(-)-Epigallocatechin-3-gallate induced apoptosis by dissociation of c-FLIP/Ku70 complex in gastric cancer cells

Anti-cancer properties of (-)-epigallocatechin-3-gallate (EGCG) are mediated via apoptosis induction, as well as inhibition of cell proliferation and histone deacetylase. Accumulation of stabilized cellular FLICE-inhibitory protein (c-FLIP)/Ku70 complex in the cytoplasm inhibits apoptosis through interruption of extrinsic apoptosis pathway. In this study, we evaluated the anti-cancer role of EGCG in gastric cancer (GC) cells through dissociation of c-FLIP/Ku70 complex. MKN-45 cells were treated with EGCG or its antagonist MG149 for 24 h. Apoptosis was evaluated by flow cytometry and quantitative RT-PCR. Protein expression of c-FLIP and Ku70 was analysed using western blot and immunofluorescence. Dissociation of c-FLIP/Ku70 complex as well as Ku70 translocation were studied by sub-cellular fractionation and co-immunoprecipitation. EGCG induced apoptosis in MKN-45 cells with substantial up-regulation of P53 and P21, down-regulation of c-Myc and Cyclin D1 as well as cell cycle arrest in S and G2/M check points. Moreover, EGCG treatment suppressed the expression of c-FLIP and Ku70, decreased their interaction while increasing the Ku70 nuclear content. By dissociating the c-FLIP/Ku70 complex, EGCG could be an alternative component to the conventional HDAC inhibitors in order to induce apoptosis in GC cells. Thus, its combination with other cancer therapy protocols could result in a better therapeutic outcome.     

Wound dressing based on PVA nanofiber containing silk fibroin modified with GO/ZnO nanoparticles for superficial wound healing: In vitro and in vivo evaluations

Silk fibroin (SF), extracted from Bombyx mori, has unique physicochemical properties to achieve an efficient wound dressing. In this study, reduced graphene oxide (RGO)/ZnO NPs/silk fibroin nanocomposite was made, and an innovative nanofiber of SF/polyvinyl alcohol (PVA)/RGO/ZnO NPs was ready with the electrospinning technique and successfully characterized. The results of MIC and OD analyses were used to investigate the synthesized materials' antibacterial effects and displayed that the synthesized materials could inhibit growth against Staphylococcus aureus and Escherichia coli bacteria. However, both in vitro cytotoxicity (MTT) and scratch wound studies have shown that RGO/ZnO NPs and SF/PVA/RGO/ZnO NPs are not only non-toxic to NIH 3T3 fibroblasts, but also can cause cell viability, cell proliferation, and cell migration. Furthermore, improving the synthesized nanofiber's structural properties in the presence of RGO and ZnO NPs has been confirmed by performing tensile strength, contact angle, and biodegradation analyses. Also, in a cell attachment analysis, fibroblast cells had migrated and expanded well in the nanofibrous structures. Moreover, in vivo assay, SF/PVA/RGO/ZnO NPs nanofiber treated rats and has been shown significant healing activity and tissue regeneration compared with other treated groups. Therefore, this study suggests that SF/PVA/RGO/ZnO NPs nanofiber is a hopeful wound dressing for preventing bacteria growth and improving superficial wound repair.     

Optical PCR: Genomic analysis by long-range PCR and optical mapping

Optical mapping is an approach for the rapid, automated, non-electrophoretic construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genomic loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors. Received: 9 February 1999 / Accepted: 26 May 1999     

Conjugation of tetanus toxoid with Salmonella typhimurium PTCC 1735 O-specific polysaccharide and its effects on production of opsonizing antibodies in a mouse model

Serious enteric and extra-intestinal infections with Salmonella typhimurium are very common in many human populations. Phagocytosis is the main defense mechanism against this bacterium; however, the unique structure of S. typhimurium lipopolysaccharide (LPS) makes it resistant to opsonization by complement components. In the present study, the S. typhimurium LPS O-chain was purified and conjugated with tetanus toxoid and the effects of the conjugated vaccine (O-specific polysaccharide tetanus toxoid (O-SP-TT)) on induction of specific antibodies were investigated in a mouse model. In vitro assays measuring phagocytosis in the presence of opsonizing antibodies were performed. Three subcutaneous injections of the O-SP-TT conjugate conferred protection against the intraperitoneal challenge with S. typhimurium and the LD50 was greater for immunized animals than for controls. The mean number of ingested S. typhimirium / mouse peritoneal cell in the presence of sera obtained from immunized mice with purified O-chain, O-SP-TT conjugate, heat-killed bacteria, and negative control were 6.96, 14.24, 15.96, and 6.67, respectively. In summary, we developed an O-SP-TT conjugate that induced opsonizing antibodies that increased phagocytosis, as determined by in vitro assays. In addition, chemiluminescence results, an indicator of oxidative burst, indicated that peritoneal cells respond better to live S. typhimurium cells in the presence of sera obtained from O-SP-TT conjugate immunized mice.     

Repulsive guidance molecule (RGMa), a DRAGON homologue, is a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta (TGF-beta) superfamily of ligands, which regulate many mammalian physiologic and pathophysiologic processes. BMPs exert their effects through type I and type II serine/threonine kinase receptors and the Smad intracellular signaling pathway. Recently, the glycosylphosphatidylinositol (GPI)-anchored protein DRAGON was identified as a co-receptor for BMP signaling. Here, we investigate whether a homologue of DRAGON, repulsive guidance molecule (RGMa), is similarly involved in the BMP signaling pathway. We show that RGMa enhances BMP, but not TGF-beta, signals in a ligand-dependent manner in cell culture. The soluble extracellular domain of RGMa fused to human Fc (RGMa.Fc) forms a complex with BMP type I receptors and binds directly and selectively to radiolabeled BMP-2 and BMP-4. RGMa mediates BMP signaling through the classical BMP signaling pathway involving Smad1, 5, and 8, and it up-regulates endogenous inhibitor of differentiation (Id1) protein, an important downstream target of BMP signals. Finally, we demonstrate that BMP signaling occurs in neurons that express RGMa in vivo. These data are consistent with a role for RGMa as a BMP co-receptor.     

Milk progesterone profiles in various reproductive states in dairy buffaloes under field conditions

Fifty-one dairy buffaloes in the last two months of gestation were selected at seven private peri-urban farms in the Peshawar district. Observations were recorded in buffaloes during normal (NBS, August to January) and low breeding seasons (LBS, February to July). After parturition, rectal examination of reproductive organs was carried out. Estrus detection was made through visual observation and the use of intact bull. Postpartum ovulation was confirmed by ovarian palpation per rectum and milk progesterone levels (MPL), determined through radio-immunoassay. MPL was higher (p < 0.01) at various intervals in NBS calves (1.97 +/- 0.30 ng/ml) as compared to LBS calves (0.68 +/- 0.08 ng/ml). During LBS, MPL remained < 0.30 ng/ml up to the third fortnight and started rising later, reaching a peak of 1.27 ng/ml during the sixth fortnight. During NBS, there was a sharp rise in MPL during the second fortnight, reaching 3.64 ng/ml during the sixth fortnight. MPL was significantly different on different experimental farms (p < 0.01). MPL reached the lowest levels on the day of estrus (0.10 ng/ml), reached it's peak on day 7 and started declining on day 17 of estrus. MPL showed two postpartum elevations. In true anestrus buffaloes, MPL remained consistently low. However, in the anestrus period, silent ovulations were also noted, as reflected by increasing MPL without estrus signs. In pregnant buffaloes, MPL remained > 1 ng/ml. Results of the study showed that the low postpartum reproductive performance in dairy buffaloes during LBS was primarily due to inadequate functioning of the corpus luteum in secreting optimum concentrations of progesterone. The higher incidence of silent estrus during LBS indicated improved management for the detection of estrus.     

DRAGON, a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)beta superfamily of ligands that regulate many crucial aspects of embryonic development and organogenesis. Unlike other TGFbeta ligands, co-receptors for BMP ligands have not been described. Here we show that DRAGON, a glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, which is expressed early in the developing nervous system, enhances BMP but not TGFbeta signaling. DRAGON binds directly to BMP2 and BMP4 but not to BMP7 or other TGFbeta ligands. The enhancing action of DRAGON on BMP signaling is also reduced by administration of Noggin, a soluble BMP antagonist, indicating that the action of DRAGON is ligand-dependent. DRAGON associates directly with BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In the Xenopus embryo, DRAGON both reduces the threshold of the ability of Smad1 to induce mesodermal and endodermal markers and alters neuronal and neural crest patterning. The direct interaction of DRAGON with BMP ligands and receptors indicates that it is a BMP co-receptor that potentiates BMP signaling.     

Hydrogeochemistry of seasonal variation of Urmia Salt Lake, Iran

Urmia Lake has been designated as an international park by the United Nations. The lake occupies a 5700 km2 depression in northwestern Iran. Thirteen permanent rivers flow into the lake. Water level in the lake has been decreased 3.5 m in the last decade due to a shortage of precipitation and progressively dry climate. Geologically the lake basin is considered to be a graben of tectonic origin. Na, K, Ca, Li and Mg are the main cations with Cl, SO4, and HCO3 as the main anions. F & Br are the other main elements in the lake. A causeway crossing the lake is under construction, which may affect the lake's annual geochemistry. The main object of this project is mainly to consider the potential of K-mineral production along with ongoing salt production. Seven hundred and four samples were taken and partially analyzed for the main cations and anions. Surface water (0.5 m. depth) was analyzed for Na, K, Mg, Ca, Br and Li, and averaged 87.118 g/lit, 1.48 g/lit, 4.82 g/lit, 4.54 g/lit, 1.19 ppm and 12.7 ppm respectively for the western half of the lake. Sodium ranged between 84 to 91.2 g/lit, and showed higher concentrations in the south than in the north. This unexpected result may be caused by shallower depth in the south and a higher net evaporation effect. Calcium ranged between 4.2 to 5 g/lit, apparently slightly higher in the north. K is higher in the south, possibly due to rivers entering from south that may carry slightly higher K in solution. In the middle-range samples (0.5–5 m.), K averaged 1.43 g/lit and ranged from 1.40 to 1.46 g/lit. At this intermediate depth the distribution of K is clearly higher to the south of the causeway that is currently under construction. It is not clear whether this increase is the effect of the causeway or the effect of the salty Aji-Chay River to the east, and the Khoy salt domes to the north of the lake. At depth (5 m–10 m), K averaged 1.48 g/lit and ranged from 1.4 to 1.49 g/lit, differing only in the second decimal from the average of the middle and surface samples. Ignoring the small difference between the averages of the three sample depths, the distribution of K is highly homogeneous in the lake water due to the mixing process. Therefore causeway construction has not yet strongly affected K distribution, or it may be at the starting point. Magnesium concentration ranged from 4.6 to 5-g/lit, and was elevated in the south. This differs somewhat compared to calcium. Lithium, with an average of 12–13 ppm, is slightly higher in the south, and has not shown any significant variation in all three seasons. Iodine was below the detection limit in the lake. Urmia Lake, geochemically, is highly uniform both to the south and north of the causeway, in both the surface and deep brines. K and Mg, which average 1.48 and 6.6 g/lit in order, could be elements worth production in addition to the NaCl currently being produced from the lake. Br, F, Li and B in the limit of <50 ppm don't look to be in the economical range.     

Biomarkers of Plasmodium falciparum Infection during Pregnancy in Women Living in Northeastern Tanzania

In pregnant women, Plasmodium falciparum infections are an important cause of maternal morbidity as well as fetal and neonatal mortality. Erythrocytes infected by these malaria-causing parasites accumulate through adhesive interactions in placental intervillous spaces, thus evading detection in peripheral blood smears. Sequestered infected erythrocytes induce inflammation, offering the possibility of detecting inflammatory mediators in peripheral blood that could act as biomarkers of placental infection. In a longitudinal, prospective study in Tanzania, we quantified a range of different cytokines, chemokines and angiogenic factors in peripheral plasma samples, taken on multiple sequential occasions during pregnancy up to and including delivery, from P. falciparum-infected women and matched uninfected controls. The results show that during healthy, uninfected pregnancies the levels of most of the panel of molecules we measured were largely unchanged except at delivery. In women with P. falciparum, however, both comparative and longitudinal assessments consistently showed that the levels of IL-10 and IP-10 increased significantly whilst that of RANTES decreased significantly, regardless of gestational age at the time the infection was detected. ROC curve analysis indicated that a combination of increased IL-10 and IP-10 levels and decreased RANTES levels might be predictive of P. falciparum infections. In conclusion, our data suggest that host biomarkers in peripheral blood may represent useful diagnostic markers of P. falciparum infection during pregnancy, but placental histology results would need to be included to verify these findings.     

Determining the Most Important Physiological and Agronomic Traits Contributing to Maize Grain Yield through Machine Learning Algorithms: A New Avenue in Intelligent Agriculture

Prediction is an attempt to accurately forecast the outcome of a specific situation while using input information obtained from a set of variables that potentially describe the situation. They can be used to project physiological and agronomic processes; regarding this fact, agronomic traits such as yield can be affected by a large number of variables. In this study, we analyzed a large number of physiological and agronomic traits by screening, clustering, and decision tree models to select the most relevant factors for the prospect of accurately increasing maize grain yield. Decision tree models (with nearly the same performance evaluation) were the most useful tools in understanding the underlying relationships in physiological and agronomic features for selecting the most important and relevant traits (sowing date-location, kernel number per ear, maximum water content, kernel weight, and season duration) corresponding to the maize grain yield. In particular, decision tree generated by C&RT algorithm was the best model for yield prediction based on physiological and agronomical traits which can be extensively employed in future breeding programs. No significant differences in the decision tree models were found when feature selection filtering on data were used, but positive feature selection effect observed in clustering models. Finally, the results showed that the proposed model techniques are useful tools for crop physiologists to search through large datasets seeking patterns for the physiological and agronomic factors, and may assist the selection of the most important traits for the individual site and field. In particular, decision tree models are method of choice with the capability of illustrating different pathways of yield increase in breeding programs, governed by their hierarchy structure of feature ranking as well as pattern discovery via various combinations of features.