Effective pH pretreatment and cell disruption method for real-time intracellular enzyme activity assay of a marine fungus covered with pigments

<p>Filamentous fungi are capable producers of many bioactive compounds, and real-time intracellular enzyme activity assay is an essential guidance for their bioprocess developments. However, there are many difficulties in preparing homogenate for enzyme activity assay, such as disrupting fungal cell with complicated cellular structure and solid cell wall, removing abundant extracellular metabolites accumulating on mycelia, and so on. <i>Halorosellinia</i> sp. (No. 1403) was a marine-derived filamentous fungus producing a potential antitumor compound 1403C, and the deep red pigments (with main component of 1403C) covering on its mycelia showed strong absorption in a wide range, which critically affected the measurement of many enzyme activities. In this study, we developed an effective pH pretreatment and cell disruption method to prepare homogenate for enzyme activity assay. When mycelia were washed by the solution with pH 5.0 for 3 min, most pigments could be removed without severe loss on enzyme activities. Afterward, grinding with mini bead for 15 min with alternating cooling could effectively disrupt both cell wall and mitochondrial membrane. These methods have been successfully applied on real-time intracellular enzyme activity assay of <i>Halorosellinia</i> sp. (No. 1403) and can offer enlightenment for other filamentous fungi with similar problems.</p

Recombinant Human Parathyroid Hormone Related Protein 1-34 and 1-84 and Their Roles in Osteoporosis Treatment

<div><p>Osteoporosis is a common disorder characterized by compromised bone strength that predisposes patients to increased fracture risk. Parathyroid hormone related protein (PTHrP) is one of the candidates for clinical osteoporosis treatment. In this study, GST Gene Fusion System was used to express recombinant human PTHrP (hPTHrP) 1-34 and 1-84. To determine whether the recombinant hPTHrP1-34 and 1-84 can enhance renal calcium reabsorption and promote bone formation, we examined effects of recombinant hPTHrP1-34 and 1-84 on osteogenic lineage commitment in a primary bone marrow cell culture system and on osteoporosis treatment. Results revealed that both of recombinant hPTHrP1-34 and 1-84 increased colony formation and osteogenic cell differentiation and mineralization in vitro; however, the effect of recombinant hPTHrP1-84 is a little stronger than that of hPTHrP1-34. Next, ovariectomy was used to construct osteoporosis animal model (OVX) to test activities of these two recombinants in vivo. HPTHrP1-84 administration elevated serum calcium by up-regulating the expression of renal calcium transporters, which resulted in stimulation of osteoblastic bone formation. These factors contributed to augmented bone mass in hPTHrP1-84 treated OVX mice but did not affect bone resorption. There was no obvious bone mass alteration in hPTHrP1-34 treated OVX mice, which may be, at least partly, associated with shorter half-life of hPTHrP1-34 compared to hPTHrP1-84 in vivo. This study implies that recombinant hPTHrP1-84 is more effective than hPTHrP1-34 to enhance renal calcium reabsorption and to stimulate bone formation in vivo.</p></div

Molecular Insights into Multiphase Transport through Realistic Kerogen-Based Nanopores

Water is ubiquitous within organic-rich shale in cases of connate water occurrence and during hydraulic fracturing treatment. Understanding multiphase transport behaviors in organic nanopores is crucial for the efficient development of shale gas reservoirs. However, current studies have predominantly focused on single-phase or two-phase transport behaviors in ideal graphite nanopores, leaving the understanding of multiphase transport processes within realistic kerogen-based nanopores limited. In this study, we conducted molecular dynamic simulations to investigate shale gas transport behaviors through organic nanopores constructed with realistic kerogen. The results reveal that, due to the complex composition in the chemistry and physics of kerogen macromolecules, gas transport through kerogen nanopores manifests parabolic-shaped velocity distributions with a negligible slip length at the walls, in contrast to the observations of fast mass transport in previous studies using smooth carbon-based skeleton nanopores. In water-saturated nanopores, H2O molecules tend to aggregate at the walls, forming water clusters, and eventually, a water pillar across the pore can be observed. As a result, a water blockage is formed, while the water film or water bridge dominates in some inorganic minerals. The presence of H2O molecules has a dramatic impact on shale gas transport capacity. On this basis, an analytical model was proposed to quantitatively characterize shale gas transport behaviors under different water saturations. The results demonstrate that the traditional continuous model with no-slip assumption remains applicable because of the rough kerogen surface and hindrance of water clusters, advancing the understanding of multiphase transport behaviors in shale nanopores and exploitation of shale gas reservoirs

Effects of recombinant hPTHrP1-34 and 1-84 on osteoclastic bone resorption parameters and half-life analyses of recombinant hPTHrP1-34 and 1-84 in vivo.

<p>(a) Representative micrographs of paraffin embedded sections of vertebrae stained histochemically for TRAP and photographed at a magnification of 200. Scale bars in a represents 50 µm. (b) Number of TRAP positive osteoclasts per mm bone parameter (N.Oc/B.Pm, #/mm) and (c) the surface of osteoclasts relative to the bone surface (Oc.S/B.S, %) were determined in the trabeculae of TRAP-stained vertebrae. Each value is the mean ± SEM of determinations in 6 animals of each group. (d) Real-time RT-PCR was performed on long bone extracts for <i>RANKL</i> and <i>OPG</i> mRNA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088237#s2" target="_blank">Materials and Methods</a>. Messenger RNA expression assessed by real-time RT-PCR analysis is calculated as a ratio to the <i>GAPDH</i> mRNA level and expressed relative to levels of Sham group. Ratio of <i>RANKL/OPG</i> relative mRNA levels was calculated and was presented as the mean ± SEM of determinations in six animals of each group. (e) Plasma immunoreactive PTHrP determined using ELISA after subcutaneous injection of vehicle or hPTHrP1-34 or hPTHrP1-84. Blood samples were drawn at the following time points after injection of study drugs: 15 and 30 minutes, then 1, 2, 4, 6 hours. Each value is the mean ± SEM of determinations in 6 animals of each group. *, P<0.05; **, P<0.01 compared with Sham group.</p

Effects of recombinant hPTHrP1-34 and 1-84 on serum chemistry and on expression of calcium transporters in kidney.

<p>(a) Serum calcium, (b) phosphorus and (c) ALP ratio were determined after 4-week administration in vehicle-treated sham-operated mice (Sham), vehicle-treated OVX mice (Control), hPTHrP1-34-treated OVX mice (hPTHrP1-34) and hPTHrP1-84-treated OVX mice (hPTHrP1-84). (d) Western blots of renal extracts for expression of CB_28K, CB_9K, NCX1 and TRPV5. β-tubulin was used as loading control for Western blots. (e) CB_28K, CB_9K, NCX1 and TRPV5 protein levels relative to β-tubulin protein level and expressed relative to levels of Sham group. Each value is the mean ± SEM of determinations in six mice of each group.*, P<0.05; ***, P<0.001 compared with Sham group; #, P<0.05 compared with Control group; △, p<0.05 compared with hPTHrP1-34 group.</p

Effects of recombinant hPTHrP1-34 and 1-84 on BMCs.

<p>Cells from 18-day primary BMC cultures incubated in the absence (Control) or presence of 10⁻⁷ M hPTHrP1-34 (hPTHrP1-34) or presence of 10⁻⁷ M hPTHrP1-84 (hPTHrP1-84) on day 0 were stained cytochemically for ALP (a. CFU-f _ALP) and with sirius red for total collagen(c. CFU-f _Col) and with the von Kossa method for calcified colonies(e. CFU-f _Ca) and with methylene blue to show total CFU-f (g. Total CFU-f) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088237#s2" target="_blank">Materials and Methods</a>. The positive number of CFU-f _ALP (b), CFU-f _Col (d), CFU-f _Ca (f) and Total CFU-f number (h) per dish are the mean±SEM of triplicate determinations from three replicate experiments, respectively. (i) Real-time RT-PCR of cells extracts from 14-day primary BMC cultures for the expression of Cbfa I, ALP, Col I and OCN. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio to the GAPDH mRNA level and expressed relative to levels of Control group. Each value is the mean ± SEM of three trials. *, P<0.05; **, P<0.01; ***, P<0.001 compared with Control group; #, P<0.05 compared with hPTHrP1-34 group.</p

Preparation of Recombinant hPTHrP1-34 and 1-84.

<p>(a) Determination of the recombinants by PCR and double-enzyme (EcoR I+ BamH I) digestion. LaneM1, l-Hind III digest DNA marker; Lane1-3, double-enzyme digestion patterns of recombinant <i>hPTHrP1-34</i>, <i>hPTHrP1-84</i> and <i>pGEX-2TK</i>; LaneM2, DL2000 DNA marker; Lane4-5, PCR band patterns of recombinant hPTHrP1-34 and hPTHrP1-84. SDS-PAGE analysis of the GST-hPTHrP1-34 (b) or GST-hPTHrP1-84 (d) fusion protein under inducing condition and stained with coomassie blue. LaneM, molecular weight marker; Lane1, total cell soluble protein of BL21, control; Lanes2–6, cells with <i>pGEX-2TK/hPTHrP1-34</i> or <i>pGEX-2TK/hPTHrP1-84</i> after inducing with IPTG 0 h, 1 h, 2 h, 3 h, 4 h respectively. SDS-PAGE analyses of hPTHrP1-34 (c) or hPTHrP1-84 (e) peptide expression and purification. LaneM, molecular weight marker; Lane1, IPTG induction of BL21 transformed with <i>pGEX-2TK</i> without insert (expression control); Lane2, BL21 transformed with <i>pGEX-2TK/hPTHrP1-34</i> or <i>pGEX-2TK/hPTHrP1-84</i> without IPTG induction (induction control); Lane3, IPTG induction of BL21 transformed with <i>pGEX-2TK/hPTHrP1-34</i> or <i>pGEX-2TK/hPTHrP1-84</i>; Lane4, The proteins not bound to GSTrap FF column; Lane5, Purified hPTHrP1-34 or hPTHrP1-84 obtained after binding to GSTrap FF column and cleaved by thrombin; Lane6, GST-tag eluted from GSTrap FF column. (f) Western blot analysis of purified recombinant hPTHrP1-34 and hPTHrP1-84 with N-terminal hPTHrP antibody.</p

Effects of recombinant hPTHrP1-34 and 1-84 on osteoblastic bone formation parameters.

<p>(a) Representative micrographs of calcein double labeling and (c) sections stained with the von Kossa procedure in the trabeculae were imaged from ethanol fixed and undecalcified LR white resin embedded sections of vertebrae. (b) MAR of trabeculae was determined. (d) Osteoid volume was determined in undecalcified von Kossa-stained sections and is presented as a percent of bone volume (OV/BV, %) of trabeculae. Micrographs of decalcified paraffin sections of vertebrae stained with H&E (e) and histochemically for ALP (g). (f) Number of osteoblasts per mm bone parameter (N.Ob/B.Pm, #/mm) and (h) ALP positive area as a percent of the tissue area were determined in the vertebrae. Scale bars represent 25 µm in a and c and 50 µm in e and g. (i) Real-time RT-PCR of long bone extracts for the expression of <i>Cbfa I</i>, <i>ALP</i>, <i>Col I</i> and <i>OCN</i>. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio to the <i>GAPDH</i> mRNA level and expressed relative to levels of Sham group. Each value is the mean ± SEM of determinations in six mice of each group. *, P<0.05; **, P<0.01 compared with Sham group; #, P<0.05; ##, P<0.01 compared with Control group; △, P<0.05 compared with hPTHrP1-34 group.</p

Converting a Microarray Signature into a Diagnostic Test: A Trial of Custom 74 Gene Array for Clarification and Prediction the Prognosis of Gastric Cancer

<div><p>Background</p><p>Gastric cancer (GC) is associated with high mortality rates and an unfavorable prognosis at advanced stages. In addition, there are no effective methods for diagnosing gastric cancer at an early stage or for predicting the outcome for the purpose of selecting patient-specific treatment options. Therefore, it is important to investigate new methods for GC diagnosis. </p> <p>Methodology/Principal Findings</p><p>To facilitate its use in a diagnostic setting, a group of 74 genes with diagnostic and prognostic information was translated into a customized microarray containing a reduced set of 1,042 probes suitable for high throughput processing. In this report, we demonstrate for the first time that the custom mini-array can be used as a reliable diagnostic tool in gastric cancer. With an AUC value of 0.565 (95% CI 0.305-0.825) indicating a perfect test, the sensitivity and specificity of diagnosis from the ROC curve were calculated to be 70% and 80%, respectively. </p> <p>Conclusions/Significance</p><p>The data clearly demonstrate the reproducibility and robustness of the small custom-made microarray. The array is an excellent tool for classifying and predicting the outcome of disease in gastric cancer patients.</p> </div

Physiological and Biochemical Changes Imposed by CeO₂ Nanoparticles on Wheat: A Life Cycle Field Study

Interactions of <i>n</i>CeO₂ with plants have been mostly evaluated at seedling stage and under controlled conditions. In this study, the effects of <i>n</i>CeO₂ at 0 (control), 100 (low), and 400 (high) mg/kg were monitored for the entire life cycle (about 7 months) of wheat plants grown in a field lysimeter. Results showed that at high concentration <i>n</i>CeO₂ decreased the chlorophyll content and increased catalase and superoxide dismutase activities, compared with control. Both concentrations changed root and leaf cell microstructures by agglomerating chromatin in nuclei, delaying flowering by 1 week, and reduced the size of starch grains in endosperm. Exposed to low concentration produced embryos with larger vacuoles, while exposure to high concentration reduced number of vacuoles, compared with control. There were no effects on the final biomass and yield, Ce concentration in shoots, as well as sugar and starch contents in grains, but grain protein increased by 24.8% and 32.6% at 100 and 400 mg/kg, respectively. Results suggest that more field life cycle studies are needed in order to better understand the effects of <i>n</i>CeO₂ in crop plants