Improve the clinical effective decision of the oral feeding readiness in preterm infants: Revise and validate the TC-POFRAS.

BACKGROUND: Currently there is limited information to guide health professionals regarding the optimal time frame to initiate safe and effective oral feedings to preterm infants. The study aims to revise and validate a streamlined version of the Traditional Chinese-Preterm Oral Feeding Readiness Assessment Scale, the TC-POFRAS®, and evaluate its construct validity in the clinical decisions regarding feeding readiness of preterm infants. METHODS: Eighty-one clinically stable preterm infants were assessed using the TC-POFRAS for oral feeding readiness. Item-total correlation analysis was used to check if any item was inconsistent with the averaged TC-POFRAS scores. Cronbach's α coefficient was used to evaluate the inter-item consistency. Exploratory factor analysis was used to determine the coherence of variables to reorganize assessment domains. The revised version of TC-POFRAS (TC-POFRAS®) was developed and a new cut-off score based on discriminant accuracy was established. RESULTS: Based on the results from statistical analysis, five items ("lips posture," "tongue posture," "biting reflex," "gag reflex," and "tongue cupping") were deleted from the original TC-POFRAS to form the TC-POFRAS®. The TC-POFRAS®'s global accuracy was 92.1%. The cut-off value of 19 was the one that presented the most optimization of sensitivity based on specificity. The TC-POFRAS® was reconstructed into corrected gestational age and five behavioral domains. CONCLUSIONS: The TC-POFRAS® is considered a valid, safe, and accurate objective instrument to assist health professionals to initiate oral feeding of preterm infants.

Cell proliferation of human bone marrow mesenchymal stem cells on biodegradable microcarriers enhances in vitro differentiation potential.

OBJECTIVES: For reasons of provision of highly-specific surface area and three-dimensional culture, microcarrier culture (MC) has garnered great interest for its potential to expand anchorage-dependent stem cells. This study utilizes MC for in vitro expansion of human bone marrow mesenchymal stem cells (BMMSCs) and analyses its effects on BMMSC proliferation and differentiation. MATERIALS AND METHODS: Effects of semi-continuous MC compared to control plate culture (PC) and serial bead-to-bead transfer MC (MC bead-T) on human BMMSCs were investigated. Cell population growth kinetics, cell phenotypes and differentiation potential of cells were assayed. RESULTS: Maximum cell density and overall fold increase in cell population growth were similar between PCs and MCs with similar starting conditions, but lag period of BMMSC growth differed substantially between the two; moreover, MC cells exhibited reduced granularity and higher CXCR4 expression. Differentiation of BMMSCs into osteogenic and adipogenic lineages was enhanced after 3 days in MC. However, MC bead-T resulted in changes in cell granularity and lower osteogenic and adipogenic differentiation potential. CONCLUSIONS: In comparison to PC, MC supported expansion of BMMSCs in an up-scalable three-dimensional culture system using a semi-continuous process, increasing potential for stem cell homing ability and osteogenic and adipogenic differentiation.

Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster.

Streptomyces lavendulae produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein-protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an alpha-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning ca. 50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or alpha-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.

G1 phase dependent nuclear localization of relaxed-circular hepatitis B virus DNA and aphidicolin-induced accumulation of covalently closed circular DNA.

During chronic hepatitis B virus (HBV) infection, virus persistence relies on the maintenance of a pool of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. To achieve this, HBV DNA has to be transported from the cytoplasm to the nucleus. By carrying out subcellular fractionation experiment, both of the relaxed-circular (RC) and single-stranded (SS) HBV DNA were found in the cytoplasm whereas only RC form could be detected in the nucleus of a hepatoblastoma cell line (HepG2) stably producing HBV. This fraction of nuclear RC viral DNA was clearly demonstrated in the G1 but not S phase of synchronized HepG2 cells. Conversely, the relative amount of cytoplasmic RC viral DNA in the S phase was larger than that in the G1 phase. Although no cccDNA could be detected in HepG2 cells without synchronization, an increasing amount of cccDNA in the nucleus was demonstrated after prolonged incubation of the cells in aphidicolin. Finally, by undertaking in situ hybridization using a probe specific to plus-strand HBV DNA, nuclear viral DNA was detected predominantly in the G1 phase of HepG2 cells. In summary, the results indicated that only RC but not SS form of HBV DNA was localized to the nuclei of HepG2 cells. The nuclear localization occurred preferentially in the G1 but not S phase and prolonged treatment with aphidicolin resulted in accumulation of nuclear cccDNA.

Pre-steady-state kinetic analysis of the trichodiene synthase reaction pathway.

The pre-steady-state kinetics of the trichodiene synthase reaction were investigated by rapid chemical quench methods. The single-turnover rate was found to be 3.5-3.8 s-1, a rate 40 times faster than the steady-state catalytic rate (kcat = 0.09 s-1) for trichodiene synthase-catalyzed conversion of farnesyl diphosphate (FPP) to trichodiene at 15 degrees C. In a multiturnover experiment, a burst phase (kb = 4.2 s-1) corresponding to the accumulation of trichodiene on the surface of the enzyme was followed by a slower, steady-state release of products (klin = 0.086 s-1) which corresponds to kcat. These results strongly suggest that the release of trichodiene from the enzyme active site is the rate-limiting step in the overall reaction, while the consumption of FPP is the step which limits chemical catalysis at the active site. Single-turnover experiments with trichodiene synthase mutant D101E, for which the steady-state rate constant kcat is 1/3 of that of wild type, revealed that the mutation actually depresses the rate of FPP consumption by a factor of 100. The deuterium isotope effect on the consumption of [1-2H,1,2-14C]FPP was found to be 1.11 +/- 0.06. Single turnover reactions of [1,2-14C]FPP catalyzed by trichodiene synthase were carried out at 4, 15, or 30 degrees C in an effort to provide direct observation of the proposed intermediate nerolidyl diphosphate (NPP). However, no NPP was detected, indicating that the conversion of NPP must be too fast to be observed within the detection limits of the assay. Taken together, these observations suggest that the isomerization of FPP to NPP is the step which limits the rate of chemical catalysis in the trichodiene synthase reaction pathway.