Development of a novel integrated isothermal amplification system for detection of bacteria-spiked blood samples.

Bloodstream infection (BSI) caused by bacteria is highly pathogenic and lethal, and easily develops whole-body inflammatory state. Immediate identification of disease-causing bacteria can improve patient prognosis. Traditional testing methods are not only time-consuming, but such tests are limited to laboratories. Recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) holds great promise for rapid nucleic acid detection, but the uncapping operation after amplification easily contaminates laboratories. Therefore, the establishment of a more effective integrated isothermal amplification system has become an urgent problem to be solved. In this study, we designed and fabricated a hermetically sealed integrated isothermal amplification system. Combining with this system, a set of RPA-LFD assays for detecting S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI were established and evaluated. The whole process could be completed in less than 15 min and the results can be visualized by the naked eye. The developed RPA-LFD assays displayed a good sensitivity, and no cross-reactivity was observed in seven similar bacterial genera. The results obtained with 60 clinical samples indicated that the developed RPA-LFD assays had high specifcity and sensitivity for identifying S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI. In conclusion, our results showed that the developed RPA-LFD assay is an alternative to existing PCR-based methods for detection of S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI in primary hospitals.

Advances in liquid biopsy-based markers in NSCLC.

Lung cancer is the second most-frequently occurring cancer and the leading cause of cancer-associated deaths worldwide. Non-small cell lung cancer (NSCLC), the most common type of lung cancer is often diagnosed in middle or advanced stages and have poor prognosis. Diagnosis of disease at an early stage is a key factor for improving prognosis and reducing mortality, whereas, the currently used diagnostic tools are not sufficiently sensitive for early-stage NSCLC. The emergence of liquid biopsy has ushered in a new era of diagnosis and management of cancers, including NSCLC, since analysis of circulating tumor-derived components, such as cell-free DNA (cfDNA), circulating tumor cells (CTCs), cell-free RNAs (cfRNAs), exosomes, tumor-educated platelets (TEPs), proteins, and metabolites in blood or other biofluids can enable early cancer detection, treatment selection, therapy monitoring and prognosis assessment. There have been great advances in liquid biopsy of NSCLC in the past few years. Hence, this chapter introduces the latest advances on the clinical application of cfDNA, CTCs, cfRNAs and exosomes, with a particular focus on their application as early markers in the diagnosis, treatment and prognosis of NSCLC.

Rapid detection of EGFR mutation in CTCs based on a double spiral microfluidic chip and the real-time RPA method.

Circulating tumor cells (CTCs) are cells shed from primary or metastatic tumors and spread into the peripheral bloodstream. Mutation detection in CTCs can reveal vital genetic information about the tumors and can be used for "liquid biopsy" to indicate cancer treatment and targeted medication. However, current methods to measure the mutations in CTCs are based on PCR or DNA sequencing which are cumbersome and time-consuming and require sophisticated equipment. These largely limited their applications especially in areas with poor healthcare infrastructure. Here we report a simple, convenient, and rapid method for mutation detection in CTCs, including an example of a deletion at exon 19 (Del19) of the epidermal growth factor receptor (EGFR). CTCs in the peripheral blood of NSCLC patients were first sorted by a double spiral microfluidic chip with high sorting efficiency and purity. The sorted cells were then lysed by proteinase K, and the E19del mutation was detected via real-time recombinase polymerase amplification (RPA). Combining the advantages of microfluidic sorting and real-time RPA, an accurate mutation determination was realized within 2 h without professional operation or complex data interpretation. The method detected as few as 3 cells and 1% target variants under a strongly interfering background, thus, indicating its great potential in the non-invasive diagnosis of E19del mutation for NSCLC patients. The method can be further extended by redesigning the primers and probes to detect other deletion mutations, insertion mutations, and fusion genes. It is expected to be a universal molecular diagnostic tool for real-time assessment of relevant mutations and precise adjustments in the care of oncology patients.

Centrifugal microfluidic-based multiplex recombinase polymerase amplification assay for rapid detection of SARS-CoV-2.

The COVID-19 pandemic has spread worldwide, and rapid detection of the SARS-CoV-2 virus is crucial for infection surveillance and epidemic control. This study developed a centrifugal microfluidics-based multiplex reverse transcription recombinase polymerase amplification (RT-RPA) assay for endpoint fluorescence detection of the E, N, and ORF1ab genes of SARS-CoV-2. The microscope slide-shaped microfluidic chip could simultaneously accomplish three target genes and one reference human gene (i.e., ACTB) RT-RPA reactions in 30 min, and the sensitivity was 40 RNA copies/reaction for the E gene, 20 RNA copies/reaction for the N gene, and 10 RNA copies/reaction for the ORF1ab gene. The chip demonstrated high specificity, reproducibility, and repeatability. Chip performance was also evaluated using real clinical samples. Thus, this rapid, accurate, on-site, and multiplexed nucleic acid test microfluidic chip would significantly contribute to detecting patients with COVID-19 in low-resource settings and point-of-care testing (POCT) and, in the future, could be used to detect emerging new variants of SARS-CoV-2.

A Resistance-Based Microfluidic Chip for Deterministic Single Cell Trapping Followed by Immunofluorescence Staining.

Microchips are fundamental tools for single-cell analysis. Although various microfluidic methods have been developed for single-cell trapping and analysis, most microchips cannot trap single cells deterministically for further analysis. In this paper, we describe a novel resistance-based microfluidic chip to implement deterministic single-cell trapping followed by immunofluorescence staining based on the least flow resistance principle. The design of a large circular structure before the constriction and the serpentine structure of the main channel made the flow resistance of the main channel higher than that of the trapping channel. Since cells preferred to follow paths with lower flow resistance, this design directed cells into the capture sites and improved single-cell trapping efficiency. We optimized the geometric parameters using numerical simulations. Experiments using A549 and K562 cell lines demonstrated the capability of our chip with (82.7 ± 2.4)% and (84 ± 3.3)% single-cell trapping efficiency, respectively. In addition, cells were immobilized at capture sites by applying the pulling forces at the outlet, which reduced the cell movement and loss and facilitated tracking of the cell in real time during the multistep immunofluorescence staining procedure. Due to the simple operation, high-efficiency single-cell trapping and lower cell loss, the proposed chip is expected to be a potential analytical platform for single tumor cell heterogeneity studies and clinical diagnosis.

Tetrahedral framework nucleic acids-based delivery of microRNA-155 inhibits choroidal neovascularization by regulating the polarization of macrophages.

Choroidal neovascularization (CNV) is a common pathological feature of various eye diseases and an important cause of visual impairment in middle-aged and elderly patients. In previous studies, tetrahedral framework nucleic acids (tFNAs) showed good carrier performance. In this experiment, we developed microRNA-155-equipped tFNAs (T-155) and explored its biological effects on CNV. Based on the results of in-vitro experiments, T-155 could regulate macrophages into the antiangiogenic M1 type. Then, we injected T-155 into the vitreous of laser-induced CNV model mice and found that T-155 significantly reduced the size and area of CNV, inhibited blood vessel leakage. In summary, we prove that T-155 could regulate the inflammatory process of CNV by polarizing macrophages, thereby improving the symptoms of CNV. Thus, T-155 might become a new DNA-based drug with great potential for treating CNV.

Enhanced Separation Efficiency and Purity of Circulating Tumor Cells Based on the Combined Effects of Double Sheath Fluids and Inertial Focusing.

Circulating tumor cells (CTCs) play a crucial role in solid tumor metastasis, but obtaining high purity and viability CTCs is a challenging task due to their rarity. Although various works using spiral microchannels to isolate CTCs have been reported, the sorting purity of CTCs has not been significantly improved. Herein, we developed a novel double spiral microchannel for efficient separation and enrichment of intact and high-purity CTCs based on the combined effects of two-stage inertial focusing and particle deflection. Particle deflection relies on the second sheath to produce a deflection of the focused sample flow segment at the end of the first-stage microchannel, allowing larger particles to remain focused and entered the second-stage microchannel while smaller particles moved into the first waste channel. The deflection of the focused sample flow segment was visualized. Testing by a binary mixture of 10.4 and 16.5 µm fluorescent microspheres, it showed 16.5 µm with separation efficiency of 98% and purity of 90% under the second sheath flow rate of 700 µl min-1. In biological experiments, the average purity of spiked CTCs was 74% at a high throughput of 1.5 × 108 cells min-1, and the recovery was more than 91%. Compared to the control group, the viability of separated cells was 99%. Finally, we validated the performance of the double spiral microchannel using clinical cancer blood samples. CTCs with a concentration of 2-28 counts ml-1 were separated from all 12 patients' peripheral blood. Thus, our device could be a robust and label-free liquid biopsy platform in inertial microfluidics for successful application in clinical trials.

Nonocular Influencing Factors for Primary Glaucoma: An Umbrella Review of Meta-Analysis.

INTRODUCTION: Glaucoma is the main cause of irreversible blindness worldwide. Still, little is known about nonocular risk factors. We use an umbrella review to examine the meta-analytic evidence of the correlation between nonocular factors and glaucoma. METHOD: We searched PubMed and Embase databases up to July 24, 2020. Eligible meta-analyses (MAs) included cohort, case-control, and randomized controlled study designs. Two authors independently extracted the data and evaluated the methodological quality of the MAs. AMSTAR 2 was used to assess the methodological quality of each included MA. RESULTS: This umbrella review contains 22 MAs with 22 unique nonocular factors in total. We identified 11 factors that increase the risk of glaucoma: hyperlipidemia, nocturnal dip in blood pressure, infection with Helicobacter pylori, myopia, obstructive sleep apnea syndrome, corneal properties, diabetes, hypertension, hypothyroidism, migraine, and plasma homocysteine. We identified 3 factors that reduce the risk of glaucoma: dietary intake of vitamin A, dietary intake of vitamin C, and short-term statin use. We identified 8 factors that had no association with glaucoma: dietary intake of vitamin B, dietary intake of vitamin E, cigarette smoking, Alzheimer's disease, serum folic acid, serum vitamin B6, serum vitamin B12, and serum vitamin D. CONCLUSIONS: In this umbrella review of MAs, evidence was found for associations of various nonocular factors with glaucoma to different degrees. However, risk factors were only mildly associated, suggesting low impact of systemic risk factors. Additional higher quality studies are needed to provide robust evidence.

Genome sequence of the white-rot fungus Irpex lacteus F17, a type strain of lignin degrader fungus.

Irpex lacteus, a cosmopolitan white-rot fungus, degrades lignin and lignin-derived aromatic compounds. In this study, we report the high-quality draft genome sequence of I. lacteus F17, isolated from a decaying hardwood tree in the vicinity of Hefei, China. The genome is 44,362,654 bp, with a GC content of 49.64% and a total of 10,391 predicted protein-coding genes. In addition, a total of 18 snRNA, 842 tRNA, 15 rRNA operons and 11,710 repetitive sequences were also identified. The genomic data provides insights into the mechanisms of the efficient lignin decomposition of this strain.

Ion concentration effect (Na+ and Cl-) on lipid vesicle formation.

Lipid vesicle formation is known to be suppressed in salt solutions, but the mechanism of this phenomenon remains unclear. In order to better understand this issue, the effect of salt concentrations (0-800mM) of sodium chloride on the behavior of L-α-phosphatidylcholine (PC) in aqueous solution was investigated in this work. The results showed that fusion among vesicles, micelles and bilayers may be essential for vesicle formation. With addition of ions and an increase in ion concentration, the lipids became constrained in lateral movement and packed increasingly tightly. The resulted hard supported phospholipid bilayers (SPBs) were thus more difficult to detach from the substrate to form vesicles. These phenomena were tried to be explained at molecular level. Hydrophobic effect is the original cause of lipid vesicle formation, which in fact is absence of attraction between the involved substances. That is to say, the stronger the 3D network was bounded in the medium, the stronger the hydrophobic repulsion on the lipids would be. This might be one reason for the suppression of vesicle formation in salt solution.

Impacts of electrical parameters on the electroformation of giant vesicles on ITO glass chips.

A "sandwich" structure device consisted of ITO electrodes and PDMS spacer was designed and used to explore the impact of different electrical parameters (intensity and frequency) on the electroformation of GUVs (giant unilamellar vesicles). Theoretical analysis of the dielectrophoretic effect (DEPE) and the electrohydrodynamic effect (EHE) on the electroformation process indicated that the characteristic frequency of the system could maximize the mutual effect of the both, which might benefit the formation of GUVs. The calculated value of the characteristic frequency (13.3 kHz) was very close to the experimental one (11 kHz). We demonstrated that for a given electroformation system, large amount of well-distributed GUVs can be obtained by optimizing the electrical parameters. In this paper, when choosing the optimal electrical parameters (11 kHz frequency and 5V/mm intensity), the amount of GUVs could reach 65/mm(2), and the diameters of most GUVs (>70%) were 50-70 µm.