Analysis of soluble factors in conditioned media derived from primary cultures of cirrhotic liver of biliary atresia

Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and β chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-β1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3–4 wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient’s own cells for the treatment of BA.     

Cryopreservation of fruit germplasm

Most temperate fruit species are genetically heterozygous and vegetatively propagated. Active collections of fruit genetic resources in Germany are generally maintained in the field, e.g., as potted plants for Fragaria and as trees for pome and stone fruit species. The plant material in active collections should be kept in duplicate to ensure security in case of disease or environmental disaster. The aim of this study was to develop an efficient complementary conservation strategy for fruit genetic resources. Although costly, fruit tree cultivars can be duplicated as field collections at a second site within the framework of the German Fruit Genebank, which is a decentralized fruit-specific network. Wild species accessions, particularly those of the genera Malus spp. (apple) and Fragaria spp. (strawberry) as well as strawberry cultivars, can also be duplicated by means of cryopreservation. In the current study, long-term cryopreservation was initiated for 194 Fragaria genotypes. A protocol combining vitrification with cold acclimation was effective and highly reproducible, with an average regrowth rate of 86%. In Malus, a general cryopreservation protocol based on dormant winter buds was adopted. Based on the results provided in this study, a combination of traditional ex situ conservation and cryopreservation can greatly improve the stability and security of fruit germplasm conservation.     

Concentration of Zinc,Copper, Iron,Calcium, and Magnesium in the Serum,Tissues, and Urine of Streptozotocin-Induced Mild Diabetic Rat Model

A survey carried out in Lake Tana in 2015 found that Hg levels in some fish species exceeded internationally accepted safe levels for fish consumption. The current study assesses human exposure to Hg through fish consumption around the Lake Tana. Of particular interest was that a dietary intake of fishes is currently a health risk for Bihar Dar residents and anglers. Hair samples were collected from three different groups: anglers, college students and teachers, and daily laborers. A questionary includes gender, age, weight, activity. Frequency of fish consumption and origin of the eaten fish were completed by each participant. Mercury concentrations in hair were significantly higher (P value <0.05) for anglers (mean?±?standard deviation 0.120?±?0.199 μg/g) than college students (mean?±?standard deviation 0.018?±?0.039 μg/g) or daily workers (mean?±?standard deviation 16?±?9.5 ng/g). Anglers consumed fish more often than daily workers and college group. Moreover, there was also a strong correlation (P value <0.05) between the logarithms of total mercury and age associated with mercury concentration in scalp hair. Mercury concentrations in the hair of men were on average twice the value of the women. Also, users of skin lightening soap on a daily basis had 2.5 times greater mercury in scalp hair than non-users. Despite the different sources of mercury exposure mentioned above, the mercury concentrations of the scalp hair of participants of this study were below levels deemed to pose a threat to health.     

Hypoglycemic and Hypolipidemic Effects of Leucine,Zinc, and Chromium,Alone and in Combination,in Rats with Type 2 Diabetes

The main objective was to determine the elemental profile of the lung lining fluid of rats which are used as model animals in various experiments. Lung lining fluid elemental constitution obtained after bronchoalveolar lavage fluid (BALF) was analyzed by inductively coupled plasma mass spectrometry (ICP-MS) to determine the biological trace elements along with calcium and magnesium. BALF was collected from healthy rats using a tracheal cannula. However, cells in BALF were counted to monitor any underlying inflammatory lung condition. Cell free BALF samples were processed and analyzed for the elements including magnesium (Mg), calcium (Ca), chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), selenium (Se), bromine (Br), and iodine (I). In view of this, calcium concentration was the highest (6318.08 ± 3094.3 μg/L) and copper concentration was the lowest (0.89 ± 0.21 μg/L). The detected elements, from high to low concentration, include Ca > Mg > Fe > Br > I > Cr > Ni > Zn > Mn > Se > Cu. Pearson’s correlation analysis revealed no significant correlation between cell count and concentration of any of the element detected in BALF. Correlation analysis also revealed significant positive correlation among Fe, I, Cr, Ni, and Mn. Ca was found to be correlated negatively with Cu and positively with Se. Br and Mg found to be positively correlated with each other. Zn remained the only element that was not found to be correlated with any of the elements in the rat BALF.     

Assessment of Zinc Status in School-Age Children from Rural Areas in China Nutrition and Health Survey 2002 and 2012

Zinc is an essential trace element for growth and development in children, but zinc deficiency is a serious nutritional problem worldwide. Our study aimed to assess the zinc status of school-age children living in rural areas of China and to examine the change of zinc status based on the China Nutrition and Health Survey 2002 and 2012. We used the probability proportional to size sampling method for subject selection, and a total of 3407 school-age children were included in this study. Zinc status was assessed by three items of indicators recommended by the World Health Organization (WHO), the United Nations Children’s Fund (UNICEF), the International Atomic Energy Agency (IAEA), and the International Zinc Nutrition Consultative Group (IZiNCG). The concentration of serum zinc was 718.2 μg/L, and 44.4% of children being zinc deficiency in 2002, while 846.8 μg/L and 10.4% in 2012. Zinc intake was 7.8 mg/day with a 7.6% inadequate zinc intake in 2002, together with 6.9 mg/day and 38.2% in 2012. Height-for-age Z score was ?1.06 and 19.1% of children being stunting in 2002, as well as ?0.15 and 6.8% in 2012. In conclusion, the zinc status of school-age children living in rural areas of China has been significantly improved in addition to zinc intake over the past 10 years. However, the zinc deficiency still observed in poor rural areas of China in 2012. In addition, we suggested that the zinc bioavailability should be taken into account when assessing zinc status in population.     

Magnesium Ions Promote the Biological Behaviour of Rat Calvarial Osteoblasts by Activating the PI3K/Akt Signalling Pathway

Magnesium has been investigated as a biodegradable metallic material. Increased concentrations of Mg²⁺ around magnesium implants due to biodegradation contribute to its satisfactory osteogenic capacity. However, the mechanisms underlying this process remain elusive. We propose that activation of the PI3K/Akt signalling pathway plays a role in the Mg²⁺-enhanced biological behaviours of osteoblasts. To test this hypothesis, 6, 10 and 18 mM Mg²⁺ was used to evaluate the stimulatory effect of Mg²⁺ on osteogenesis, which was assessed by evaluating cell adhesion, cell viability, ALP activity, extracellular matrix mineralisation and RT-PCR. The expression of p-Akt was also determined by western blotting. The results showed that 6 and 10 mM Mg²⁺ elicited the highest stimulatory effect on cell adhesion, cell viability and osteogenic differentiation as evidenced by cytoskeletal staining, MTT assay results, ALP activity, extracellular matrix mineralisation and expression of osteogenic differentiation-related genes. In contrast, 18 mM Mg²⁺ had an inhibitory effect on the behaviour of osteoblasts. Furthermore, 10 mM Mg²⁺ significantly increased the phosphorylation of Akt in osteoblasts. Notably, the aforementioned beneficial effects produced by 10 mM Mg²⁺ were abolished by blocking the PI3K/Akt signalling pathway through the addition of wortmannin. In conclusion, these results demonstrate that 6 mM and 10 mM Mg²⁺ can enhance the behaviour of osteoblasts, which is at least partially attributed to activation of the PI3K/Akt signalling pathway. Furthermore, a high concentration (18 mM Mg²⁺) showed an inhibitory effect on the biological behaviour of osteoblasts. These findings advance the understanding of cellular responses to biodegradable metallic materials and may attract greater clinical interest in magnesium.     

Association of Iodine and Iron with Thyroid Function

Iodine and iron are essential elements for healthy thyroid function. However, little is known about the association of iron and iodine with thyroid function in the general US population. We investigated iron and iodine status in relation to concentrations of thyroid hormones. We included 7672 participants aged 20 and older from three surveys (2007–2008, 2009–2010, and 2011–2012) of the National Health and Nutrition Examination Survey. Serum thyroid measures (including free and total T3 and T4, and TSH), serum iron concentration, and urinary iodine concentrations were measured. Multivariate linear regression models were conducted with serum thyroid measures as dependent variables and combinations of serum iron concentration and urinary iodine concentration as predictors with covariate adjustment. Logistic regression models were performed with TSH levels (low, normal, and high) and combinations of serum iron concentration and urinary iodine concentration. Overall, 10.9% of the study population had low iron; 32.2 and 18.8% had low or high iodine levels, respectively. Compared with normal levels of iron and iodine, normal iron and high iodine were associated with reduced free T3 and increased risk of abnormal high TSH. Combined low iron and low iodine was associated with reduced free T3 and increased TSH. In addition, high iodine was associated with increased risk of abnormal high TSH in females but not in males. Thyroid function may be disrupted by low levels of iron or abnormal iodine, and relationships are complex and sex-specific. Large prospective studies are needed to understand the mechanisms by which iron interacts with iodine on thyroid function.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.