Electrochemical Enzyme Immunoassay Using Sequential Saturation Technique in A 20-μl Capillary: Digoxin as A Model Analyte

Capillary enzyme immunoassay with flow-injection analysis for digoxin using the sequential saturation technique has been developed. Glass capillary tubes (10 cm × 0.53 mm i.d.) with immobilized digoxin antibody were used as the immunoassay reactor. The product of enzymatic reaction. 4-aminophenol, was detected amperometrically. The digoxin and the labeled digoxin binding reaction with the immobilized digoxin antibody were completed in 2 and 10 min, respectively. Digoxin was determined in a 20-μl sample with a detection limit of 10 pg ml−1 (200 fg or 260 attomoles) and a 3 orders of magnitude range

Genome-wide parent-of-origin DNA methylation analysis reveals the intricacies of human imprinting and suggests a germline methylation-independent mechanism of establishment

Differential methylation between the two alleles of a gene has been observed in imprinted regions, where the methylation of one allele occurs on a parent-of-origin basis, the inactive X-chromosome in females, and at those loci whose methylation is driven by genetic variants. We have extensively characterized imprinted methylation in a substantial range of normal human tissues, reciprocal genome-wide uniparental disomies, and hydatidiform moles, using a combination of whole-genome bisulfite sequencing and high-density methylation microarrays. This approach allowed us to define methylation profiles at known imprinted domains at base-pair resolution, as well as to identify 21 novel loci harboring parent-of-origin methylation, 15 of which are restricted to the placenta. We observe that the extent of imprinted differentially methylated regions (DMRs) is extremely similar between tissues, with the exception of the placenta. This extra-embryonic tissue often adopts a different methylation profile compared to somatic tissues. Further, we profiled all imprinted DMRs in sperm and embryonic stem cells derived from parthenogenetically activated oocytes, individual blastomeres, and blastocysts, in order to identify primary DMRs and reveal the extent of reprogramming during preimplantation development. Intriguingly, we find that in contrast to ubiquitous imprints, the majority of placenta-specific imprinted DMRs are unmethylated in sperm and all human embryonic stem cells. Therefore, placental-specific imprinting provides evidence for an inheritable epigenetic state that is independent of DNA methylation and the existence of a novel imprinting mechanism at these loci

IODP workshop: Core-Log Seismic Investigation at Sea – Integrating legacy data to address outstanding research questions in the Nankai Trough Seismogenic Zone Experiment

The first International Ocean Discovery Program (IODP) Core-Log-Seismic Integration at Sea (CLSI@Sea) workshop, held in January–February 2018, brought together an international, multidisciplinary team of 14 early-career scientists and a group of scientific mentors specialized in subduction zone processes at the Nankai Trough, one of the Earth's most active plate-subduction zones located off the southwestern coast of Japan. The goal of the workshop was to leverage existing core, log, and seismic data previously acquired during the IODP's Nankai Trough Seismogenic Zone Experiment (NanTroSEIZE), to address the role of the deformation front of the Nankai accretionary prism in tsunamigenic earthquakes and slow slip in the shallow portion of the subduction interface. The CLSI@Sea workshop was organized onboard the D/V Chikyu concurrently with IODP Expedition 380, allowing workshop participants to interact with expedition scientists installing a long-term borehole monitoring system (LTBMS) at a site where the workshop's research was focused. Sedimentary cores from across the deformation front were brought onboard Chikyu, where they were made available for new description, sampling, and analysis. Logging data, drilling parameters, and seismic data were also available for investigation by workshop participants, who were granted access to Chikyu laboratory facilities and software to perform analyses at sea.Multi-thematic presentations facilitated knowledge transfer between the participants across field areas, and highlighted the value of multi-disciplinary collaboration that integrates processes across different spatiotemporal scales. The workshop resulted in the synthesis of existing geophysical, geologic, and geochemical data spanning IODP Sites C0006, C0007, C0011 and C0012 in the NanTroSEIZE area, the identification of key outstanding research questions in the field of shallow subduction zone seismogenesis, and fostered collaborative and individual research plans integrating new data analysis techniques and multidisciplinary approaches.</p

JNK interacting protein 1 (JIP-1) protects LNCaP prostate cancer cells from growth arrest and apoptosis mediated by 12-0-tetradecanoylphorbol-13-acetate (TPA)

12-0-tetradecanoylphorbol-13-acetate (TPA) stimulates protein kinase C (PKC) which mediates apoptosis in androgen-sensitive LNCaP human prostate cancer cells. The downstream signals of PKC that mediate TPA-induced apoptosis in LNCaP cells are unclear. In this study, we found that TPA activates the c-Jun NH2-terminal kinase (JNK)/c-Jun/AP-1 pathway. To explore the possible role that the JNK/c-Jun/AP-1 signal pathway has on TPA-induced apoptosis in LNCaP cells, we stably transfected the scaffold protein, JNK interacting protein 1 (JIP-1), which binds to JNK inhibiting its ability to phosphorylate c-Jun. TPA (10(-9)-10(-7) mol l(-1)) caused phosphorylation of JNK in both wild-type and JIP-1-transfected (LNCaP-JIP-1) cells. It resulted in phosphorylation and upregulation of expression of c-Jun protein in the wild-type LNCaP cells, but not in the JIP-1-transfected LNCaP cells. In addition, upregulation of AP-1 reporter activity by TPA (10(-9) mol l(-1)) occurred in LNCaP cells but was abrogated in LNCaP-JIP-1 cells. Thus, TPA stimulated c-Jun through JNK, and JIP-1 effectively blocked JNK. TPA (10(-12)-10(-8) mol l(-1)) treatment of LNCaP cells caused their growth inhibition, cell cycle arrest, upregulation of p53 and p21waf1, and induction of apoptosis. All of these effects were significantly attenuated when LNCaP-JIP-1 cells were similarly treated with TPA. A previous study showed that c-Jun/AP-1 blocked androgen receptor (AR) signaling by inhibiting AR binding to AR response elements (AREs) of target genes including prostate-specific antigen (PSA). Therefore, we hypothesised that TPA would not be able to disrupt the AR signal pathway in LNCaP-JIP-1 cells. Contrary to expectation, TPA (10(-9)-10(-8) mol l(-1)) inhibited DHT-induced AREs reporter activity and decreased levels of PSA in the LNCaP-JIP-1 cells. Taken together, TPA, probably by stimulation of PKC, phosphorylates JNK, which phosphorylates and increases expression of c-Jun leading to AP-1 activity. Growth control of prostate cancer cells can be mediated through the JNK/c-Jun pathway, but androgen responsiveness of these cells can be independent of this pathway, suggesting that androgen independence in progressive prostate cancer may not occur through activation of this pathway

Increased expression of phosphorylated forms of RNA-dependent protein kinase and eukaryotic initiation factor 2α may signal skeletal muscle atrophy in weight-losing cancer patients

Previous studies suggest that the activation (autophosphorylation) of dsRNA-dependent protein kinase (PKR) can stimulate protein degradation, and depress protein synthesis in skeletal muscle through phosphorylation of the translation initiation factor 2 (eIF2) on the α-subunit. To understand whether these mediators are important in muscle wasting in cancer patients, levels of the phospho forms of PKR and eIF2α have been determined in rectus abdominus muscle of weight losing patients with oesophago-gastric cancer, in comparison with healthy controls. Levels of both phospho PKR and phospho eIF2α were significantly enhanced in muscle of cancer patients with weight loss irrespective of the amount and there was a linear relationship between phosphorylation of PKR and phosphorylation of eIF2α (correlation coefficient 0.76, P=0.005). This suggests that phosphorylation of PKR led to phosphorylation of eIF2α. Myosin levels decreased as the weight loss increased, and there was a linear relationship between myosin expression and the extent of phosphorylation of eIF2α (correlation coefficient 0.77, P=0.004). These results suggest that phosphorylation of PKR may be an important initiator of muscle wasting in cancer patients

Peak bone mineral density in Vietnamese women

While the prevalence of osteoporosis and risk factors for low bone mineral density (BMD) has been well documented in Caucasian populations, there is a lack of data from Asia. This work was designed to clarify to what extent osteoporosis could be regarded as a major public health problem in Vietnam. Furthermore, to elucidate the prevalence of certain risk factors, such as vitamin D deficiency and other determinants of bone mass as a basis to indentify high-risk individuals among the Vietnamese women and men. The clinical studies were designed as cross-sectional investigations using a multistage sampling scheme. Within the setting of northern Vietnam (latitude 21°N), districts were selected to represent urban and rural areas. In total 612 healthy women and 222 men aged 13-83 years were investigated. BMD was measured at the lumbar spine, femoral neck and total hip in all qualified subjects with dual energy X-ray absortiometry. Serum concentrations of 25(OH)D, parathyroid hormone, estrogen and testosterone were quantified by electrochemiluminescence immunoassay. Data on clinical history and lifestyle were collected by individual face-to-face interviews. Reference values for peak BMD were defined. These data allowed the calculation of T-scores and thus for the first time, an accurate identification of osteoporosis in a Vietnamese population. As determined at the femoral neck, the prevalence of osteoporosis was 17-23% in women and 9% in men. The results clearly suggest that osteoporosis is an important public health problem. Postmenopausal women living in urban areas experienced osteoporosis more than rural residents. Serum levels of 25(OH)D and estrogen were significantly associated with bone mass in both women and men. The prevalence of vitamin D deficiency (<20 ng/mL) was very high, 30% in women and 16% in men. An experimental study on the isoflavone content of different soymilk preparations was performed by HPLC (high pressure liquid chromatography). Values of isoflavones were very low, around 60-80 mg/L, and there were only 10-20% of bioactive aglycones. This is far below the reported threshold levels to exert significant effects on bone. In the future these data will be useful as a valuable reference base to diagnose osteoporosis and for the clinical management of its consequences. The high prevalence of vitamin D deficiency should raise the awareness of potentially important health issues such as osteoporosis but also about other serious diseases within the Vietnamese society

Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes.

The natriuretic peptides, Atrial-, B-type and C-type natriuretric peptides (ANP, BNP, CNP), are regulators of many endocrine tissues and exert their effects predominantly through the activation of their specific guanylyl cyclase receptors (GC-A and GC-B) to generate cGMP. Whereas cGMP-independent signalling has been reported in response to natriuretic peptides, this is mediated via either the clearance receptor (Npr-C) or a renal-specific NPR-Bi isoform, which both lack intrinsic guanylyl cyclase activity. Here, we report evidence of GC-B-dependent cGMP-independent signalling in pituitary GH3 cells. Stimulation of GH3 cells with CNP resulted in a rapid and sustained enhancement of ERK1/2 phosphorylation (P-ERK1/2), an effect that was not mimicked by dibutryl-cGMP. Furthermore, CNP-stimulated P-ERK1/2 occurred at concentrations below that required for cGMP accumulation. The effect of CNP on P-ERK1/2 was sensitive to pharmacological blockade of MEK (U0126) and Src kinases (PP2). Silencing of the GC-B1 and GC-B2 splice variants of the GC-B receptor by using targeted short interfering RNAs completely blocked the CNP effects on P-ERK1/2. CNP failed to alter GH3 cell proliferation or cell cycle distribution but caused a concentration-dependent increase in the activity of the human glycoprotein α-subunit promoter (αGSU) in a MEK-dependent manner. Finally, CNP also activated the p38 and JNK MAPK pathways in GH3 cells. These findings reveal an additional mechanism of GC-B signalling and suggest additional biological roles for CNP in its target tissues