霍乱弧菌CT毒素环介导等温扩增技术快速检测方法的建立

目的将一种新颖的核酸扩增技术——环介导等温扩增技术(LAMP)应用于霍乱弧菌毒素基因ctxA的快速检测。方法针对霍乱弧菌毒素基因ctxA设计6条特异性引物(两条内引物、两条外引物和两条环引物)进行LAMP扩增,对扩增反应进行优化。并考核方法的敏感性和特异性。结果最佳反应时间为60 min,反应温度为65℃。对8种相关肠道细菌进行LAMP扩增,仅含毒素基因ctxA的霍乱弧菌得到阳性扩增结果,证明引物具有很高的特异性。霍乱弧菌基因组DNA和纯培养物的检测灵敏度分别约为68 fg和42 cfu/ml。对模拟样品进行直接检测,检测限为72 cfu/g。结论该方法检测霍乱弧菌毒素基因ctxA特异性强、灵敏度高,并且操作简便、检测成本低,1 h即可完成,适合基层检验部门使用。     

The utility of cerebrospinal fluid for the molecular diagnosis of toxoplasmic encephalitis

The aim of this study was to assess the efficacy of nested polymerase chain reaction (PCR) and the loop-mediated isothermal amplification (LAMP) assay, which were developed to detect and identify toxoplasma parasites in human cerebrospinal fluid (CSF). Nested PCR was performed using primers generated by Dr. L.D. Sibley to target the 18S rDNA instead of the conventionally used primers which target the B1 gene. We also designed Toxoplasma gondii–specific LAMP primers targeting both genes. In vitro detection sensitivity was evaluated using 10-fold serially diluted genomic DNA purified from RH tachyzoites, and clinical sensitivity and specificity were evaluated using clinical CSF samples from 16 patients with toxoplasmic encephalitis (TE) and from 12 patients with other diseases. The 18S rDNA nested PCR showed the highest detection sensitivity limit with a minimum of 1.0 × 10⁻⁸ ng/μL. However, sensitivity and specificity of nested PCR with clinical specimens were 50% and 100%, respectively. The sensitivity of molecular diagnosis of TE is not sufficient; therefore, patients clinically suspected of having TE should be treated promptly. Our molecular diagnostic tool would restrictively facilitate a definitive diagnosis of TE at an early stage in approximately 50% of patients.     

辣椒碱-羟丙基-β-环糊精包合物制备鉴定及 热力学稳定性研究

目的:制备及鉴定辣椒碱-HP-β-CD包合物,并考察辣椒碱和HP-β-CD之间的包合摩尔比及包合过程的热力学常数.方法:采用饱和水溶液法制备辣椒碱-HP-β-CD包合物,以差示扫描量热法(DSC)、X射线衍射法(XRD)和红外光谱法(IR)对包合物进行鉴定,通过表观溶解度法考察包合物中主客分子之间的包合摩尔比及包合过程的热力学常数.结果:25,35,45℃下辣椒碱和HP-β-CD能形成1:1摩尔比包合物,相溶解度图呈A_L型.结论:辣椒碱-羟丙基-β-环糊精包合物能显著增大药物的溶解度.     

Influence of Cement Replacement with Sewage Sludge Ash (SSA) on the Heat of Hydration of Cement Mortar

The amount of fly ash from the incineration of sewage sludge is increasing all over the world, and its utilization is becoming a serious environmental problem. In the study, a type of sewage sludge ash (SSA) collected directly from the municipal sewage treatment plant was used. Five levels of cement replacement (2.5%, 5%, 7.5%, 10% and 20%) and unchanged water-to-binder (w/b) ratio (0.55) were used. The purpose of the study was to evaluate the effect of sewage sludge ash (SSA) on the hydration heat process of cement mortars. The heat of the hydration of cement mortars was monitored by the isothermal calorimetric method for 7 days at 23 °C. The analysis of chemical composition and particle size distribution was performed on the tested material. The tests carried out have shown that SSA particles have irregular grain morphology and, taking into account the chemical composition consists mainly of oxides such as CaO, P₂O₅, SiO₂ and Al₂O₃. The concentration of these compounds affects the hydration process of cement mortars doped with SSA. In turn, the content of selected heavy metals in the tested ash should not pose a threat to the environment. Calorimetric studies proved that the hydration process is influenced by the presence of SSA in cement mortars. The studies showed that the rate of heat generation decreased (especially in the initial setting period) with the increasing replacement of cement by SSA, which also reduced the amount of total heat compared to the control cement mortar. With increasing mass of the replacement of cement with SSA up to 20%, the 7-day compressive strength of the mortar samples decreases.     

Comparative Evaluation of Rapid Isothermal Amplification and Antigen Assays for Screening Testing of SARS-CoV-2

Human transmission of SARS-CoV-2 and emergent variants of concern continue to occur globally, despite mass vaccination campaigns. Public health strategies to reduce virus spread should therefore rely, in part, on frequent screening with rapid, inexpensive, and sensitive tests. We evaluated two digitally integrated rapid tests and assessed their performance using stored nasal swab specimens collected from individuals with or without COVID-19. An isothermal amplification assay combined with a lateral flow test had a limit of detection of 10 RNA copies per reaction, and a positive percent agreement (PPA)/negative percent agreement (NPA) during the asymptomatic and symptomatic phases of 100%/100% and 95.83/100%, respectively. Comparatively, an antigen-based lateral flow test had a limit of detection of 30,000 copies and a PPA/NPA during the asymptomatic and symptomatic phases of 82.86%/98.68% and 91.67/100%, respectively. Both the isothermal amplification and antigen-based lateral flow tests had optimized detection of SARS-CoV-2 during the peak period of transmission; however, the antigen-based test had reduced sensitivity in clinical samples with qPCR Ct values greater than 29.8. Low-cost, high-throughput screening enabled by isothermal amplification or antigen-based techniques have value for outbreak control.     

Structural Basis of the Avian Influenza NS1 Protein Interactions with the Cell Polarity Regulator Scribble

Scribble is a highly conserved regulator of cell polarity, a process that enables the generation of asymmetry at the cellular and tissue level in higher organisms. Scribble acts in concert with Disc-large (Dlg) and Lethal-2-giant larvae (Lgl) to form the Scribble polarity complex, and its functional dysregulation is associated with poor prognosis during viral infections. Viruses have been shown to interfere with Scribble by targeting Scribble PDZ domains to subvert the network of interactions that enable normal control of cell polarity via Scribble, as well as the localisation of the Scribble module within the cell. The influenza A virus NS1 protein was shown to bind to human Scribble (SCRIB) via its C-terminal PDZ binding motif (PBM). It was reported that the PBM sequence ESEV is a virulence determinant for influenza A virus H5N1 whilst other sequences, such as ESKV, KSEV and RSKV, demonstrated no affinity towards Scribble. We now show, using isothermal titration calorimetry (ITC), that ESKV and KSEV bind to SCRIB PDZ domains and that ESEV unexpectedly displayed an affinity towards all four PDZs and not just a selected few. We then define the structural basis for the interactions of SCRIB PDZ1 domain with ESEV and ESKV PBM motifs, as well as SCRIB PDZ3 with the ESKV PBM motif. These findings will serve as a platform for understanding the role of Scribble PDZ domains and their interactions with different NS1 PBMs and the mechanisms that mediate cell polarity within the context of the pathogenesis of influenza A virus.     

Loop mediated isothermal amplification (LAMP) for the detection and subtyping of human papillomaviruses (HPV) in oropharyngeal squamous cell carcinoma (OPSCC)

BackgroundHuman papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) is a growing problem that presents a significant challenge to Otolaryngologist-Head and Neck Surgeons. Knowledge of HPV status yields critical prognostic information, with potential for treatment selection based on tumour HPV status. The current gold standard of diagnosis, PCR, is expensive, demanding and time consuming. Alternatives such as p16 immunohistochemistry are subjective and potentially inaccurate. Loop-mediated isothermal amplification (LAMP) is a rapid, robust and inexpensive molecular diagnostic technique.ObjectivesOur aim was to verify LAMP as a potential bedside diagnostic assay for subtyping of HPV in OPSCC.Study designDNA from 72 formalin-fixed paraffin embedded (FFPE) OPSCC patient samples was tested. PCR and LAMP were then performed to specifically identify HPV 16, 18, 31, 33 and 35.Results and conclusionsFor these high-risk subtypes, LAMP had an overall sensitivity of 99.4% and specificity of 93.2% relative to PCR. LAMP is a promising technology that can accurately diagnose high-risk HPV infection.     

Cyrene™ as an Alternative Sustainable Solvent for the Preparation of Poly(lactic-co-glycolic acid) Nanoparticles

Toxic and environmental harmful organic solvents are widely applied to prepare poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NP) in standard preparation methods. Alternative non-toxic solvents suffer from disadvantages like high viscosity and plasticizing effects. To overcome these hurdles, Cyrene? as a new sustainable, non-toxic and low viscous solvent was used to formulate PLGA NPs. A new preparation method was developed and optimized. Small sized blank NPs around 220 nm with a narrow size distribution and highly negative charge (<?23 mV) were obtained. To test the application for drug delivery, the lipophilic model drug atorvastatin was encapsulated in high drug loads with comparable physicochemical characteristics as the blank NPs, and a total drug release within 24 h. No changes of the crystallinity or plasticizing effects could be observed. Highly purified NPs were obtained with a residual Cyrene? content <2.5%. Finally, the biocompatibility of Cyrene? itself and of the NPs formed in the presence of Cyrene? was demonstrated in a hen's egg test. Conclusively, the use of Cyrene? as solvent offers a simple, fast and non-toxic procedure for preparation of PLGA NPs as drug delivery systems circumventing the downsides of standard methods.     

Loading of Tetanus Toxoid to Biodegradable Nanoparticles from Branched Poly(Sulfobutyl-Polyvinyl Alcohol)-g-(Lactide-Co-Glycolide) Nanoparticles by Protein Adsorption: A Mechanistic Study

Purpose. Mucosal delivery of vaccine-loaded nanoparticles (NP) is an attractive proposition from an immunologic perspective. Although numerous NP preparation methods are known, sufficient antigen loading of NP remains a challenge. The aim of this study was to evaluate adsorptive loading of NP with a negatively charged surface structure using tetanus toxoid (TT) as a model vaccine. Methods. Blank NP, consisting of poly(sulfobutyl-polyvinyl alcohol)-g-(lactide-co-glycolide), as well as poly(lactide-co-glycolide) NP were prepared by a solvent displacement technique. The use of polymers with different degrees of substitution resulted in NP with different negative surfaces charges. Adsorption of TT to NP was performed varying to NP surface properties, protein equilibrium concentration, and loading conditions. Results. The protein adsorption was controlled by NP surface properties, and maximum TT adsorption occurred at highly negatively charged NP surfaces. Results from isothermal titration calorimetry and -potential measurement suggest an adsorption process governed by electrostatic interactions. The adsorption followed the Langmuir isotherm in the concentration ranges studied. TT withstood this gentle loading procedure in a nonaggregated, enzyme-linked immunoabsorbant assay-active form. Conclusions. The results demonstrate that negatively charged NP consisting of poly(sulfobutyl-polyvinyl alcohol)-g-(lactide-co-glycolide) are suitable for adsorptive loading with TT and may have potential for mucosal vaccination.     

The influence of charge clustering on the anti-HIV-1 activity and in vivo distribution of negatively charged albumins

The substitution of human serum albumin with negatively charged molecules, such as succinic acid (Suc-HSA) or aconitic acid (Aco-HSA), resulted in proteins with potent anti-HIV activities, by binding to viral gp120 (V3 loop). The aim of the present study was to investigate whether the distribution of negative charges on the albumin backbone influences the anti-HIV activity. Therefore, we prepared albumins with clusters of negatively charged groups by coupling of heparin. The effects of this substitution on anti-HIV activity, in vivo distribution and the protein structure as compared to random succinylation were assessed. In vitro studies indicated that HSA-modified with heparin 6 or 13 kD displayed anti-HIV activity (IC50=660 and 37 nM, respectively) and exhibited affinity for gp120-V3 loop, although the activity was lower than that of Suc-HSA. Combined derivatization of HSA with heparin 13 kD and aconitic acid groups resulted in significantly increased inhibitory actions (IC50=2.8 nM). Structural analysis showed that modification of HSA with heparin did not lead to extensive unfolding of the protein, meaning that these modified proteins were still globular in structure. In contrast, succinylation of HSA resulted in a highly randomly coiled conformation. Dynamic light scattering experiments revealed that, at neutral pH, the heparin fragments attached to the protein were wrapped around the molecule rather than sticking out into the solution. In conclusion, coupling of sufficient clustered negative charges, by coupling of Hep-fragments, on HSA resulted in a clear anti-HIV activity of the protein. Yet, random distribution of anionic groups in the albumin seemed more optimal for in vitro anti-HIV activity. The higher plasma and lymphatic concentrations of Hep-HSA compared to Suc-HSA seemed more favorable for an anti-HIV activity in vivo.