Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia.

Consistent with recent reports indicating that neurons differentiated in vitro from human-induced pluripotent stem cells (hiPSCs) are immature relative to those in the human brain, gene expression comparisons of our hiPSC-derived neurons to the Allen BrainSpan Atlas indicate that they most resemble fetal brain tissue. This finding suggests that, rather than modeling the late features of schizophrenia (SZ), hiPSC-based models may be better suited for the study of disease predisposition. We now report that a significant fraction of the gene signature of SZ hiPSC-derived neurons is conserved in SZ hiPSC neural progenitor cells (NPCs). We used two independent discovery-based approaches-microarray gene expression and stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomic mass spectrometry analyses-to identify cellular phenotypes in SZ hiPSC NPCs from four SZ patients. From our findings that SZ hiPSC NPCs show abnormal gene expression and protein levels related to cytoskeletal remodeling and oxidative stress, we predicted, and subsequently observed, aberrant migration and increased oxidative stress in SZ hiPSC NPCs. These reproducible NPC phenotypes were identified through scalable assays that can be applied to expanded cohorts of SZ patients, making them a potentially valuable tool with which to study the developmental mechanisms contributing to SZ.

Filling up the heme pocket stabilizes apomyoglobin and speeds up its folding.

Wild type apomyoglobin folds in at least two steps: the ABGH core rapidly, followed much later by the heme-binding CDEF core. We hypothesize that the evolved heme-binding function of the CDEF core frustrates its folding: it has a smaller contact order and is no more complex topologically than ABGH, and thus, it should be able to fold faster. Therefore, filling up the empty heme cavity of apomyoglobin with larger, hydrophobic side chains should significantly stabilize the protein and increase its folding rate. Molecular dynamics simulations allowed us to design four different mutants with bulkier side chains that increase the native bias of the CDEF region. In vitro thermal denaturation shows that the mutations increase folding stability and bring the protein closer to two-state behavior, as judged by the difference of fluorescence- and circular dichroism-detected protein stability. Millisecond stopped flow measurements of the mutants exhibit refolding kinetics that are over 4 times faster than the wild type's. We propose that myoglobin-like proteins not evolved to bind heme are equally stable, and find an example. Our results illustrate how evolution for function can force proteins to adapt frustrated folding mechanisms, despite having simple topologies.

Relationship between chlorine consumption and chlorination by-products formation for model compounds.

The objective of this research is to investigate the relationship between chlorine decay and the formations of disinfection by-products (DBP), including trichloromethane (TCM) and chloroacetic acid (CAA) in the presence of four model compounds, i.e., resorcinol, phloroglucinol, p-hydroxybenzoic acid, and m-hydroxybenzoic acid. The chlorine degradation in model compounds with OH and/or COOH functional groups were rapid after chlorination. The TCM yields of carboxylic group substituted compounds (3-hydroxybenzoic acid [3-HBA], 4-hydroxybenzoic acid [4-HBA]) were found to be lower than that of the m-dihydroxy substituted compounds. Phloroglucinol, with one more OH substitution group than resorcinol, tends to form significant amounts of CAA after chlorination. However, it was observed that with the COOH substitution of 3-HBA and 4-HBA tend to exhibit more CAA formation potential than resorcinol. The developed parallel second and first-order reaction model for chlorine demand has been successfully utilized for TCM, CAA and DBP formation modeling. A high correlation between CAA and TCM was observed for the model compounds.

Surface modification and initial adhesion events for intestinal epithelial cells.

Rapid resealing of the mucosal epithelia is imperative following injuries to the small intestine because the mucosa is responsible for the adsorption of nutrients as well as providing a barrier to noxious agents present in the lumen. Tissue engineering may provide a possible solution for treating intestinal erosions, ulcerations, inflammatory bowel disease, and infection. Cell-biomaterial interaction is a critical component in tissue engineering that can determine the success of the tissue construct. Cell-biomaterial interactions can be enhanced by various types of surface modification, which promote integrin ligation leading to increased cell function. In order to relate the effect of surface adhesion molecules to signaling events and macroscopic cell response, an intestinal epithelial cell line, IEC-6, was plated on fibronectin (receptor-mediated) and poly-L-lysine (non-specific) surfaces. Focal adhesion kinase (FAK) phosphorylation, cell spreading, and cell adhesion strength were measured. Results showed increases in FAK phosphorylation generally corresponded to increases in cell spreading and adhesion strength for IEC-6 cells. Therefore, in a simplified system, initial adhesion and signaling mechanisms appeared to correspond to subsequent physical responses in IEC-6 cells relevant to tissue engineering applications.

Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo.

Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor in clinical trials as a cancer therapy that has been recently shown to block human immunodeficiency virus Tat transactivation and viral replication through inhibition of positive transcription elongation factor b (P-TEFb). Flavopiridol is the most potent P-TEFb inhibitor reported and the first Cdk inhibitor that is not competitive with ATP. We examined the ability of flavopiridol to inhibit P-TEFb (Cdk9/cyclin T1) phosphorylation of both RNA polymerase II and the large subunit of the 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and found that the IC(50) determined was directly related to the concentration of the enzyme. We concluded that the flavonoid associates with P-TEFb with 1:1 stoichiometry even at concentrations of enzyme in the low nanomolar range. These results indicate that the apparent lack of competition with ATP could be caused by a very tight binding of the drug. We developed a novel immobilized P-TEFb assay and demonstrated that the drug remains bound for minutes even in the presence of high salt. Flavopiridol remained bound in the presence of a 1000-fold excess of the commonly used inhibitor DRB, suggesting that the immobilized P-TEFb could be used in a simple screening assay that would allow the discovery or characterization of compounds with binding properties similar to flavopiridol. Finally, we compared the ability of flavopiridol and DRB to inhibit transcription in vivo using nuclear run-on assays and concluded that P-TEFb is required for transcription of most RNA polymerase II molecules in vivo.

Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription.

The C-terminal domain (CTD) of the large subunit of RNA polymerase II plays a role in transcription and RNA processing. Yeast ESS1, a peptidyl-prolyl cis/trans isomerase, is involved in RNA processing and can associate with the CTD. Using several types of assays we could not find any evidence of an effect of Pin1, the human homolog of ESS1, on transcription by RNA polymerase II in vitro or on the expression of a reporter gene in vivo. However, an inhibitor of Pin1, 5-hydroxy-1,4-naphthoquinone (juglone), blocked transcription by RNA polymerase II. Unlike N-ethylmaleimide, which inhibited all phases of transcription by RNA polymerase II, juglone disrupted the formation of functional preinitiation complexes by modifying sulfhydryl groups but did not have any significant effect on either initiation or elongation. Both RNA polymerases I and III, but not T7 RNA polymerase, were inhibited by juglone. The primary target of juglone has not been unambiguously identified, although a site on the polymerase itself is suggested by inhibition of RNA polymerase II during factor-independent transcription of single-stranded DNA. Because of its unique inhibitory properties juglone should prove useful in studying transcription in vitro.

Flavopiridol inhibits P-TEFb and blocks HIV-1 replication.

Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor that is in clinical trials as a cancer treatment because of its antiproliferative properties. We found that the flavonoid potently inhibited transcription by RNA polymerase II in vitro by blocking the transition into productive elongation, a step controlled by P-TEFb. The ability of P-TEFb to phosphorylate the carboxyl-terminal domain of the large subunit of RNA polymerase II was inhibited by flavopiridol with a K(i) of 3 nm. Interestingly, the drug was not competitive with ATP. P-TEFb composed of Cdk9 and cyclin T1 is a required cellular cofactor for the human immunodeficiency virus (HIV-1) transactivator, Tat. Consistent with its ability to inhibit P-TEFb, flavopiridol blocked Tat transactivation of the viral promoter in vitro. Furthermore, flavopiridol blocked HIV-1 replication in both single-round and viral spread assays with an IC(50) of less than 10 nm.

The Bloch equations in high-gradient magnetic resonance force microscopy: theory and experiment.

We report theory and observations of paramagnetic resonance in a measured field gradient of 44,000 T per meter by the technique of magnetic resonance force microscopy (MRFM). Resonance was induced in a dilute solid solution of diphenylpicrylhydrazyl in polystyrene at 77 and 10 K by an amplitude-modulated microwave field. This modulated the force between resonant sample spins and a micrometer-scale SmCo magnetic tip on a force microscope cantilever. The force signals were typically of order 10 fN, and were detected above a thermal noise floor of 80 aN per root hertz at 10 K, equivalent to a magnetic moment noise of 200 micro(B) per root hertz of bandwidth. Resonance saturation was readily observed. Starting with the Bloch equations, we derived simple analytic expressions for the predicted cantilever signal amplitudes and T(1)-dependent phase lags, valid at low microwave power levels. For power levels below saturation, the data were in good agreement with the Bloch equation predictions, while above saturation the measured force increased more slowly with power than predicted. Several ESR mechanisms which might lead to non-Bloch dynamics in the MRFM environment are reviewed. Spin-relaxation mechanisms are also reviewed. A detailed description of the experimental apparatus is offered.

Effects of polydiallyldimethyl ammonium chloride coagulant on formation of chlorinated by products in drinking water.

The objectives of this research work was to evaluate the reduction of THM precursors by cationic p-DADMAC and determine the correlations between the chlorine demand and trihalomethane formation in the presence of electrolyte solutions and ambient light. The chlorine demand was found to be significantly reduced provided that the H2SO4 electrolyte was fed to the sample solutions. The amount of CHCl3 formation was also decreased when the Na2SO4 electrolyte was introduced in spite of the levels of light intensity. The p-DADMAC can not only effectively remove the turbidity but also reduce the formation of CHCl3. The optimum dosage of p-DADMAC for reducing the turbidity, TOC and CHCl3 in the humic acid and source water samples was determined and depended upon the nature of organics.

Surgical management of refractory exit-site/tunnel infection of Tenckhoff catheter: technical innovations of partial replantation.

OBJECTIVE: Catheter-related infection has been the major cause of catheter removal for peritoneal dialysis (PD) patients. A salvage technique--partial replantation of the infected catheter--was developed in our hospital to rescue catheters with refractory exit-site or tunnel infection. PATIENTS: We performed 26 partial replantations of Tenckhoff catheters for 23 patients with refractory exit-site or tunnel infection and 2 patients with near-cuff perforation of the catheter. Their problems were all resolved successfully without interruption of PD. INTERVENTIONS: We removed the infected portion of the catheter and preserved the still-functioning internal conduit, connecting it to a divided new catheter. All of the patients resumed PD immediately after the advancement of the new catheter through a new subcutaneous tunnel and exit site on the opposite side. RESULTS: No technical complications such as disconnection of the catheter or leakage of dialysate were noted. Repeated partial replantation of the catheter was done for 1 patient with a new refractory exit-site infection. Tunnel infection was not an absolute contraindication for this procedure. About one third (34.6%) of our patients had preoperative tunnel infection. CONCLUSION: Partial replantation of a Tenckhoff catheter is a simple and effective procedure for patients with refractory exit-site/tunnel infection and patients with near-cuff perforation of the catheter. Repeated partial replantation is also feasible for repeat exit-site infections.

Immunocytochemical localization of ribonuclease in yolk granules of adult Rana catesbeiana oocytes.

To determine the localization of the pyrimidine-guanine sequence-specific ribonuclease in Rana catesbeiana (bullfrog) oocytes, the RNase was first isolated and used to prepare a specific rabbit antiserum. Only one protein of similar molecular size to the RNase was immunoprecipitated from ovary homogenate by the antiserum, but two bands were observed by Western blotting analysis. These two proteins were shown by further purification of antibody and Western blotting analysis to have similar antigenicity. Immunoprecipitation and Western blotting of tissue homogenates showed that the RNase was found predominantly in the ovary, but not in other tissues. The specific localization of the RNase was determined by immuno-electron microscopy of oocyte sections incubated with the specific antiserum; the yolk granules, but not other organelles, were found to contain the RNase. Most of the RNase was evenly distributed in the lateral amorphous area of the yolk granule but not in the central yolk crystal area which contains stored vitellogenin proteins. Our results indicate that the RNase is compartmentalized in the yolk granules of oocytes, which might prevent damage to cellular RNAs.

Stimulation of myosin light-chain kinase by Cd2+ and Pb2+.

The effect of Cd2+ on myosin light chain kinase (MLCK) reported in the literature is controversial, apparently because the level of Ca2+ contaminating the reaction mixture could not be accurately controlled by the addition of a metal chelator when Cd2+ was also present. In the present study, we have reduced the contaminating Ca2+ to a trace level that did not interfere with the enzyme activity; thus the use of a metal chelator was not necessary. We showed that Cd2+, or Pb2+ had a biphasic effect on MLCK isolated from chicken gizzard: stimulation at low and inhibition at high concentrations. (The stimulatory effect of on the enzyme activity isolated from chicken gizzard: stimulation at low and inhibition at high concentrations). The stimulatory effect of Cd2+ or Pb2+ on MLCK activity was not seen in the absence of calmodulin, and was abolished by trifluoperazine, a calmodulin antagonist, indicating that the heavy metals exert their activation via calmodulin. The inhibition of the enzyme activity by Cd2+ or Pb2+ at higher concentrations was also seen with the calmodulin-independent catalytic fragment of MLCK, suggesting that the inhibition is probably through their binding to sulfhydryl groups that are essential for catalytic activity. Pb2+ was more effective than Cd2+ in stimulating the enzyme activity, but less potent in inhibition. The extent of stimulation by heavy metals most likely resulted from a combination of the biphasic effects. Dithiothreitol and N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine selectively chelated Cd2+ and Pb2+ over Ca2+, and reversed their stimulatory or inhibitory effect on MLCK.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmural suture technique for trocar-site bleeding following laparoscopic cholecystectomy.

Laparoscopic cholecystectomy was performed in a cirrhotic patient who had cholelithiasis. Despite the existence of coagulopathy, excessive bleeding from the gallbladder and nodular liver was avoidable. Dissection and extraction of the gallbladder went smoothly. However, serious bleeding from the trocar site occurred following the withdrawal of the trocar/cannula. The bleeding was not controllable by electrocauterization. A novel attempt using a transmural suture technique was tried, and hemostasis was achieved satisfactorily. Our patient enjoyed an uneventful postoperative recovery and was discharged 2 days after surgery.