The structure of Escherichia coli heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism.

The heat-stable enterotoxin b (STb) is secreted by enterotoxigenic Escherichia coli that cause secretory diarrhea in animals and humans. It is a 48-amino acid peptide containing two disulfide bridges, between residues 10 and 48 and 21 and 36, which are crucial for its biological activity. Here, we report the solution structure of STb determined by two- and three-dimensional NMR methods. Approximate interproton distances derived from NOE data were used to construct structures of STb using distance-geometry and simulated annealing procedures. The NMR-derived structure shows that STb is helical between residues 10 and 22 and residues 38 and 44. The helical structure in the region 10-22 is amphipathic and exposes several polar residues to the solvent, some of which have been shown to be important in determining the toxicity of STb. The hydrophobic residues on the opposite face of this helix make contacts with the hydrophobic residues of the C-terminal helix. The loop region between residues 21 and 36 has another cluster of hydrophobic residues and exposes Arg 29 and Asp 30, which have been shown to be important for intestinal secretory activity. CD studies show that reduction of disulfide bridges results in a dramatic loss of structure, which correlates with loss of function. Reduced STb adopts a predominantly random-coil conformation. Chromatographic measurements of concentrations of native, fully reduced, and single-disulfide species in equilibrium mixtures of STb in redox buffers indicate that the formation of the two disulfide bonds in STb is only moderately cooperative. Similar measurements in the presence of 8 M urea suggest that the native secondary structure significantly stabilizes the disulfide bonds.     

双歧杆菌三联活菌散治疗儿童抗生素相关性腹泻的疗效及安全性观察

目的探讨双歧杆菌三联活菌散治疗儿童抗生素相关性腹泻的疗效及安全性观察。方法选取抗生素相关性腹泻的患儿86例,采用随机数字表将纳入患者分为观察组（n=43例）和对照组（n=43例）。两组患儿均予以调整饮食、口服或静脉补液及口服蒙脱石散等常规治疗。观察组患儿加用双歧杆菌三联活菌散1.0 g/次,3次/d,溶解于温水中口服。对照组患儿除不使用双歧杆菌三联活菌散外余治疗同观察组。观察并记录两组患儿治疗后大便次数恢复时间和性状改变时间,并比较其临床疗效及不良反应。结果观察组患儿大便次数恢复时间和性状改变时间明显短于对照组（P〈0.05）;治疗72 h后,观察组患儿的临床总有效率为明显优于对照组（95.35%vs.81.40%）（χ^2=4.07,P〈0.05）,两组患儿治疗中均未出现明显的不良反应。结论双歧杆菌三联活菌散治疗抗生素相关性腹泻具有较好效果及安全性,能明显减少患儿腹泻次数及程度,改善患儿大便性状,较快缓解患儿的病情。     

Characterization of Thrombate III®, a pasteurized and nanofiltered therapeutic human antithrombin concentrate

Thrombate III^® is a highly purified antithrombin concentrate that has been used by clinicians worldwide for more than two decades for the treatment of hereditary antithrombin deficiency. The manufacturing process is based on heparin-affinity chromatography and pasteurization. To modernize the process and to further enhance the pathogen safety profile of the final product, despite the absence of infectious disease transmission, a nanofiltration step was added. The biochemical characterization and pathogen safety evaluation of Thrombate III^® manufactured using the modernized process are presented. Bioanalytical data demonstrate that the incorporation of nanofiltration has no impact on the antithrombin content, potency, and purity of the product.Scaledown models of the manufacturing process were used to assess virus and prion clearance under manufacturing setpoint conditions. Additionally, robustness of virus clearance was evaluated at or slightly outside the manufacturing operating limits. The results demonstrate that pasteurization inactivated both enveloped and non-enveloped viruses. The addition of nanofiltration substantially increased clearance capacities for both enveloped and non-enveloped viruses by approximately 4–6 log₁₀. In addition, the process achieves 6.0 log₁₀ ID₅₀ prion infectivity clearance. Thus, the introduction of nanofiltration increased the pathogen safety margin of the manufacturing process without impacting the key biochemical characteristics of the product.     

The ORF4a protein of human coronavirus 229E functions as a viroporin that regulates viral production

In addition to a set of canonical genes, coronaviruses encode additional accessory proteins. A locus located between the spike and envelope genes is conserved in all coronaviruses and contains a complete or truncated open reading frame (ORF). Previously, we demonstrated that this locus, which contains the gene for accessory protein 3a from severe acute respiratory syndrome coronavirus (SARS-CoV), encodes a protein that forms ion channels and regulates virus release. In the current study, we explored whether the ORF4a protein of HCoV-229E has similar functions. Our findings revealed that the ORF4a proteins were expressed in infected cells and localized at the endoplasmic reticulum/Golgi intermediate compartment (ERGIC). The ORF4a proteins formed homo-oligomers through disulfide bridges and possessed ion channel activity in both Xenopus oocytes and yeast. Based on the measurement of conductance to different monovalent cations, the ORF4a was suggested to form a non-selective channel for monovalent cations, although Li⁺ partially reduced the inward current. Furthermore, viral production decreased when the ORF4a protein expression was suppressed by siRNA in infected cells. Collectively, this evidence indicates that the HCoV-229E ORF4a protein is functionally analogous to the SARS-CoV 3a protein, which also acts as a viroporin that regulates virus production. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.     

Microvillus Inclusion Disease: Loss of Myosin Vb Disrupts Intracellular Traffic and Cell Polarity

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by loss of apical microvilli and formation of cytoplasmic inclusions lined by microvilli in enterocytes. MVID is caused by mutations in the MYO5B gene, coding for the myosin Vb motor protein. Although myosin Vb is implicated in the organization of intracellular transport and cell surface polarity in epithelial cells, its precise role in the pathogenesis of MVID is unknown. We performed correlative immunohistochemistry analyses of sections from duodenal biopsies of a MVID patient, compound heterozygous for two novel MYO5B mutations, predicting loss of function of myosin Vb in duodenal enterocytes together with a stable MYO5B CaCo2 RNAi cell system. Our findings show that myosin Vb‐deficient enterocytes display disruption of cell polarity as reflected by mislocalized apical and basolateral transporter proteins, altered distribution of certain endosomal/lysosomal constituents including Rab GTPases. Together, this severe disturbance of epithelial cell function could shed light on the pathology and symptoms of MVID.      

Comparison of three commercial vaccines for preventing persistent infection with bovine viral diarrhea virus

Eighty crossbred beef heifers were randomly allocated to four groups to evaluate the efficacy of vaccination in preventing development of calves persistently infected with bovine viral diarrhea virus (BVDV). Group 1 (n = 11) was non-vaccinated controls, whereas three groups were vaccinated with commercially available multivalent BVDV vaccines at weaning (∼7 mo of age), 28 d post-weaning, ∼1 y of age, and 28 d later. Groups 2 (n = 23) and 3 (n = 23) were given a modified-live BVDV vaccine, whereas Group 4 was given an inactivated BVDV vaccine. Heifers were bred by AI and subsequently exposed to two bulls. At 61 d after AI, 70 heifers were pregnant (n = 10 for Group 1 and n = 20/group for Groups 2, 3, and 4). Three cattle persistently infected with BVDV were commingled with the pregnant heifers (in an isolated pasture) from 68 to 126 d after AI. Thereafter, viremias were detected in pregnant heifers from Groups 1, 3, and 4 (10/10, 1/20, and 10/20, respectively), but not in pregnant heifers from Group 2 (0/20). Resulting calves were assessed for persistent infection using serum PCR, ear notch antigen capture-ELISA, and immunohistochemistry. Persistently infected calves were only produced in Group 1 (10/10) and Group 4 (2/18). In conclusion, commercial vaccines provided effective fetal protection despite prolonged natural exposure to BVDV. Given that viremias were detected in 11 vaccinated heifers after BVDV exposure, and two vaccinated heifers gave birth to persistently infected calves, there is continued need for biosecurity and diagnostic surveillance, in addition to vaccination, to ensure effective BVDV control.     

Saccharomyces boulardii produces a soluble anti-inflammatory factor that inhibits NF-kappaB-mediated IL-8 gene expression

Saccharomyces boulardii (Sb) is a non-pathogenic yeast that ameliorates intestinal injury and inflammation caused by a wide variety of enteric pathogens. We hypothesized that Sb may exert its probiotic effects by modulation of host cell signaling and pro-inflammatory gene expression. Human HT-29 colonocytes and THP-1 monocytes were stimulated with IL-1beta, TNFalpha or LPS in the presence or absence of Sb culture supernatant (SbS). IL-8 protein and mRNA levels were measured by ELISA and RT-PCR, respectively. The effect of SbS on IkappaB alpha degradation was studied by Western blotting and on NF-kappaB-DNA binding by EMSA. NF-kappaB-regulated gene expression was evaluated by transient transfection of THP-1 cells with a NF-kappaB-responsive luciferase reporter gene. SbS inhibited IL-8 protein production in IL-1beta or TNFalpha stimulated HT-29 cells (by 75% and 85%, respectively; P<0.001) and prevented IL-1beta-induced up-regulation of IL-8 mRNA. SbS also inhibited IL-8 production, prevented IkappaB alpha degradation, and reduced both NF-kappaB-DNA binding and NF-kappaB reporter gene up-regulation in IL-1beta or LPS-stimulated THP-1 cells. Purification and characterization studies indicate that the S. boulardii anti-inflammatory factor (SAIF) is small (<1 kDa), heat stable, and water soluble. The probiotic yeast Saccharomyces boulardii exerts an anti-inflammatory effect by producing a low molecular weight soluble factor that blocks NF-kappaB activation and NF-kappaB-mediated IL-8 gene expression in intestinal epithelial cells and monocytes. SAIF may mediate, at least in part, the beneficial effects of Saccharomyces boulardii in infectious and non-infectious human intestinal disease.     

贝飞达治疗小儿迁延性腹泻的临床研究

目的评价贝飞达治疗小儿迁延性腹泻的临床疗效,为合理应用贝飞达治疗小儿迁延性腹泻提供科学依据。方法选取60例1个月～2岁的迁延性腹泻患儿为研究对象,把他们随机分为贝飞达组和常规组。另30例1个月-2岁的健康儿作为对照组;3组均进行治疗前后粪便中的4种常驻的正常菌群的测定和60例迁廷性腹泻患儿疗效、住院时间及费用的观察。结果贝飞达组总有效率明显高于常规组,且住院时间及费用也明显减少。治疗前60例迁延性腹泻患儿的粪便菌群中双歧杆菌、乳杆菌明显减少,肠球菌、肠杆菌增加,与健康同龄儿比t〈0．05,治疗后贝飞达组粪便菌群中的4种菌群数比常规组增加明显结论贝飞达具有调整肠道菌群失调的作用,从而提高疗效。     

Comparison of efficacy of moxidectin and ivermectin in the treatment of Strongyloides fulleborni infection in rhesus macaques

BACKGROUND: Strongyloides infection may result in clinical disease or confound experimental protocols that utilize non-human primates. There is presently a Strongyloides fulleborni infection rate of approximately 27% in the Tulane National Primate Research Center's breeding colonies despite the routine therapeutic and prophylactic use of ivermectin. METHODS: A study was conducted to determine if moxidectin treatment offers advantages to the intestinal parasite control program. A total of 150 rhesus macaques (Macaca mulatta) that were removed from the breeding colonies due to illness were selected for the study. The animals were randomly assigned to treatment groups with 75 receiving ivermectin and 75 receiving moxidectin. Egg counts were performed on fecal samples collected pre- and post-treatment. RESULTS: Both treatments resulted in decreases in the number of eggs/g in the post-treatment sample as compared with the pre-treatment sample; however, no significant difference was found between treatment groups. CONCLUSIONS: With the data demonstrating a similar efficacy in both ivermectin and moxidectin treated macaques, the benefit of moxidectin treatment relates to biosafety and topical application.