The addition of a pH-sensitive gel improves microemulsion stability for the targeted removal of colonic ammonia

<p>Abstract</p> <p>Background</p> <p>We prepared an oral W/O microemulsion for the removal of colonic ammonia (ME-RCA). The effect of this microemulsion was influenced by the digestion process in the gastrointestinal tract. In this paper, we aim to show that stability was improved by using a microemulsion-based gel for the removal of colonic ammonia (MBG-RCA).</p> <p>Methods</p> <p>MBG-RCA was prepared by adding sodium alginate to the ME-RCA. MBG-RCA and ME-RCA were passed through a simulated gastrointestinal environment, and the amount of colonic ammonia present was then determined by titration with a standard solution of hydrochloric acid. The pH of the gastrointestinal fluid was measured using a pH test paper and the size and form of the microemulsions were examined under the microscope. 18 healthy rats were randomly divided into three groups, fasted for 24 hours and allowed to drink normally. Three-way pipes were placed at the gastroduodenal junction in Group I, and at the terminal ileum in Group II. After the intragastric administration of ME-RCA, the stomach contents in Group I, the effluent from the terminal ileum in Group II and discharge from the anus in Group III were collected. The pH values of the gastrointestinal juice were measured by the pH test paper and those of the colon were determined by a universal indicator. These animal experiments were also used to test the effect of MBG-RCA.</p> <p>Results</p> <p>MBG-RCA showed a better removal rate of artificial colonic ammonia than ME-RCA (P < 0.05). The decrease in pH value of the artificial small intestinal fluid due to ME-RCA did not occur when MBG-RCA was used. In the simulated gastrointestinal process, MBG-RCA maintained greater stability and released the emulsion (ME-RCA) in the colonic fluid. In the gastrointestinal tract of normal SD rats, ME-RCA decreased in size and lost its stable form after entering the small intestine, while MBG-RCA remained stable and intact emulsion-drops were observed from the anus. Neither substance had any effect on the pH of the stomach or colon of normal rats (partly because normal rats were fasted for 24 hours and allowed to drink normally, which resulted in a low level of ammonia production in the colon). Unlike ME-RCA, MBG-RCA did not reduce the pH of the small intestine.</p> <p>Conclusions</p> <p>MBG-RCA was more stable in the gastrointestinal tract and more effective at removing colonic ammonia when a higher concentration of ammonia was present. This made it possible to achieve the targeted removal of colonic ammonia and is a promising method to prevent hepatic encephalopathy (HE) in future studies.</p

Locked Nucleic Acid Pentamers as Universal PCR Primers for Genomic DNA Amplification

Background: Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping. Results: We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 specie

Cell Surface Sialylation and Fucosylation Are Regulated by L1 via Phospholipase Cγ and Cooperate to Modulate Neurite Outgrowth, Cell Survival and Migration

BACKGROUND: Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using a panel of carbohydrate surface markers, we have shown that cell surface sialylation and fucosylation were downregulated in L1(-/y) neurons versus L1(+/y) neurons. Consistently, mRNA levels of sialyltransferase ST6Gal1, and fucosyltransferase FUT9 were significantly reduced in L1(-/y) neurons. Moreover, treatment of L1(+/y) neurons with L1 antibodies, triggering signal transduction downstream of L1, led to an increase in cell surface sialylation and fucosylation compared to rat IgG-treated cells. ShRNAs for both ST6Gal1 and FUT9 blocked L1 antibody-mediated enhancement of neurite outgrowth, cell survival and migration. A phospholipase Cgamma (PLCgamma) inhibitor and shRNA, as well as an Erk inhibitor, reduced ST6Gal1 and FUT9 mRNA levels and inhibited effects of L1 on neurite outgrowth and cell survival. CONCLUSIONS: Neuronal surface sialylation and fucosylation are regulated via PLCgamma by L1, modulating neurite outgrowth, cell survival and migration

A mass vaccination campaign targeting adults and children to prevent typhoid fever in Hechi; Expanding the use of Vi polysaccharide vaccine in Southeast China: A cluster-randomized trial

BACKGROUND: One of the goals of this study was to learn the coverage, safety and logistics of a mass vaccination campaign against typhoid fever in children and adults using locally produced typhoid Vi polysaccharide (PS) and group A meningococcal PS vaccines in southern China. METHODS: The vaccination campaign targeted 118,588 persons in Hechi, Guangxi Province, aged between 5 to 60 years, in 2003. The study area was divided into 107 geographic clusters, which were randomly allocated to receive one of the single-dose parenteral vaccines. All aspects regarding vaccination logistics, feasibility and safety were documented and systematically recorded. Results of the logistics, feasibility and safety are reported. RESULTS: The campaign lasted 5 weeks and the overall vaccination coverage was 78%. On average, the 30 vaccine teams gave immunizations on 23 days. Vaccine rates were higher in those aged ≤ 15 years (90%) than in adolescents and young adults (70%). Planned mop-up activities increased the coverage by 17%. The overall vaccine wastage was 11%. The cold chain was maintained and documented. 66 individuals reported of adverse events out of all vaccinees, where fever (21%), malaise (19%) and local redness (19%) were the major symptoms; no life-threatening event occurred. Three needle-sharp events were reported. CONCLUSION: The mass immunization proved feasible and safe, and vaccine coverage was high. Emphasis should be placed on: injection safety measures, community involvement and incorporation of mop-up strategies into any vaccination campaign. School-based and all-age Vi mass immunizations programs are potentially important public health strategies for prevention of typhoid fever in high-risk populations in southern China

Host factors do not influence the colonization or infection by fluconazole resistant Candida species in hospitalized patients

Nosocomial yeast infections have significantly increased during the past two decades in industrialized countries, including Taiwan. This has been associated with the emergence of resistance to fluconazole and other antifungal drugs. The medical records of 88 patients, colonized or infected with Candida species, from nine of the 22 hospitals that provided clinical isolates to the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) program in 1999 were reviewed. A total of 35 patients contributed fluconazole resistant strains [minimum inhibitory concentrations (MICs) ≧ 64 mg/l], while the remaining 53 patients contributed susceptible ones (MICs ≦ 8 mg/l). Fluconazole resistance was more frequent among isolates of Candida tropicalis (46.5%) than either C. albicans (36.8%) or C. glabrata (30.8%). There was no significant difference in demographic characteristics or underlying diseases among patients contributing strains different in drug susceptibility

Reduction of Plasma Gelsolin Levels Correlates with Development of Multiple Organ Dysfunction Syndrome and Fatal Outcome in Burn Patients

BACKGROUND: Depletion of the circulating actin-binding protein, plasma gelsolin (pGSN) has been described in critically ill surgical patients. We hypothesized that the extent of pGSN reduction might correlate with different outcome of burn patients. The study was performed to evaluate the prognostic implications of pGSN levels on the development of multiple organ dysfunction syndrome (MODS) and fatal outcome in a group of severely burn patients. METHODS AND FINDINGS: 95 patients were included, and they were divided into three groups with different burn area: group I (n = 33), group II (n = 32) and group III (n = 30). According to whether there was development of MODS or not, patients were divided into MODS group (n = 28) and none-MODS group (n = 67); then the patients with MODS were further divided into non-survivor group (n = 17) and survivor group (n = 11). The peripheral blood samples were collected on postburn days (PBD) 1, 3, 7, 14, and 21. The levels of pGSN were determined and T cells were procured from the blood. The contents of cytokines (IL-2, IL-4 and IFN-γ) released by T cells were also measured. The related factors of prognosis were analyzed by using multivariate logistic regression analysis. The results showed that pGSN concentrations, as well as the levels of IL-2 and IFN-γ, decreased markedly on PBD 1-21, whereas, the levels of IL-4 increased markedly in all burn groups as compared with normal controls (P<0.05 or P<0.01), and there were obviously differences between group I and group III (P<0.05 or P<0.01). The similar results were found in MODS patients and the non-survivor group as compared with those without MODS and the survival group on days 3-21 postburn (P<0.05 or P<0.01). Moreover, as the pGSN levels decreased, the incidence of septic complication as well as MODS remarkably increased. CONCLUSIONS: pGSN levels appear to be an early prognostic marker in patients suffering from major burns

Impact of the Herbal Medicine Sophora flavescens on the Oral Pharmacokinetics of Indinavir in Rats: The Involvement of CYP3A and P-Glycoprotein

Sophora flavescens is a Chinese medicinal herb used for the treatment of gastrointestinal hemorrhage, skin diseases, pyretic stranguria and viral hepatitis. In this study the herb-drug interactions between S. flavescens and indinavir, a protease inhibitor for HIV treatment, were evaluated in rats. Concomitant oral administration of Sophora extract (0.158 g/kg or 0.63 g/kg, p.o.) and indinavir (40 mg/kg, p.o.) in rats twice a day for 7 days resulted in a dose-dependent decrease of plasma indinavir concentrations, with 55%–83% decrease in AUC0-∞ and 38%–78% reduction in Cmax. The CL (Clearance)/F (fraction of dose available in the systemic circulation) increased up to 7.4-fold in Sophora-treated rats. Oxymatrine treatment (45 mg/kg, p.o.) also decreased indinavir concentrations, while the ethyl acetate fraction of Sophora extract had no effect. Urinary indinavir (24-h) was reduced, while the fraction of indinavir in faeces was increased after Sophora treatment. Compared to the controls, multiple dosing of Sophora extract elevated both mRNA and protein levels of P-gp in the small intestine and liver. In addition, Sophora treatment increased intestinal and hepatic mRNA expression of CYP3A1, but had less effect on CYP3A2 expression. Although protein levels of CYP3A1 and CYP3A2 were not altered by Sophora treatment, hepatic CYP3A activity increased in the Sophora-treated rats. All available data demonstrated that Sophora flavescens reduced plasma indinavir concentration after multiple concomitant doses, possibly through hepatic CYP3A activity and induction of intestinal and hepatic P-gp. The animal study would be useful for predicting potential interactions between natural products and oral pharmaceutics and understanding the mechanisms prior to human studies. Results in the current study suggest that patients using indinavir might be cautioned in the use of S. flavescens extract or Sophora-derived products

How Protein Stability and New Functions Trade Off

Numerous studies have noted that the evolution of new enzymatic specificities is accompanied by loss of the protein's thermodynamic stability (ΔΔG), thus suggesting a tradeoff between the acquisition of new enzymatic functions and stability. However, since most mutations are destabilizing (ΔΔG>0), one should ask how destabilizing mutations that confer new or altered enzymatic functions relative to all other mutations are. We applied ΔΔG computations by FoldX to analyze the effects of 548 mutations that arose from the directed evolution of 22 different enzymes. The stability effects, location, and type of function-altering mutations were compared to ΔΔG changes arising from all possible point mutations in the same enzymes. We found that mutations that modulate enzymatic functions are mostly destabilizing (average ΔΔG = +0.9 kcal/mol), and are almost as destabilizing as the “average” mutation in these enzymes (+1.3 kcal/mol). Although their stability effects are not as dramatic as in key catalytic residues, mutations that modify the substrate binding pockets, and thus mediate new enzymatic specificities, place a larger stability burden than surface mutations that underline neutral, non-adaptive evolutionary changes. How are the destabilizing effects of functional mutations balanced to enable adaptation? Our analysis also indicated that many mutations that appear in directed evolution variants with no obvious role in the new function exert stabilizing effects that may compensate for the destabilizing effects of the crucial function-altering mutations. Thus, the evolution of new enzymatic activities, both in nature and in the laboratory, is dependent on the compensatory, stabilizing effect of apparently “silent” mutations in regions of the protein that are irrelevant to its function

Clinical and molecular characterization of Wilson's disease in China: identification of 14 novel mutations

<p>Abstract</p> <p>Background</p> <p>Wilson's disease (WND) is a rare autosomal recessive disorder. Here we have evaluated 62 WND cases (58 probands) from the Chinese Han population to expand our knowledge of <it>ATP7B </it>mutations and to more completely characterize WND in China.</p> <p>Methods</p> <p>The coding and promoter regions of the <it>ATP7B </it>gene were analyzed by direct sequencing in 62 Chinese patients (58 probands) with WND (male, n = 37; female, n = 25; age range, 2 ~ 61 years old).</p> <p>Results</p> <p>Neurologic manifestations were associated with older age at diagnosis (p < 0.0001) and longer diagnostic delay (p < 0.0001). Age at diagnosis was also correlated with urinary copper concentration (r = 0.58, p < 0.001). Forty different mutations, including 14 novel mutations, were identified in these patients. Common mutations included p.Arg778Leu (31.9%) and p.Pro992Leu (11.2%). Homozygous p.Arg778Leu and nonsense mutation/frameshift mutations were more often associated with primary hepatic manifestations (p = 0.0286 and p = 0.0383, respectively) and higher alanine transaminase levels at diagnosis (p = 0.0361 and p = 0.0047, respectively). Nonsense mutation/frameshift mutations were also associated with lower serum ceruloplasmin (p = 0.0065).</p> <p>Conclusions</p> <p>We identified 14 novel mutations and found that the spectrum of mutations of <it>ATP7B </it>in China is quite distinct from that of Western countries. The mutation type plays a role in predicting clinical manifestations. Genetic testing is a valuable tool to detect WND in young children, especially in patients younger than 8 years old. Four exons (8, 12, 13, and 16) and two mutations (p.Arg778Leu, p.Pro992Leu) should be considered high priority for cost-effective testing in China.</p