Immobilization of Lipase B from Candida antarctica in Octyl-Vinyl Sulfone Agarose: Effect of the Enzyme-Support Interactions on Enzyme Activity,Specificity, Structure and Inactivation Pathway

Lipase B from Candida antarctica was immobilized on heterofunctional support octyl agarose activated with vinyl sulfone to prevent enzyme release under drastic conditions. Covalent attachment was established, but the blocking step using hexylamine, ethylenediamine or the amino acids glycine (Gly) and aspartic acid (Asp) altered the results. The activities were lower than those observed using the octyl biocatalyst, except when using ethylenediamine as blocking reagent and p-nitrophenol butyrate (pNPB) as substrate. The enzyme stability increased using these new biocatalysts at pH 7 and 9 using all blocking agents (much more significantly at pH 9), while it decreased at pH 5 except when using Gly as blocking agent. The stress inactivation of the biocatalysts decreased the enzyme activity versus three different substrates (pNPB, S-methyl mandelate and triacetin) in a relatively similar fashion. The tryptophane (Trp) fluorescence spectra were different for the biocatalysts, suggesting different enzyme conformations. However, the fluorescence spectra changes during the inactivation were not too different except for the biocatalyst blocked with Asp, suggesting that, except for this biocatalyst, the inactivation pathways may not be so different.     

Modeling RTT Syndrome by iPSC-Derived Neurons from Male and Female Patients with Heterogeneously Severe Hot-Spot MECP2 Variants

Rett syndrome caused by MECP2 variants is characterized by a heterogenous clinical spectrum accounted for in 60% of cases by hot-spot variants. Focusing on the most frequent variants, we generated in vitro iPSC-neurons from the blood of RTT girls with p.Arg133Cys and p.Arg255*, associated to mild and severe phenotype, respectively, and of an RTT male harboring the close to p.Arg255*, p.Gly252Argfs*7 variant. Truncated MeCP2 proteins were revealed by Western blot and immunofluorescence analysis. We compared the mutant versus control neurons at 42 days for morphological parameters and at 120 days for electrophysiology recordings, including girls’ isogenic clones. A precocious reduced morphological complexity was evident in neurons with truncating variants, while in p.Arg133Cys neurons any significant differences were observed in comparison with the isogenic wild-type clones. Reduced nuclear size and branch number show up as the most robust biomarkers. Patch clamp recordings on mature neurons allowed the assessment of cell biophysical properties, V-gated currents, and spiking pattern in the mutant and control cells. Immature spiking, altered cell capacitance, and membrane resistance of RTT neurons, were particularly pronounced in the Arg255* and Gly252Argfs*7 mutants. The overall results indicate that the specific markers of in vitro cellular phenotype mirror the clinical severity and may be amenable to drug testing for translational purposes.     

Enlarging the Scope of 5-Aminolevulinic Acid-Mediated Photodiagnosis towards Breast Cancers

Today, most research on treating cancers targets one single cancer, often because of the very specific operation principle of the therapy. For instance, immunotherapies require the expression of a particular antigen, which might not be expressed in all cancers or in all patients. What about metastases? Combination therapies are promising but require treatment personalization and are an expensive approach that many health systems are not willing to pay for. Resection of cancerous tissues may be conducted beforehand. However, the precise location and removal of tumors are in most cases, hurdles that require margins to prevent recurrence. Herein, we further demonstrate the wide application of aminolevulinate-based photodynamic diagnosis and therapy toward breast cancers. By selecting four breast cancer cell lines that represent the main breast tumor subtypes, we investigated their ability to accumulate the fluorescent protoporphyrin IX upon treatment with the marketed 5-aminolevulinic acid hexyl ester (ALA-Hex) or our new and more stable derivative PSI-ALA-Hex. We found that all cell lines were able to accumulate PpIX under a few hours independent of their hormonal status with both treatments. Additionally, this accumulation was less dose-dependent with PSI-ALA-Hex and induced similar or higher fluorescence intensity than ALA-Hex in three out of four cell lines. The toxicity of the two molecules was not different up to 0.33 mM. However, PSI-ALA-Hex was more toxic at 1 mM, even though lower concentrations of PSI-ALA-Hex led to the same PpIX accumulation level. Additional illumination with blue light to induce cell death by generating reactive oxygen species was also considered. The treatments led to a dramatic death of the BT-474 cells under all conditions. In SK-BR-3 and MCF-7, ALA-Hex was also very efficient at all concentrations. However, increasing doses of PSI-ALA-Hex (0.33 and 1 mM) surprisingly led to a higher viability rate. In contrast, the triple-negative breast cancer cells MDA-MB-231 showed a higher death induction with higher concentrations of ALA-Hex or PSI-ALA-Hex. Derivatives of ALA seem promising as fluorescence-guided resection tools and may enable subsequent completion of cancer cell destruction by blue light irradiation.     

Precision Killing of Sinoporphyrin Sodium-Mediated Photodynamic Therapy against Malignant Tumor Cells

Photodynamic therapy (PDT) has significant advantages in the treatment of malignant tumors, such as high efficiency, minimal invasion and less side effects, and it can preserve the integrity and quality of the organs. The power density, irradiation time and photosensitizer (PS) concentration are three main parameters that play important roles in killing tumor cells. However, until now, the underlying relationships among them for PDT outcomes have been unclear. In this study, human malignant glioblastoma U-118MG and melanoma A375 cells were selected, and the product of the power density, irradiation time and PS concentration was defined as the total photodynamic parameter (TPP), in order to investigate the mechanisms of PS sinoporphyrin sodium (DVDMS)-mediated PDT (DVDMS-PDT). The results showed that the survival rates of the U-118MG and A375 cells were negatively correlated with the TPP value in the curve, and the correlation exactly filed an e-exponential function. Moreover, according to the formula, we realized controllable killing effects of the tumor cells by randomly adjusting the three parameters, and we finally verified the accuracy and repeatability of the formula. In conclusion, the establishment and implementation of a newly functional relationship among the PDT parameters are essential for predicting PDT outcomes and providing personalized precise treatment, and they are contributive to the development of PDT dosimetry.     

三种卟啉类光敏剂的荧光量子产率

实验研究了喜泊分(Hiporfin),血啉甲醚(HMME)和癌光啉(PsD-007)等3种国产卟啉类光敏剂在水溶液中的吸收和荧光光谱特性,并利用对照法首次测定了它们在水溶液中的荧光量子产率.Hiporfin,HMME和PsD-007具有相似的吸收光谱和荧光发射特性,它们的荧光量子产率分别为0.013,0.026和0.020.实验表明光敏剂的荧光量子产率还与溶液中的氧含量有关.实验结果为光动力学诊断中光敏剂的选择,以及相应最佳荧光激发和检测波长的选取提供了理论依据.     

填充床放电反应器灭活铜绿微囊藻的机理

为了得到一种合理有效的铜绿微囊藻去除方法,研究了填充床放电反应器灭活铜绿微囊藻的机理,主要考察了放电对藻细胞数的影响、细胞形态的影响和生长的间接影响,以及过氧化氢在藻细胞外产物溶液中的浓度和稳定性,外加过氧化氢对藻生长的影响。结果表明:放电处理当天藻细胞数下降不太明显,但在放置培养过程中下降明显;藻处理后的扫描电镜图显示细胞表面受到严重破坏,细胞内物质溢出,细胞出现孔洞;处理后的藻细胞外产物溶液对藻生长率的影响随着放置时间的增加而下降,整个放置培养期间,离心前藻细胞的光密度较离心后的略有降低;放电产生的过氧化氢浓度为μmol/L量级,且随着放电处理时间的增加而增加,但放置培养期间呈一级下降趋势;填充床放电反应器灭活铜绿微囊藻主要机理或原因是放电产生的物理和化学的综合作用导致铜绿微囊藻细胞死亡,过氧化氢的相对稳定存在加强了一些本已受到放电破坏的藻细胞损伤程度,使这部分细胞不能自身修复而逐渐死亡。     

光敏剂在光动力治疗中的应用研究进展

光动力疗法（PDT）以其超高时空分辨率、非侵入性及低毒副作用的优点,被认为是治疗癌症和各种非恶性疾病的有效疗法之一。本文主要综述了几类光敏剂发展历史、主要结构、特点及研究进展,分析了高性能光敏剂的开发动态,包括化学修饰;与具有特定细胞受体的其他配体缀合成复合光敏剂;采取纳米技术,如纳米颗粒输送,基于富勒烯的光敏剂等。基于此,指出具有临床应用前景的高性能光敏剂的基本特征、设计原则及发展趋势。     

NIR-Triggered Generation of Reactive Oxygen Species and Photodynamic Therapy Based on Mesoporous Silica-Coated LiYF4 Upconverting Nanoparticles

To date, the increase in reactive oxygen species (ROS) production for effectual photodynamic therapy (PDT) treatment still remains challenging. In this study, a facile and effective approach is utilized to coat mesoporous silica (mSiO₂) shell on the ligand-free upconversion nanoparticles (UCNPs) based on the LiYF₄ host material. Two kinds of mesoporous silica-coated UCNPs (UCNP@mSiO₂) that display green emission (doped with Ho³⁺) and red emission (doped with Er³⁺), respectively, were successfully synthesized and well characterized. Three photosensitizers (PSs), merocyanine 540 (MC 540), rose bengal (RB), and chlorin e6 (Ce6), with the function of absorption of green or red emission, were selected and loaded into the mSiO₂ shell of both UCNP@mSiO₂ nanomaterials. A comprehensive study for the three UCNP@mSiO₂/PS donor/acceptor pairs was performed to investigate the efficacy of fluorescence resonance energy transfer (FRET), ROS generation, and in vitro PDT using a MCF-7 cell line. ROS generation detection showed that as compared to the oleate-capped and ligand-free UCNP/PS pairs, the UCNP@mSiO₂/PS nanocarrier system demonstrated more pronounced ROS generation due to the UCNP@mSiO₂ nanoparticles in close vicinity to PS molecules and a higher loading capacity of the photosensitizer. As a result, the three LiYF₄ UCNP@mSiO₂/PS nanoplatforms displayed more prominent therapeutic efficacies in PDT by using in vitro cytotoxicity tests.     

Application of Antimicrobial Photodynamic Therapy for Inactivation of Acinetobacter baumannii Biofilms

Acinetobacter baumannii is a dangerous hospital pathogen primarily due to its ability to form biofilms on different abiotic and biotic surfaces. The present study investigated the effect of riboflavin- and chlorophyllin-based antimicrobial photodynamic therapy, performed with near-ultraviolet or blue light on the viability of bacterial cells in biofilms and their structural stability, also determining the extent of photoinduced generation of intracellular reactive oxygen species as well as the ability of A. baumannii to form biofilms after the treatment. The efficacy of antimicrobial photodynamic therapy was compared with that of light alone and the role of the photosensitizer type on the photosensitization mechanism was demonstrated. We found that the antibacterial effect of riboflavin-based antimicrobial photodynamic therapy depends on the ability of photoactivated riboflavin to generate intracellular reactive oxygen species but does not depend on the concentration of riboflavin and pre-incubation time before irradiation. Moreover, our results suggest a clear interconnection between the inactivation efficiency of chlorophyllin-based antimicrobial photodynamic therapy and the sensitivity of A. baumannii biofilms to used light. In summary, all the analyzed results suggest that riboflavin-based antimicrobial photodynamic therapy and chlorophyllin-based antimicrobial photodynamic therapy have the potential to be applied as an antibacterial treatment against A. baumannii biofilms or as a preventive measure against biofilm formation.     

高静压处理对预制绿芦笋品质的影响

绿芦笋嫩茎含水量高,呼吸作用较强,采收后不耐贮藏。高静压技术(High hydrostatic pressure,HHP)是一种新兴的最少加工技术,为解决绿芦笋采后损失问题提供了有效途径。本文研究了高静压处理对预制绿芦笋(Asparagus officinalis L)杀菌效果与品质的影响。分别在300、400、500、600 MPa压力和室温(25℃)下,研究了HHP处理绿芦笋1、3、5、10、15、20 min对微生物的杀灭效果。在保证杀菌效果的基础上,筛选出3组HHP处理参数,研究了高静压处理后预制绿芦笋贮藏品质的变化。结果表明:随着压力升高及保压时间延长,HHP的杀菌效果逐渐增强;霉菌和酵母对压力比较敏感,300 MPa处理3 min就能全部杀灭;300 MPa处理15 min、400 MPa处理10 min、500 MPa处理5 min、600 MPa处理3 min后,绿芦笋菌落总数含量均低于100 cfu/g。Weibull模型在压力300~600 MPa范围内拟合度较高(相关系数R~2>0.970)。贮藏期间,与传统热处理相比,3组高静压处理较好的保留了绿芦笋的色泽、质构、维生素含量等品质。综上所述,500 MPa处理5 min的预制绿芦笋贮藏期可达21 d,品质优于其它两组高静压处理。