Pulp tissues from permanent premolars and deciduous molars aged 11–14 years were extirpated and mRNA was isolated for cDNA microarray analysis, and quantitative real-time PCR (qPCR). Other teeth were used for immunohistochemical analysis (IHC). Microarray analysis identified 263 genes with a twofold or greater difference in expression level between the two types of pulp tissue, 43 and 220 of which were more abundant in deciduous and permanent pulp tissues, respectively. qPCR analysis was conducted for eight randomly selected genes, and the findings were consistent with the cDNA microarray results. IHC confirmed that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was broadly expressed in deciduous dental pulp tissue, but minimally expressed in permanent dental pulp tissue. Immunohistochemical analysis showed that calbindin 1 (CALB1), leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and gamma-aminobutyric acid A receptor beta 1 (GABRB1) were abundantly expressed in permanent predentin/odontoblasts, but only minimally expressed in deciduous dental pulp tissue. These results show that deciduous and permanent pulp tissues have different characteristics and gene expression, suggesting that they may have different functions and responses to therapies focused on pulp or dentin regeneration.     

Analyses of the Erosive Effect of Dietary Substances and Medications on Deciduous Teeth

This study aimed at analysing the erosive potential of 30 substances (drinks, candies, and medicaments) on deciduous enamel, and analyse the associated chemical factors with enamel dissolution. We analysed the initial pH, titratable acidity (TA) to pH 5.5, calcium (Ca), inorganic phosphate (P_i), and fluoride (F) concentration, and degree of saturation ((pK -pI)_HAP, (pK -pI)_FAP, and (pK−pI)_CaF2) of all substances. Then, we randomly distributed 300 specimens of human deciduous enamel into 30 groups (n = 10 for each of the substances tested. We also prepared 20 specimens of permanent enamel for the sake of comparison between the two types of teeth, and we tested them in mineral water and Coca-Cola^®. In all specimens, we measured surface hardness (VHN: Vickers hardness numbers) and surface reflection intensity (SRI) at baseline (SH_baseline and SRI_baseline), after a total of 2 min (SH_2min) and after 4 min (SH_4min and SRI_4min) erosive challenges (60 ml of substance for 6 enamel samples; 30°C, under constant agitation at 95 rpm). There was no significant difference in SH_baseline between deciduous and permanent enamel. Comparing both teeth, we observed that after the first erosive challenge with Coca-Cola^®, a significantly greater hardness loss was seen in deciduous (−90.2±11.3 VHN) than in permanent enamel (−44.3±12.2 VHN; p = 0.007), but no differences between the two types of teeth were observed after two challenges (SH_4min). After both erosive challenges, all substances except for mineral water caused a significant loss in relative surface reflectivity intensity, and most substances caused a significant loss in surface hardness. Multiple regression analyses showed that pH, TA and Ca concentration play a significant role in initial erosion of deciduous enamel. We conclude that drinks, foodstuffs and medications commonly consumed by children can cause erosion of deciduous teeth and erosion is mainly associated with pH, titratable acidity and calcium concentration in the solution.     

Concentrations of Metals in Maxilla and Mandible Deciduous and Permanent Human Teeth

The study shows the results of research on the concentrations of metals (Cd, Pb, Mn, Cu, Cr, Fe, Zn, Na, K, Mg, and Ca) in deciduous and permanent teeth with respect to their location in the oral cavity (maxilla, mandible). It has been found that the concentrations of metals were significantly higher in the deciduous teeth than in permanent ones. Regression analysis and principal component analysis revealed an increased dynamics of the processes of binding the elements by the hydroxyapatite of deciduous teeth. Higher concentrations of the metals were found in the maxilla permanent and deciduous teeth than in the mandible ones.     

Evaluation of Mineral Concentrations in Maternal Serum Before and After Birth and in Newborn Cord Blood Postpartum—Preliminary Study

The mineral levels in maternal serum change during pregnancy and may be correlated with those of newborn cord blood. The aim of this study was to evaluate the concentrations of calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), and copper (Cu) in maternal blood before and after delivery and in umbilical cord vein and artery serum. The study was carried out in 64 Caucasian pregnant women who delivered in a district hospital in Greater Poland region, aged 28.1 ± 5.4 years, with a mean gestational age of 39.2 ± 1.3 weeks. Blood samples were taken from women 2–8 h before delivery and immediately after childbirth. The umbilical cord artery and vein blood of newborns was obtained immediately after childbirth. The levels of minerals in serum were determined by flame atomic absorption spectrometry. A significant drop in the concentrations of Mg (17.71 ± 1.51 vs 17.07 ± 1.61 μg/ml; p < 0.007), Fe (1.08 ± 0.46 vs 0.82 ± 0.35 μg/ml; p < 0.0004), and Zn (0.63 ± 0.17 vs 0.46 ± 0.16; p < 0.0001) in maternal serum was found after delivery. Moreover, higher levels of Ca, Fe, and Zn and lower levels of Cu were observed in the umbilical vein (Ca: 102.80 ± 7.80 μg/ml; p < 0.0001, Fe: 1.96 ± 0.43 μg/ml; p < 0.0001, Zn: 0.65 ± 0.16 μg/ml; p < 0.0001, Cu: 0.36 ± 0.09 μg/ml; p < 0.0001) and in the umbilical artery cord blood (Ca: 98.07 ± 8.18 μg/ml; p < 0.0001, Fe: 1.63 ± 0.30 μg/ml; p < 0.0001, Zn: 0.65 ± 0.15 μg/ml; p < 0.0001, and Cu: 0.36 ± 0.10 μg/ml; p < 0.0001) compared to the maternal serum (Ca: 85.05 ± 10.76 μg/ml, Fe: 0.82 ± 0.35 μg/ml, Zn: 0.46 ± 0.16 μg/ml, and Cu: 1.90 ± 0.35 μg/ml). Fe levels in the cord artery serum negatively correlated with blood loss during delivery (R = ?0.48; p = 0.01), while the Ca concentration in the maternal serum after birth decreased with the age of the women (R = ?0.25; p = 0.03). In conclusion, it seems that the process of birth alters the mineral levels in pregnant women’s blood. Moreover, it was found that blood loss and the age of the mother are associated with mineral concentrations in the maternal serum and cord artery blood.     

The Rho kinase inhibitor Y-27632 facilitates the differentiation of bone marrow mesenchymal stem cells

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, but technical challenges have limited cell yield and thus the success of these potential treatments. The Rho GTPases and downstream Rho kinases (Rho coiled-coil kinases or ROCKs) are central regulators of cytoskeletal dynamics during the cell cycle and thus help determine the balance between stem cells self-renewal, lineage commitment, and apoptosis. Here, we examined if suppression of ROCK signaling enhances the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) differentiation into neurons and neuroglial cells. BMSCs were cultured in epidermal growth factor (EGF, 10 µg/l) and basic fibroblastic growth factor (bFGF, 10 µg/l) in the presence or absence of the Rho kinase inhibitor Y-27632 (10 µM). The expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence and Western blotting. The average number of NSE-positive cells increased from 83.20 ± 8.677 (positive ratio 0.2140 ± 0.0119) to 109.20 ± 8.430 (positive ratio 0.3193 ± 0.0161) per visual field in the presence of Y-27632, while GFAP-positive cell number increased from 96.30 ± 8.486 (positive ratio 0.18 ± 0.0152) to 107.50 ± 8.683 (positive ratio 0.27 ± 0.0115) (P < 0.05 for both). Both NSE and GFAP protein expression levels were enhanced significantly by Y-27632 treatment (NSE: 0.74 ± 0.05 vs. 1.03 ± 0.06; GFAP: 0.64 ± 0.08 vs. 0.97 ± 0.05, both P < 0.01) as indicated by Western blots. The Rho kinase inhibitor Y-27632 concomitant with EGF and bFGF stimulation promotes BMSC differentiation into neural cells. Control of Rho kinase activity may enhance the efficiency of stem cell-based treatments for neurodegenerative diseases.     

Differential Effects of Fluoride During Osteoblasts Mineralization in C57BL/6J and C3H/HeJ Inbred Strains of Mice

The behavior of fluoride ions in biological systems has advantages and problems. On one hand, fluoride could be a mitogenic stimulus for osteoblasts. However, high concentrations of this element can cause apoptosis in rat and mouse osteoblasts. Toward an understanding of this effect, we examined the role of sodium fluoride (NaF) in two mouse calvaria osteoblasts during the mineralization process. The animals used were C3H/HeJ (C3) and C57BL/6J (B6) mice. The calvaria cells were cultured for 28 days in the presence of several doses of NaF (0, 5, 10, 25, 50, and 75 μM), and we performed the assays: mineralized nodule measurements, alkaline phosphatase (ALP) activity, determination of type I collagen, and matrix metalloproteinase-2 (MMP-2) activity. The results showed no effects on alkaline phosphatase activity but decreased mineralized nodule formation. In B6 cells, the NaF effect was already seen with 10 μM of NaF and a greater increase of cellular type I collagen, and MMP-2 activity was upregulated after 7 days of NaF exposure. C3 osteoblasts showed a reduction in the mineralization pattern only after 50 μM of NaF with a slight increase of type I collagen and downregulation of MMP-2 activity during the mineralization period. In conclusion, fluoride affects the production and degradation of the extracellular matrix during early onset and probably during the mineralization period. Additionally, the genetic factors may contribute to the variation in cell response to fluoride exposure, and the differences observed between the two strains could be explained by an alteration of the bone matrix metabolism (synthesis and degradation).     

High-Precision (MC-ICPMS) Isotope Ratio Analysis Reveals Contrasting Sources of Elevated Blood Lead Levels of an Adult with Retained Bullet Fragments,and of His Child,in Milwaukee,Wisconsin

This study aimed at evaluation of a relationship between blood selenium concentration (Se-B) and blood cystatin C concentration (CST) in a randomly selected population of healthy children, environmentally exposed to lead and cadmium. The studies were conducted on 172 randomly selected children (7.98 ± 0.97 years). Among participants, the subgroups were distinguished, manifesting marginally low blood selenium concentration (Se-B 40–59 μg/l), suboptimal blood selenium concentration (Se-B: 60–79 μg/l) or optimal blood selenium concentration (Se-B ≥ 80 μg/l). At the subsequent stage, analogous subgroups of participants were selected separately in groups of children with BMI below median value (BMI <16.48 kg/m²) and in children with BMI ≥ median value (BMI ≥16.48 kg/m²). In all participants, values of Se-B and CST were estimated. In the entire group of examined children no significant differences in mean CST values were detected between groups distinguished on the base of normative Se-B values. Among children with BMI below 16.48 kg/m², children with marginally low Se-B manifested significantly higher mean CST values, as compared to children with optimum Se-B (0.95 ± 0.07 vs. 0.82 ± 0.15 mg/l, p < 0.05). In summary, in a randomly selected population of healthy children no relationships could be detected between blood selenium concentration and blood cystatin C concentration. On the other hand, in children with low body mass index, a negative non-linear relationship was present between blood selenium concentration and blood cystatin C concentration.