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.     

Multimodal imaging for a theranostic approach in a murine model of B-cell lymphoma with engineered nanoparticles

Nanoparticles (NPs) are a promising tool for in vivo multimodality imaging and theranostic applications. Hyaluronic acid (HA)-based NPs have numerous active groups that make them ideal as tumor-targeted carriers. The B-lymphoma neoplastic cells express on their surfaces a clone-specific immunoglobulin receptor (Ig-BCR). The peptide A20-36 (pA20-36) selectively binds to the Ig-BCR of A20 lymphoma cells. In this work, we demonstrated the ability of core-shell chitosan-HA-NPs decorated with pA20-36 to specifically target A20 cells and reduce the tumor burden in a murine xenograft model. We monitored tumor growth using high-frequency ultrasonography and demonstrated targeting specificity and kinetics of the NPs via in vivo fluorescent reflectance imaging. This result was also confirmed by ex vivo magnetic resonance imaging and confocal microscopy. In conclusion, we demonstrated the ability of NPs loaded with fluorescent and paramagnetic tracers to act as multimodal imaging contrast agents and hence as a non-toxic, highly specific theranostic system.     

Targeted-gene silencing of BRAF to interrupt BRAF/MEK/ERK pathway synergized photothermal therapeutics for melanoma using a novel FA-GNR-siBRAF nanosystem

Melanoma is significantly associated with mutant BRAF gene, a suitable target for siRNA-based anti-melanoma therapy. However, a tumor-specific delivery system is a major hurdle for clinical applications. Here, we developed a novel nano-carrier, FA-GNR-siBRAF for safe topical application, which consists of folic acid (FA) as the tumor-targeting moiety, golden nanorods (GNR) providing photothermal capability to kill tumor cells under laser irradiation, and siRNA specifically silencing BRAF (siBRAF). The in vitro and in vivo results revealed that FA-GNR-siBRAF displayed high transfection rates, and subsequently induced remarkable gene knockdown of BRAF, resulting in suppression of melanoma growth due to the interruption of the MEK/ERK pathway. Combinatorial photothermal effects and BRAF knockdown by FA-GNR-siBRAF effectively killed tumor cells through apoptosis, with enhanced efficiency than individual treatments. Therefore, the FA-GNR-siBRAF simultaneously induced BRAF gene silencing and photothermal effects which achieved synergistic efficacy in the treatment of melanoma, paving a new path for developing clinical treatment methods for melanoma.     

Nano in nano: Biosynthesized gold and iron nanoclusters cargo neoplastic exosomes for cancer status biomarking

Timely detection is crucial for successful treatment of cancer. The current study describes a new approach that involves utilization of the tumor cell environment for bioimaging with in-situ biosynthesized nanoscale gold and iron probes and subsequent dissemination of Au-Fe nanoclusters from cargo exosomes within the circulatory system. We have isolated the Au-Fe cargo exosomes from the blood of the treated murine models after in situ biosyntheses from their respective pre-ionic solutions (HAuCl₄, FeCl₂), whereas Na₂SeO₃ supplementation added into Au lethal effect. The microarray data of various differentially expressed genes revealed the up-regulated tumor ablation and metal binding genes in SGC-7901 cell lines after treatment with Au-Fe-Se triplet ionic solution. The isolation of Au-Fe nanoclusters cargo exosomes (nano in nano) after secretion from deeply seated tumors may help in early diagnosis and reveal the tumor ablation status during and after the relevant treatment like radio-chemo therapies et al.     

Nanoformulations of anticancer FGFR inhibitors with improved therapeutic index

Fibroblast growth factor receptor (FGFR) inhibitors like ponatinib and nintedanib are clinically approved for defined cancer patient cohorts but often exert dose-limiting adverse effects. Hence, we encapsulated the FGFR inhibitors ponatinib, PD173074, and nintedanib into polylactic acid nanoparticles and liposomes to enable increased tumor accumulation/specificity and reduce side effects. Different methods of drug loading were tested and the resulting formulations compared regarding average size distribution as well as encapsulation efficiency. Appropriate encapsulation levels were achieved for liposomal preparations only. Nanoencapsulation resulted in significantly decelerated uptake kinetics in vitro with clearly decreased short-term (up to 72?h) cytotoxicity at higher concentrations. However, in long-term clonogenic assays liposomal formations were equally or even more active as compared to the free drugs. Accordingly, in an FGFR inhibitor-sensitive murine osteosarcoma transplantation model (K7M2), only liposomal but not free ponatinib resulted in significant tumor growth inhibition (by 60.4%) at markedly reduced side effects.     

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。     

咬合支持丧失后大鼠海马区神经组织形态学变化的透射电镜观察

目的:研究咬合支持丧失对高龄大鼠神经组织形态学方面的影响,从而探讨咬合支持对维持生理功能的重要性.方法:24只10月龄Wistar大鼠随机分为对照组、单侧磨牙拔除组和双侧磨牙拔除组,6周后,大鼠快速断头处死,透射电镜观察大鼠海马区神经组织形态.结果:与对照组相比,拔牙组大鼠海马区神经细胞核溶解、细胞膜完整性破坏和一些重要的蛋白合成细胞器消亡,以及突触数量减少,其中以双侧磨牙拔除组变化更为明显.结论:咬合支持丧失后6周,大鼠海马区神经组织形态学发生退行性变.     

生物活性皮肤替代物的超微结构观察

目的 :观察所培养的生物活性皮肤替代物的超微结构。方法 :以 5月龄胎儿的背部皮肤作为细胞来源 ,在牛I型胶原凝胶中立体培养成纤维细胞 ,3d后在凝胶表面接种表皮细胞 ,2d后将培养的皮肤替代物移至培养液气液面 ,促进上皮层的成熟和角化。观察HE染色后的人工皮肤组织学结构以及透射电镜下的超微结构。结果 :所培养的生物活性皮肤替代物同时含有表皮层和真皮层。表皮层中含有基底层、棘层、颗粒层和角质层。透射电镜观察结果显示 ,生物活性皮肤表皮层中 ,棘层细胞之间以桥粒连接 ,并具有天然皮肤中的板层体、张力纤维和脂滴等结构。结论 :生物活性皮肤替代物具有与天然皮肤类似的各种超微结构 ,为进一步的临床应用提供了实验依据     

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.     

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。