One-Step Nucleic Acid Purification and Noise-Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow-Abundance Nucleic Acid Detection

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point-of-care diagnostics. Currently, nucleic acid (NA) purification remains time-consuming and labor-intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one-step, liquid-phase NA purification that is simpler and faster than conventional solid-phase extraction. By further re-concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non-specific amplification caused by non-optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non-optimal PCR designs, which is 10- and 1000-fold fewer than those of the standard bench-top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point-of-care diagnosis.     

A liquid chromatography coupled to quadrupole–time-of-flight–mass spectrometry (LC–QTOF–MS) method for identification analysis of saponins from Quillaja saponaria bark extracts in foot-and-mouth disease vaccines: Development,validation and applicability

Saponins from Quillaja saponaria have been commonly used as immunomodulatory adjuvants in foot-and-mouth disease vaccines (FMDVs). However, due to the lack of consensus over the possible exacerbation of local inflammatory responses in cattle and its economic impacts, their use has been discouraged by Brazilian authorities. A qualitative method intended to determine the presence of saponins from Q. saponaria bark extracts in FMDVs was developed and validated. Instrumental analysis was performed using an liquid chromatography (LC) coupled to a quadrupole–time-of-flight–mass spectrometry (TOF-MS) system. The method was validated according to the International Conference on Harmonization Harmonized Tripartite Guideline Q2 (R1) and Brazilian Ministry of Agriculture, Livestock and Food Supply Analytical Quality Assurance Guidelines. Validation parameters were determined and considered suitable to the established criteria. The validated method has been applied in routine analysis in the National Agricultural Laboratory at Rio Grande do Sul (LANAGRO-RS). All results obtained were in agreement with the vaccine's composition described by the manufacturer. The method is easy and adequate for analysis in routine laboratories. To the best of the authors' knowledge, this is the first report of a method which intends to investigate the presence of saponins from Q. saponaria bark extracts in veterinary vaccines.     

The Cytotoxic Effect of α-Synuclein Aggregates

Parkinson's disease is a neurodegenerative disorder involving a functional protein, α-synuclein, whose primary function is related to vesicle trafficking. However, α-synuclein is prone to form aggregates, and these inclusions, known as Lewy bodies, are the hallmark of Parkinson's disease. α-synuclein can alter its conformation and acquire aggregating capacity, forming aggregates containing β-sheets. This protein's pathogenic importance is based on its ability to form oligomers that impair synaptic transmission and neuronal function by increasing membrane permeability and altering homeostasis, generating a deleterious effect over cells. First, we establish that oligomers interfere with the mechanical properties of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane, as demonstrated by nanoindentation curves. In contrast, nanoindentation revealed that the α-synuclein monomer's presence leads to a much more resistant lipid bilayer. Moreover, the oligomers’ interaction with cell membranes can promote lactate dehydrogenase (LDH) release, suggesting the activation of cytotoxic events.     

High-throughput lipidomics analysis to discover lipid biomarkers and profiles as potential targets for evaluating efficacy of Kai-Xin-San against APP/PS1 transgenic mice based on UPLC–Q/TOF–MS

Lipid metabolism has a significant function in the central nervous system and Alzheimer's disease (AD) is an age-related senile disease characterized by central nerve degeneration. The pathological development of AD is closely related to lipid metabolism disorders. To reveal the influence of Kai-Xin-San (KXS) on lipid metabolism in APP/PSI transgenic mice and potential therapeutic targets for treating AD, brain tissue samples were collected and analyzed by high-throughput lipidomics based on UPLC–Q/TOF-MS. The collected raw data were processed by multivariate data analysis to discover the potential biomarkers and lipid metabolic profiles. Compared with the control wild-type mouse group, nine potential lipid biomarkers were found in the AD model group, of which seven were up-regulated and two were down-regulated. Orally administrated KXS can reverse the changes in these potential biomarkers. Compared with the model group, a total of six differential metabolites showed a recovery trend and may be potential targets for KXS to treat AD. This study showed that high-throughput lipidomics can be used to discover the perturbed pathways and lipid biomarkers as potential targets to reveal the therapeutic effects of KXS.     

Extracellular Matrix Proteins Involved in Alzheimer's Disease

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases and characterized by cognitive and memory impairments. Emerging evidence suggests that the extracellular matrix (ECM) in the brain plays an important role in the etiology of AD. It has been detected that the levels of ECM proteins have changed in the brains of AD patients and animal models. Some ECM components, for example, elastin and heparan sulfate proteoglycans, are considered to promote the upregulation of extracellular amyloid-beta (Aβ) proteins. In addition, collagen VI and laminin are shown to have interactions with Aβ peptides, which might lead to the clearance of those peptides. Thus, ECM proteins are involved in both amyloidosis and neuroprotection in the AD process. However, the molecular mechanism of neuronal ECM proteins on the pathophysiology of AD remains elusive. More investigation of ECM proteins with AD pathogenesis is needed, and this may lead to novel therapeutic strategies and biomarkers for AD.     

刀锋伪影校正技术在精神疾病患者海马MRI检查中的应用

为探讨磁共振刀锋伪影校正（BLADE）技术提升精神疾病患者海马磁共振图像质量的效果，本文分别使用结合了BLADE技术的BLADE T₂WI TSE、BLADE T₂WI FLAIR及传统T₂WI TSE、T₂WI FLAIR四种序列，对47例精神疾病患者和美国放射学院（ACR）标准模体在3.0 T磁共振成像（MRI）设备上分别进行常规海马斜冠状位扫描和ACR标准检测.患者的磁共振图像由2名放射科医师采用5分法对运动伪影、搏动伪影、颗粒度、海马磁共振图像质量进行评价，并应用Wilcoxon符号秩检验进行数据分析.模体图像通过识别图像的钻孔阵列和轮辐的数目，半定量评价各序列的高对比空间分辨力（HCSR）和低对比物体探测能力（LCD）.结果表明相比传统序列，结合BLADE技术的序列能够明显改善海马磁共振图像的运动伪影、搏动伪影（p<0.001），提高图像质量（p<0.05）；但在图像颗粒度方面，传统序列表现更优（p<0.001）.ACR模体半定量分析显示，结合BLADE技术序列与传统序列相比，在LCD检测方面结果更优、在HCSR检测方面结果相同或略逊.本文推荐将BLADE技术应用于不合作的精神疾病患者海马的MRI检查.     

A Near-Infrared-Controllable Artificial Metalloprotease Used for Degrading Amyloid-β Monomers and Aggregates

Proteolysis of amyloid-β (Aβ) is a promising approach against Alzheimer's disease. However, it is not feasible to employ natural hydrolases directly because of their cumbersome preparation and purification, poor stability, and hazardous immunogenicity. Therefore, artificial enzymes have been developed as potential alternatives to natural hydrolases. Since specific cleavage sites of Aβ are usually embedded inside the β-sheet structures that restrict access by artificial enzymes, this strongly hinders their efficiency for practical applications. Herein, we construct a NIR (near-IR) controllable artificial metalloprotease (MoS₂-Co) using a molybdenum disulfide nanosheet (MoS₂) and a cobalt complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Codota). Evidenced by detailed experimental and theoretical studies, the NIR-enhanced MoS₂-Co can circumvent the restriction by simultaneously inhibition of β-sheet formation and destroying β-sheet structures of the preformed Aβ aggregates in living cell. Furthermore, our designed MoS₂-Co is an easy to graft Aβ-target agent that prevents misdirected or undesirable hydrolysis reactions, and has been demonstrated to cross the blood brain barrier. This method can be adapted for hydrolysis of other kinds of amyloids.     

A reversed‐phase compatible thin‐layer chromatography autography for the detection of acetylcholinesterase inhibitors

A dual readout autographic assay to detect acetylcholinesterase inhibitors present in complex matrices adsorbed on reversed‐phase or normal‐phase thin‐layer chromatography plates is described. Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The effects of substrate and enzyme concentrations, pH, incubation time, and incubation temperature on the sensitivity and the detection limit of the assay were evaluated. Experimental design and response surface methodology were used to optimize conditions with a minimum number of experiments. The assay allowed the detection of 0.01% w/w of physostigmine in both a spiked Sonchus oleraceus L. extract chromatographed on normal phase and a spiked Pimenta racemosa (Mill.) J.W. Moore leaf essential oil chromatographed on reversed phase. Finally, the reversed‐phase thin‐layer chromatography assay was applied to reveal the presence of an inhibitor in the Cymbopogon citratus (DC.) Stapf essential oil. The developed assay is able to detect acetylcholinesterase inhibitors present in complex matrixes that were chromatographed in normal phase or reversed‐phase thin‐layer chromatography. The detection limit for physostigmine on both normal and reversed phase was of 1×10^?4 μg. The results can be read by a change in color and/or a change in fluorescence.