Improved Reversible Cross-Linking-Based Solid-Phase RNA Extraction for Pathogen Diagnostics

In this study, we developed an amine-functionalized, diatomaceous earth-based, dimethyl suberimidate assisted (ADD) system as a novel binding strategy to improve the solid-phase extraction method for rapid and simple purification of RNA from biological samples including human cells and pathogenic bacteria. This ADD system is based on reversible cross-linking reactions between RNA and the silica matrix. The formation of robust covalent bonds protects RNA from both the sufferance of washing steps and isolation with ribonuclease (RNase)-rich samples, leading to the extraction of higher quality RNA. This improved RNA extraction system integrated with quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is evaluated for pathogen diagnostics. Compared to standard solid-phase extraction based commercial kits, this improved method shows highly enhanced sensitivity with 1000-fold higher sensitivity for human cells and 100-fold higher sensitivity for Brucella bacteria, according to the cycle threshold value of RT-qPCR. We envision that the ADD system can be tailored for commercial applications for RNA expression analysis in forensics studies, as well as for disease diagnostics in clinical applications

Supplementary Information Files for 'Acetate production from inorganic carbon (HCO3-) in photo-assisted biocathode microbial electrosynthesis systems using WO3/MoO3/g-C3N4 heterojunctions and Serratia marcescens species.'

Supplementary Information Files for 'Acetate production from inorganic carbon (HCO3-) in photo-assisted biocathode microbial electrosynthesis systems using WO3/MoO3/g-C3N4 heterojunctions and Serratia marcescens species.'Abstract:The efficient production of acetate from HCO3− is demonstrated in a photo-assisted microbial electrosynthesis system (MES) incorporating a WO3/MoO3/g-C3N4 heterojunction photo-assisted biocathode supporting Serratia marcescens Q1 electrotroph. The WO3/MoO3/g-C3N4 structured electrode consisting of a layer of g-C3N4 coated on graphite felt decorated with W/Mo oxides nanoparticles exhibited stable photocurrents, 4.8 times higher than the g-C3N4 electrode and acetate production of 3.12 ± 0.20 mM/d with a CEacetate of 73 ± 4 % and current of 2.5 ± 0.3 A/m2. Photo-induced electrons on the conduction bands of WO3/MoO3/g-C3N4 favoured hydrogen evolution, which was metabolized by S. marcescens with HCO3− to acetate, while the holes were refilled by the electrons travelling from the anode. Such mechanism reduced the interfacial resistances creating a supplementary driving force leading to higher acetate production. The biocompatible components of WO3/MoO3/g-C3N4 synergistically couple light-harvesting and further catalyze S. marcescens to acetate from HCO3−, providing a feasible strategy for achieving sustainable high rates of acetate production.</div

Simple and Highly Sensitive Molecular Diagnosis of Zika Virus by Lateral Flow Assays

We have developed a simple, user-friendly, and highly sensitive Zika virus (ZIKV) detection method by incorporating optimized reverse transcription loop-mediated isothermal amplification (RT-LAMP) and a lateral flow assay (LFA). The optimized RT-LAMP reaction was carried out using <i>Bst</i> 3.0 polymerase, which has robust and fast isothermal amplification performance even in the presence of high concentrations of inhibitors; this permitted the amplification of ZIKV RNA in pure water and human whole blood. In addition, the strong reverse transcription activity of <i>Bst</i> 3.0 polymerase enabled specific ZIKV RNA amplification without extra addition of reverse transcriptase. The RT-LAMP condition was optimized by adjusting the Mg²⁺ and dNTP mix concentration to extirpate nontarget amplification, which is caused by nonspecific primer dimers amplification. After 30 min of RT-LAMP reaction, the resultant amplicons were simply and rapidly analyzed by the LFA test in less than 5 min. The optimized RT-LAMP combined with the LFA allowed specific ZIKV RNA detection down to the single copy level within 35 min

Large Instrument- and Detergent-Free Assay for Ultrasensitive Nucleic Acids Isolation via Binary Nanomaterial

Nucleic acid-based diagnostics are widely used for clinical applications due to their powerful recognition of biomolecule properties. Isolation and purification of nucleic acids such as DNA and RNA in the diagnostic system have been severely hampered in point-of-care testing because of low recovery yields, degradation of nucleic acids due to the use of chaotropic detergent and high temperature, and the requirement of large instruments such as centrifuges and thermal controllers. Here, we report a novel large instrument- and detergent-free assay via binary nanomaterial for ultrasensitive nucleic acid isolation and detection from cells (eukaryotic and prokaryotic). This binary nanomaterial couples a zinc oxide nanomultigonal shuttle (ZnO NMS) for cell membrane rupture without detergent and temperature control and diatomaceous earth with dimethyl suberimidate complex (DDS) for the capture and isolation of nucleic acids (NA) from cells. The ZnO NMS was synthesized to a size of 500 nm to permit efficient cell lysis at room temperature within 2 min using the biological, chemical, and physical properties of the nanomaterial. By combining the ZnO NMS with the DDS and proteinase K, the nucleic acid extraction could be completed in 15 min with high quantity and quality. For bacterial cells, DNA isolation with the binary nanomaterial yielded 100 times more DNA, than a commercial spin column based reference kit, as determined by the NanoDrop spectrophotometer. We believe that this binary nanomaterial will be a useful tool for rapid and sensitive nucleic acid isolation and detection without large instruments and detergent in the field of molecular diagnostics

Use of Dimethyl Pimelimidate with Microfluidic System for Nucleic Acids Extraction without Electricity

The isolation of nucleic acids in the lab on a chip is crucial to achieve the maximal effectiveness of point-of-care testing for detection in clinical applications. Here, we report on the use of a simple and versatile single-channel microfluidic platform that combines dimethyl pimelimidate (DMP) for nucleic acids (both RNA and DNA) extraction without electricity using a thin-film system. The system is based on the adaption of DMP into nonchaotropic-based nucleic acids and the capture of reagents into a low-cost thin-film platform for use as a microfluidic total analysis system, which can be utilized for sample processing in clinical diagnostics. Moreover, we assessed the use of the DMP system for the extraction of nucleic acids from various samples, including mammalian cells, bacterial cells, and viruses from human disease, and we also confirmed that the quality and quantity of the nucleic acids extracted were sufficient to allow for the robust detection of biomarkers and/or pathogens in downstream analysis. Furthermore, this DMP system does not require any instruments and electricity, and has improved time efficiency, portability, and affordability. Thus, we believe that the DMP system may change the paradigm of sample processing in clinical diagnostics

Additional file 1: of The prevention of 2,4-dinitrochlorobenzene-induced inflammation in atopic dermatitis-like skin lesions in BALB/c mice by Jawoongo

Figure S1. Changes in body weight (A) and food intake (B) in DNCB-induced AD mice during treatment with Jawoongo. Values are expressed as the mean ± SEMs (n = 8). (TIF 4580 kb

Additional file 2: of The prevention of 2,4-dinitrochlorobenzene-induced inflammation in atopic dermatitis-like skin lesions in BALB/c mice by Jawoongo

Figure S2. Effects of Jawoongo on cell viability in various cell lines. HMC-1 cells were treated with the combination of ionomycin (500 ng/ml) and PMA (5 ng/ml) with varying concentrations of DMSO and Jawoongo (5–500 μg/ml) for 24 h (A). RAW264.7 cells (B) and splenocytes (C) were treated with the combination of LPS (1 mg/ml) and varying concentrations of DMSO and Jawoongo (5–500 μg/ml) for 24 h. Cell viability was measured using an MTS assay. The data were presented as mean ± SEMs of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. (TIF 18563 kb

Clinical and laboratory findings and PCR results in 10 control patients with hepatic granuloma.

<p>Clinical and laboratory findings and PCR results in 10 control patients with hepatic granuloma.</p

Agarose gel electrophoresis of <i>Coxiella burnetii IS1111a gene</i>.

<p>DNA amplification with Q-fever-IS1111a primers for the detection of <i>Coxiella burnetii</i>. Gel electrophoresis of 202 bp products by using end-point PCR. M: 50bp DNA size marker; 1–11: DNAs from the case patients with Q fever hepatitis and N: negative control (left). M: 50bp DNA size marker; PC: <i>C</i>. <i>burnetii</i> DNA control (case number 1 of case patient group); 1–10: DNAs from the control patients and N: negative control (right).</p

Representative photomicrographs of Q fever hepatitis (case no. 3 in case group, x200) and hepatic mucormycosis (case no. 6 in control group, x100).

<p>(A) Characteristic fibrin ring granulomas consisting of a central fat globule or epitheloid cells with fibrin ring (arrow) (B) A suppurative granuloma consists of multinucleated giant cells with fungal hyphae (arrow) and polymorphous lymphoid cell including eosinophils.</p