Effects of sphingomyelin/ceramide ratio on the permeability and microstructure of model stratum corneum lipid membranes

The conversion of sphingomyelin (SM) to a ceramide (Cer) by acid sphingomyelinase (aSMase) is an important event in skin barrier development. A deficiency in aSMase in diseases such as Niemann–Pick disease and atopic dermatitis coincides with impaired skin barrier recovery after disruption. We studied how an increased SM/Cer ratio influences the barrier function and microstructure of model stratum corneum (SC) lipid membranes. In the membranes composed of isolated human SC Cer (hCer)/cholesterol/free fatty acids/cholesteryl sulfate, partial or full replacement of hCer by SM increased water loss. Partial replacement of 25% and 50% of hCer by SM also increased the membrane permeability to theophylline and alternating electric current, while a higher SM content either did not alter or even decreased the membrane permeability. In contrast, in a simple membrane model with only one type of Cer (nonhydroxyacyl sphingosine, CerNS), an increased SM/Cer ratio provided a similar or better barrier against the permeation of various markers. X-ray powder diffraction revealed that the replacement of hCer by SM interferes with the formation of the long periodicity lamellar phase with a repeat distance of d = 12.7 nm. Our results suggest that SM-to-Cer processing in the human epidermis is essential for preventing excessive water loss, while the permeability barrier to exogenous compounds is less sensitive to the presence of sphingomyelin.     

Functional analysis of limb recovery following autograft treatment of volumetric muscle loss in the quadriceps femoris

Severe injuries to the extremities often result in muscle trauma and, in some cases, significant volumetric muscle loss (VML). These injuries continue to be challenging to treat, with few available clinical options, a high rate of complications, and often persistent loss of limb function. To facilitate the testing of regenerative strategies for skeletal muscle, we developed a novel quadriceps VML model in the rat, specifically addressing functional recovery of the limb. Our outcome measures included muscle contractility measurements to assess muscle function and gait analysis for evaluation of overall limb function. We also investigated treatment with muscle autografts, whole or minced, to promote regeneration of the defect area. Our defect model resulted in a loss of muscle function, with injured legs generating less than 55% of muscle strength from the contralateral uninjured control legs, even at 4 weeks post-injury. The autograft treatments did not result in significant recovery of muscle function. Measures of static and dynamic gait were significantly decreased in the untreated, empty defect group, indicating a decrease in limb function. Histological sections of the affected muscles showed extensive fibrosis, suggesting that this scarring of the muscle may be in part the cause of the loss of muscle function in this VML model. Taken together, these data are consistent with clinical findings of reduced muscle function in large VML injuries. This new model with quantitative functional outcome measures offers a platform on which to evaluate treatment strategies designed to regenerate muscle tissue volume and restore limb function.     

A systematic assessment of normalization approaches for the Infinium 450K methylation platform

The Illumina Infinium HumanMethylation450 BeadChip has emerged as one of the most popular platforms for genome wide profiling of DNA methylation. While the technology is wide-spread, systematic technical biases are believed to be present in the data. For example, this array incorporates two different chemical assays, i.e., Type I and Type II probes, which exhibit different technical characteristics and potentially complicate the computational and statistical analysis. Several normalization methods have been introduced recently to adjust for possible biases. However, there is considerable debate within the field on which normalization procedure should be used and indeed whether normalization is even necessary. Yet despite the importance of the question, there has been little comprehensive comparison of normalization methods. We sought to systematically compare several popular normalization approaches using the Norwegian Mother and Child Cohort Study (MoBa) methylation data set and the technical replicates analyzed with it as a case study. We assessed both the reproducibility between technical replicates following normalization and the effect of normalization on association analysis. Results indicate that the raw data are already highly reproducible, some normalization approaches can slightly improve reproducibility, but other normalization approaches may introduce more variability into the data. Results also suggest that differences in association analysis after applying different normalizations are not large when the signal is strong, but when the signal is more modest, different normalizations can yield very different numbers of findings that meet a weaker statistical significance threshold. Overall, our work provides useful, objective assessment of the effectiveness of key normalization methods.     

106例宫颈炎患者支原体检测及抗生素敏感性分析

目的：对本地区宫颈炎患者解脲脲原体（Uu）和人型支原体（Mh）进行监测,分析抗生素敏感情况,为临床诊疗提供实验依据。方法：无菌采集宫颈炎患者宫颈分泌物,用支原体培养、鉴定、药敏试剂盒进行培养和药敏试验。结果：支原体总检出率为71.70%。Uu检出率为52.83%,Uu合并Mh检出率16.04%,Mh检出率为2.83%。56例单纯Uu株体外抗生素敏感性显示：对四环素族多西环素100%、美满霉素98.21%高度敏感;对大环内酯类敏感性不一：克拉霉素96.43%,阿奇霉素91.07%,交沙霉素76.79%,红霉素67.86%,罗红霉素32.14%;对喹诺酮类如加替沙星83.93%较敏感,而对左氧氟沙星,司帕沙星敏感性差;对克林霉素和甲砜霉素不敏感;17例Uu合并Mh的抗生素敏感性为：美满霉素100%,多西环素100%,交沙霉素88.23%,加替沙星64.7%,对左氧氟沙星、司帕沙星、红霉素、阿奇霉素、克拉霉素,罗红霉素、甲砜霉素、克林霉素均不敏感。3株单纯Mh例数少未做统计。结论：宫颈炎患者中支原体检出率较高,其中单纯Uu检出率较Uu、Mh混合阳性率高,对四环素类、大环内酯类族中的多数抗生素敏感,治疗选择余地大。Uu、Mh混合敏感性抗生素种类少,应优先选择四环素类抗生素治疗。     

2013 年重组人干扰素alpha2b 注射剂评价性抽验质量分析

目的:评价国产重组人干扰素α2b注射剂的质量现状及存在问题。方法:采用法定检验方法结合探索性研究进行样品检验,统计分析检验结果,对国产重组人干扰素α2b注射剂的质量现状进行评价。结果:法定检验显示92批成品91批合格,合格率98.9%;24批原液20批合格(部分检验),合格率83.3%;探索性研究表明,按欧洲药典标准在原液中增加相关蛋白含量测定,则有两家企业的产品符合要求。结论:该品种总体质量状况良好,现行检验标准基本可行,建议在原液中增加相关蛋白含量检测。     

Identification, expression and characterisation of a major salivary allergen (Cul s 1) of the biting midge Culicoides sonorensis relevant for summer eczema in horses

Salivary proteins of Culicoides biting midges are thought to play a key role in summer eczema (SE), a seasonal recurrent allergic dermatitis in horses. The present study describes the identification, expression and clinical relevance of a candidate allergen of the North American midge Culicoides sonorensis. Immunoblot analysis of midge saliva revealed a 66 kDa protein (Cul s 1) that was bound by IgE from several SE-affected (SE+) horses. Further characterisation by fragmentation, mass spectrometry and bioinformatics identified Cul s 1 as maltase, an enzyme involved in sugar meal digestion. A cDNA encoding Cul s 1 was isolated and expressed as a polyhistidine-tagged fusion protein in a baculovirus/insect cell expression system. The clinical relevance of the affinity-purified recombinant Cul s 1 (rCul s 1) was investigated by immunoblotting, histamine release testing (HRT) and intradermal testing (IDT) in eight SE+ and eight control horses. Seven SE+ horses had rCul s 1-specific IgE, whereas only one control animal had IgE directed against this allergen. Furthermore, the HRT showed rCul s 1 induced basophil degranulation in samples from seven of eight SE+ horses but in none of the control animals. rCul s 1 also induced immediate (7/8), late-phase (8/8) and delayed (1/8) skin reactivity in IDT on all SE+ horses that had a positive test with the whole body extract (WBE) of C. sonorensis. None of the control horses showed immediate or delayed skin reactivity with rCul s 1, and only one control horse had a positive late-phase response, while several non-specific late-phase reactions were observed with the insect WBE. Thus, we believe rCul s 1 is the first specific salivary allergen of C. sonorensis to be described that promises to advance both in vitro and in vivo diagnosis and may contribute to the development of immunotherapy for SE in horses.     

Competitive interactions and the effect of herbivory on Bt-Brassica napus, Brassica rapa and Lolium perenne

1.  The probability of a transgenic crop establishing a feral population outside cultivated areas and possibly outcompeting naturally occurring species needs to be assessed to make an ecological risk assessment of the transgenic crop.
2.  The interaction between herbivory and competition is thought to determine the ecological success of insect-resistant plants, and this interaction was investigated in a competition experiment with transgenic insect-resistant Bt- Brassica napus , Brassica rapa , Lolium perenne , and herbivory from the large white butterfly Pieris brassicae .
3.  As expected, herbivory had a negative effect on the biomass of B. rapa at high plant densities. The competitive ability of L. perenne , when growing with B. rapa , increased significantly with the level of herbivory on B. rapa .
4.  To predict the effect of herbivory in a natural ecosystem, plant competition between the two annual Brassica species was analysed in a population ecological model. It was concluded that it is probable that transgenic Bt- B. napus plants may invade a natural habitat if herbivory is sufficiently high and the habitat is suitable for B. napus .
5.   Synthesis and applications . The results indicate that it is important to study the interaction between herbivory and competition when assessing the ecological risk of insect-resistant genetically modified crops. Furthermore, combining ecological data from manipulated experiments with population ecological modelling is a fruitful approach when conducting environmental risk assessments.     

Effect of milk on antibacterial activity of tetracycline against Escherichia coli and Staphylococcus aureus isolated from bovine mastitis

The susceptibility of mastitis-causing Escherichia coli and Staphylococcus aureus to two commonly used antibiotics, tetracycline and penicillin G, was tested in raw milk and in Muller–Hinton (MH) broth by introducing a pH indicator, bromocresol purple, which was shown to be a simple, sensitive, and rapid method. The minimum inhibitory concentration (MIC) of penicillin G in milk was the same as those in MH broth, whereas the MIC of tetracycline in milk was 4 to 32 times that in MH. An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline’s activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles.     

Drug Resistance and in Vitro Susceptibility of Plasmodium falciparum in Thailand during 1988-2003

The aim of the present study was to investigate antimalarial drug pressure resulting from the clinical use of different antimalarials in Thailand. The phenotypic diversity of the susceptibility profiles of antimalarials, i.e., chloroquine (CQ), quinine (QN), mefloquine (MQ), and artesunate (ARS) in Plasmodium falciparum isolates collected during the period from 1988 to 2003 were studied. P. falciparum isolates from infected patients were collected from the Thai-Cambodian border area at different time periods (1988-1989, 1991-1992, and 2003), during which 3 different patterns of drug use had been implemented: MQ + sulphadoxine (S) + pyrimethamine (P), MQ alone and MQ + ARS, respectively. The in vitro drug susceptibilities were investigated using a method based on the incorporation of [³H] hypoxanthine. A total of 50 isolates were tested for susceptibilities to CQ, QN, MQ, and ARS. Of these isolates, 19, 16, and 15 were adapted during the periods 1988-1989, 1991-1993, and 2003, respectively. P. falciparum isolates collected during the 3 periods were resistant to CQ. Sensitivities to MQ declined from 1988 to 2003. In contrast, the parasite was sensitive to QN, and similar sensitivity profile patterns were observed during the 3 time periods. There was a significantly positive but weak correlation between the IC₅₀ values of CQ and QN, as well as between the IC₅₀ values of QN and MQ. Drug pressure has impact on sensitivity of P. falciparum to MQ. A combination therapy of MQ and ARS is being applied to reduce the parasite resistance, and also increasing the efficacy of the drug.