Role of site-directed mutagenesis and adjuvants in the stability and potency of anthrax protective antigen

Anthrax is a zoonotic infection caused by the gram-positive, aerobic, spore-forming bacterium Bacillus anthracis. Depending on the origin of the infection, serious health problems or mortality is possible. The virulence of B. anthracis is reliant on three pathogenic factors, which are secreted upon infection: protective antigen (PA), lethal factor (LF), and edema factor (EF). Systemic illness results from LF and EF entering cells through the formation of a complex with the heptameric form of PA, bound to the membrane of infected cells through its receptor. The currently available anthrax vaccines have multiple drawbacks, and recombinant PA is considered a promising second-generation vaccine candidate. However, the inherent chemical instability of PA through Asn deamidation at multiple sites prevents its use after long-term storage owing to loss of potency. Moreover, there is a distinct possibility of B. anthracis being used as a bioweapon; thus, the developed vaccine should remain efficacious and stable over the long-term. Second-generation anthrax vaccines with appropriate adjuvant formulations for enhanced immunogenicity and safety are desired. In this article, using protein engineering approaches, we have reviewed the stabilization of anthrax vaccine candidates that are currently licensed or under preclinical and clinical trials. We have also proposed a formulation to enhance recombinant PA vaccine potency via adjuvant formulation.     

The Microstructural Evolution and Mechanical Properties of Zr-Based Metallic Glass under Different Strain Rate Compressions

In this study, the high strain rate deformation behavior and the microstructure evolution of Zr-Cu-Al-Ni metallic glasses under various strain rates were investigated. The influence of strain and strain rate on the mechanical properties and fracture behavior, as well as microstructural properties was also investigated. Before mechanical testing, the structure and thermal stability of the Zr-Cu-Al-Ni metallic glasses were studied with X-ray diffraction (XRD) and differential scanning calorimeter. The mechanical property experiments and microstructural observations of Zr-Cu-Al-Ni metallic glasses under different strain rates ranging from 10⁻³ to 5.1 × 10³ s⁻¹ and at temperatures of 25 °C were investigated using compressive split-Hopkinson bar (SHPB) and an MTS tester. An in situ transmission electron microscope (TEM) nanoindenter was used to carry out compression tests and investigate the deformation behavior arising at nanopillars of the Zr-based metallic glass. The formation and interaction of shear band during the plastic deformation were investigated. Moreover, it was clearly apparent that the mechanical strength and ductility could be enhanced by impeding the penetration of shear bands with reinforced particles.     

镧盐处理后骨髓基质干细胞内超微结构及镧元素分析

目的:探讨镧离子(La3 )能否进入体外成骨化诱导的骨髓基质干细胞(BMSCs)并对其超微结构与金属成分产生影响。方法:取杂种犬第3代体外成骨化诱导培养的BMSCs,于培养基中分别加5.564×102、5.564、5.564×10-2μg/mlLa3 干预,设无La3 干预对照组。用透射电镜(TEM)、电子探针波谱(EM-WS)及高分辨透射电镜能谱(HRTEM)观察BMSCs超微结构与金属成分。结果:TEM下观察3种浓度La3 作用后BMSCs溶酶体内有电子致密物质存在,对照组无此现象;3组BMSCs的部分细胞器有轻微变性现象,与对照组相似;EMWS检测到5.564×102μg/ml的La3 作用后BMSCs中有镧元素存在;3种浓度La3 作用后HRTEM检测BMSCs内均未测出镧元素。结论:一定浓度下La3 可能进入BMSCs。     

细菌胞外膜泡的电镜观察

本实验对分离的５株细菌的ＥＣＶ作了电镜观察，证实４株菌都能形成ＥＣＶ，但在量上有差异。以ＰｇＷ５０最为典型，菌体及其ＥＣＶ的结构层次清晰可见；ＰｅＥＣＶ的荚膜层较薄且直径相对小而均一；Ｐｍ和Ｐｉ的ＥＣＶ大小不均一，标本中破裂现象和不规碎片出现较早。     

Location,arrangement and possible function of interodontoblastic collagen fibres in association with calcium hydroxide-induced hard tissue bridges

AIM: To assess the location, arrangement and possible function of interodontoblastic collagen fibres in association with calcium hydroxide-induced hard tissue bridges by using light and transmission electron microscopy techniques and immunohistochemical staining localization. METHODOLOGY: Prior to the study, an animal use protocol form was reviewed and approved by the Screening Committee for Animal Research of the Tokyo Medical and Dental University. Exposed monkey pulps were capped with a hard-set calcium hydroxide and histopathologically evaluated at 3, 14, 21, 30 and 90 days, using light microscopy with silver staining and transmission electron microscopy to differentiate structural features of interodontoblastic collagen fibres. In addition, an attempt was made to identify and to differentiate between several types of collagen and fibronectin using immunohistochemical localization techniques. RESULTS: At 14 days, interodontoblastic collagen fibres were observed extending from the original dentine, passing through the odontoblasts, and consisted of two portions: a thick fibril and a thin fibril. At 21 days, interodontoblastic collagen fibres were seen penetrating into the predentine and becoming incorporated into the mineralized dentine. At 30 days, interodontoblastic collagen fibres reached the cell process. Although interodontoblastic collagen fibres were no longer observed near the odontoblastoid cells at the area of the newly formed tubular dentine, interodontoblastic collagen fibres were observed embedded within the primary formed dentine bridge. Immunohistochemical staining demonstrated type I collagen and fibronectin within the interodontoblastic collagen fibres. CONCLUSIONS: Interodontoblastic collagen fibres were routinely detected throughout early dentine bridges. Interodontoblastic collagen fibres are thought to be important for initial dentine bridging to induce and support a dentinogenesis framework.     

Alteration of Connexin43 expression in a rat model of obesity-related glomerulopathy

It is accepted that alteration of connexin43 (Cx43) expression in glomeruli is a common pathological response in several forms of kidney diseases. To date, however the change of the Cx43 expression in obesity-related glomerulopathy (ORG) has not been reported. In this study, the alteration of Cx43 expression in the glomeruli of rat with ORG was defined. Five-week-old rats were fed with high-fat diet for 18 weeks to establish the ORG model, then the histological change of glomeruli, the foot process effacement of podocyte, the markers for podocyte injury (nephrin,podocin and WT1) and Cx43 expression in glomeruli were examined respectively. The results demonstrated metabolic disorder, hyperinsulinemia, systemic inflammation and microalbuminuria in ORG rats. There was significant hypertrophy, glomerular expansion and inflammatory cell infiltration in the kidney of ORG rats compared to the control group. Significant foot process effacement of the podocyte in the glomeruli, nephrin loss and density reduction were shown in the ORG rats, and Cx43 expression was significant upregulated in glomeruli of ORG rats compared to the control group. The results indicate the correlation of overexpressed Cx43 with the obesity related renal inflammation and suggest that Cx43 might be a potential target in the development of obesity related glomerulopathy.     

Photodynamic therapy-mediated hypericin-loaded nanostructured lipid carriers against vulvovaginal candidiasis

Introduction and Aim: The indiscriminate use and adverse effects of the main conventional antifungal agents compromise the effectiveness of treating vulvovaginal candidiasis (VVC), mainly caused by the species Candida albicans. This study evaluated the effectiveness of photodynamic therapy (PDT) and the in vitro and in vivo anti-candida potential of the hypericin (HYP)-loaded nanostructured lipid carriers (NLC). Materials and Methods: Empty NLC and NLC-HYP were characterized by the dynamic light scattering technique and transmission electron microscopy to evaluate the average particle size distribution and its morphologies. The in vitro inhibition photodynamic effect of the systems was tested to reduce the planktonic viability of C. albicans. The therapeutic assay photodynamic of the systems was performed to treat VVC in mice. Results: Empty NLC and NLC-HYP presented values of average hydrodynamic diameter, polydispersity index, and ζ-potential from 136 to 133 nm, 0.16 to 0.22, and -18 to -30 mV, respectively, on day 30. Microscopically, the systems showed spherical morphologies and nanoscale particles. Furthermore, in the in vitro inhibition assay, the treatment of PDT with NLC-HYP (NLC-HYP+) showed a significant reduction of the C. albicans planktonic viability compared to YNB negative control after 5 min of LED light irradiation. In the in vivo therapeutic assay, the antifungal group (vaginal antifungal cream) and NLC-HYP+ evaluated in the dark and by PDT, respectively, had a significant log₁₀ reduction in fungal burden compared to the infected group on day 8 of the VVC treatment. Conclusion: Due to the in vitro and in vivo anti-candida potential, PDT-mediated systems can be an effective strategy in VVC therapy.     

Endoluminal loco-regional resection by TEM after R1 endoscopic removal or recurrence of rectal tumors

Purpose The aim of this study is to evaluate the safety and efficacy of endoluminal loco-regional resection (ELRR) by transanal endoscopic microsurgery (TEM) after R1 endoscopic resection or local recurrence of early rectal cancer after operative endoscopy. Material and methods Twenty patients with early rectal cancer were enrolled, including patients with incomplete endoscopic resection, or complete endoscopic resection of a tumor with unfavorable prognostic factors (group A, ten patients), and local recurrence after endoscopic removal (group B, ten patients). At admission, histology after endoscopic polypectomy was: TisR1(4), T1R0G3(1), T1R1(5) in group A, and TisR0(8), T1R0(2) in group B. All patients underwent ELRR by TEM with nucleotide-guided mesorectal excision (NGME). Results Mean operative time was 150?minutes. Complications occurred in two patients (10%). Definitive histology was: moderate dysplasia(4), pT0N0(3), pTisN0(5), pT1N0(6), pT2N0(2). Mean number of lymph-nodes was 3.1. Mean follow-up was 79.5 months. All patients are alive and disease-free. Conclusions ELRR by TEM after R1 endoscopic resection of early rectal cancer or for local recurrence after operative endoscopy is safe and effective. It may be considered as a diagnostic procedure, as well as a curative treatment option, instead of a more invasive TME.