DhhP,a Cyclic di-AMP Phosphodiesterase of Borrelia burgdorferi,Is Essential for Cell Growth and Virulence

Cyclic di-AMP (c-di-AMP) is a recently discovered second messenger in bacteria. Most of work on c-di-AMP signaling has been done in Gram-positive bacteria, firmicutes, and actinobacteria, where c-di-AMP signaling pathways affect potassium transport, cell wall structure, and antibiotic resistance. Little is known about c-di-AMP signaling in other bacteria. Borrelia burgdorferi, the causative agent of Lyme disease, is a spirochete that has a Gram-negative dual membrane. In this study, we demonstrated that B. burgdorferi BB0619, a DHH-DHHA1 domain protein (herein designated DhhP), functions as c-di-AMP phosphodiesterase. Recombinant DhhP hydrolyzed c-di-AMP to pApA in a Mn²⁺- or Mg²⁺-dependent manner. In contrast to c-di-AMP phosphodiesterases reported thus far, DhhP appears to be essential for B. burgdorferi growth both in vitro and in the mammalian host. Inactivation of the chromosomal dhhP gene could be achieved only in the presence of a plasmid-encoded inducible dhhP gene. The conditional dhhP mutant had a dramatic increase in intracellular c-di-AMP level in comparison to the isogenic wild-type strain. Unlike what has been observed in Gram-positive bacteria, elevated cellular c-di-AMP in B. burgdorferi did not result in an increased resistance to β-lactamase antibiotics, suggesting that c-di-AMP''s functions in spirochetes differ from those in Gram-positive bacteria. In addition, the dhhP mutant was defective in induction of the σ^S factor, RpoS, and the RpoS-dependent outer membrane virulence factor OspC, which uncovers an important role of c-di-AMP in B. burgdorferi virulence.     

Immunogenicity of recombinant human bocavirus‐1,2 VP2 gene virus‐like particles in mice

Human bocavirus (HBoV), a recently identified pathogen with a worldwide distribution is closely related to paediatric acute respiratory infection and gastroenteritis. The present study was performed to evaluate the immunogenicity of HBoV1 and HBoV2 virus‐like particles (VLPs) as vaccine candidates in mice. Both HBoV1 and HBoV2 VLPs were expressed in the bacmid virus–SF9 cell system. Mice were inoculated three times at 3‐week intervals with HBoV VLPs at one dose intramuscular (i.m.) or intradermal (i.d.) with or without the addition of the alum adjuvant. ELISA was used to detected antibody, and ELISPOT was used to test cellular immune responses. HBoV‐specific IgG antibodies were induced and alum adjuvant improved the antibody titres and avidity, while the inoculation pathway had no influence. T helper type 1/ type 2 immune responses were balanced induced by HBoV1 VLPs but not HBoV2 VLPs. Serum IgG antibody cross‐reactivity rates of the two subtypes were similar, but cross‐reactions of HBoV1 immunization groups were higher. The single i.m. group had more interferon‐γ‐secreting splenocytes. These data indicate that HBoV VP2 VLPs have good immunogenicity with induction of strong humoral and cellular immune responses, and they may be potential candidate vaccines for HBoV infection.     

‘Default’ generated neonatal regulatory T cells are hypomethylated at conserved non‐coding sequence 2 and promote long‐term cardiac allograft survival

Regulatory T (Treg) cells play an important role in the maintenance of immune self-tolerance and homeostasis. We previously reported that neonatal CD4⁺ T cells have an intrinsic ‘default’ mechanism to become Treg (neoTreg) cells in response to T-cell receptor (TCR) stimulation. However, the underlying mechanisms are unclear and the effects of neoTreg cells on regulating immune responses remain unknown. Due to their involvement in Foxp3 regulation, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b during the induction of neoTreg cells in the Foxp3^gfp mice. The function of neoTreg cells was assessed in an acute allograft rejection model established in RAG2^−/− mice with allograft cardiac transplantation and transferred with syngeneic CD4⁺ effector T cells. Following ex vivo TCR stimulation, the DNMT activity was increased threefold in adult CD4⁺ T cells, but not significantly increased in neonatal cells. However, adoptively transferred neoTreg cells significantly prolonged cardiac allograft survival (mean survival time 47 days, P < 0·001) and maintained Foxp3 expression similar to natural Treg cells. The neoTreg cells were hypomethylated at the conserved non-coding DNA sequence 2 locus of Foxp3 compared with adult Treg cells. The DNMT antagonist 5-aza-2′-deoxycytidine (5-Aza) induced increased Foxp3 expression in mature CD4⁺ T cells. 5-Aza-inducible Treg cells combined with continuous 5-Aza treatment prolonged graft survival. These results indicate that the ‘default’ pathway of neoTreg cell differentiation is associated with reduced DNMT1 and DNMT3b response to TCR stimulus. The neoTreg cells may be a strategy to alleviate acute allograft rejection.     

Mono and co-immobilization of imidazolium ionic liquids on silica: effects of the substituted groups on the adsorption behavior of 2,4-dinitrophenol

Ionic liquid modified silicas with high adsorption capacity for phenols prompt us to deeply explore the contribution of interactions between the adsorbent and adsorbate, with a particular focus on hydrophobicity, π–π, electrostatic and acid–base interactions. Herein, by introducing a series of typical substituent groups including N,N-dimethylaminopropyl (A), benzyl (B), dodecyl (D) and naphthylmethyl (N) in an imidazole ring (Im), three mono-immobilized and two co-immobilized imidazolium ionic liquid modified silicas, namely SilprAImCl, SilprBImCl, SilprNImCl, SilprDBImCl and SilprDAImCl, werre synthesized for removal and recovery of 2,4-dinitrophenol (2,4-DNP) from aqueous solutions. Adsorption kinetics, isotherms, thermodynamic analysis and desorption experiments have been carried out. The experimental results reveal that the substituent groups such as N,N-dimethylaminopropyl, benzyl and naphthylmethyl on the imidazole ring can significantly enhance the adsorption of 2,4-DNP via the acid–base interaction or π–π interaction and the adsorption capacity of 2,4-DNP follows the order: SilprNImCl > SilprAImCl > SilprBImCl. Furthermore, SilprDBImCl exhibits the largest adsorption capacity and SilprDAImCl has the lowest among the five adsorbents. These interesting finds indicate that the combination of hydrophobicity and π–π interactions lead to enhanced adsorption performance towards 2,4-DNP, while the combination of the hydrophobicity and acid–base interactions can restrain greatly adsorption of 2,4-DNP from aqueous medium. Adsorption mechanisms of 2,4-DNP on the five adsorbents have been clarified. These results will provide a deeper insight for efficient removal of phenols from water environments.

Ionic liquid modified silicas with high adsorption capacity for phenols prompt us to deeply explore the contribution of interactions between the adsorbent and adsorbate, with a particular focus on hydrophobicity, π–π, electrostatic and acid–base interactions.     

自然周期IVF/IVM、IVM和常规促排卵技术在不孕症治疗中的临床应用比较

目的探讨自然周期IVF结合未成熟卵母细胞IVM（自然周期IVF/IVM）和IVM技术与控制性促排卵技术（COH）相比,在不孕症治疗中的临床价值。方法根据筛查标准,将患者治疗方案分为以下3种类型：（A）自然周期IVF/IVM;（B）IVM;（C）COH。结果在1686个移植周期中,31.3%的周期（528个）应用自然周期IVF/IVM技术,12.8%实施IVM技术,55.9%通过COH方案治疗。自然周期IVF/IVM、IVM和COH的临床妊娠率分别为33.9%、39.8%和31.1%,IVM组高于其他两组但各组之间无显著差异,胚胎着床率也无显著差异。而IVM组的流产率（36.0%）高于其他两组（31.3%和25.9%）,无显著统计学差异。结论从本研究可以看出,自然周期IVF/IVM和IVM方案可以解决近一半周期的不孕症患者,并且可以获得与COH相近的临床妊娠率。     

超高龄原发性脑出血患者的临床特点及预后

目的探究超高龄（≥80岁）原发性脑出血患者的临床特点和预后情况。方法回顾性分析本院2011年1月至2013年1月期间收治170例原发性脑出血患者临床资料（超高龄者45例,对照组125例）,对比超高龄患者（≥80岁）和对照组（〈80岁）在临床症状、体征、影像学表现、疾病治疗等的差异,比较分析两组患者的生存情况。结果超高龄组中,女性所占的比例高于对照组,既往有高血压病史者、起病时有明确诱因或处于活动状态、入院时收缩压、舒张压低于对照组（P〈0．05）。超高龄组脑叶出血的情况多于对照组（31．1％比12．0％,P〈0．05）,基底节出血的情况则少于对照组（37．8％比62．4％,P〈0．05）;超高龄组采用手术治疗低于对照组,在住院时间上长于对照组,中位累积生存时间低于对照组（P〈0．05）。结论超高龄原发性脑出血可能是一种特殊类别的脑出血,脑血管淀粉样变性极有可能是其发病的重要病因。     

STK11 Domain XI Mutations: Candidate Genetic Drivers Leading to the Development of Dysplastic Polyps in Peutz–Jeghers Syndrome

Peutz–Jeghers syndrome (PJS) is a rare hereditary disorder resulting from mutations in serine/threonine kinase 11 (STK11) and characterized by gastrointestinal (GI) hamartomatous polyps, mucocutaneous pigmentation, and an increased risk for specific cancers. Little is known about the genetic implications of specific STK11 mutations with regard to their role in dysplastic and malignant transformation of GI polyps. Peripheral blood genomic DNA samples from 116 Chinese PJS patients from 52 unrelated families were investigated for STK11 mutations. Genotype–phenotype correlations were investigated. The mutation detection rate was 67.3% (51.9% point mutations, 15.4% large deletions). Fourteen out of the 25 point mutations identified were novel. Nearly one‐third of all mutations, 8/27 (29.6%), were in exon 7, the shortest out of the nine exons. Strikingly, mutations affecting protein kinase domain XI, encoded in part by exon 7, correlated with a 90% (9/10) incidence of GI polyp dysplasia. In contrast, only two out of 17 (11.8%) nondomain XI mutations were linked to polyp dysplasia (P = 0.0001). The extent of the association between dysplasia and the development of GI‐related cancers is currently unknown but our results highlight a novel STK11 genotype–phenotype association as the basis for future genetic counseling and basic research studies.     

Inhibitory effect of isavuconazole,ketoconazole, and voriconazole on the pharmacokinetics of methadone in vivo and in vitro

The aim of this study was to investigate the possible effect of orally administered isavuconazole, ketoconazole, or voriconazole on the pharmacokinetics of methadone in rats. Twenty Sprague–Dawley (SD) rats were divided randomly into four groups: Group A (control), group B (5 mg/kg isavuconazole), group C (5 mg/kg ketoconazole), and group D (5 mg/kg voriconazole). A single dose of methadone was administrated half an hour later. Methadone in plasma concentrations and its metabolite EDDP in microsomes were determined by ultra‐high‐performance liquid chromatography–tandem mass spectrometry method (UPLC–MS/MS), and pharmacokinetic parameters were calculated by DAS version 3.0. The C_max of methadone in groups C and D increased to 2.7‐fold and 5‐fold, respectively. While AUC increased in three groups and group D increased the most. Also, isavuconazole, ketoconazole, and voriconazole showed inhibitory effect on human and rat microsomes. The inhibition ratios of isavuconazole, ketoconazole, and voriconazole in rat liver microsome were 97.87%, 96.74% and 78.9%, respectively (p < 0.01), while in human liver microsome, inhibition ratios were 86.97%, 96.46%, and 53.11%, respectively. And the IC₅₀ for inhibition activity of isavuconazole, ketoconazole, and voriconazole in rat microsomes were 7.76 μM, 8.33 μM, and 4.45 μM, respectively. Our study indicated that taking methadone combine with ketoconazole, isavuconazole, or voriconazole could reduce the metabolism rate of methadone and prolong the pharmacological effects in vivo and in vitro.