Mkit: A mobile nucleic acid assay based on a chitosan-modified minimalistic microfluidic chip (CM3-chip) and smartphone.

Mobile sensing enabled by MS2 technology, which integrates microfluidic and smartphone components, has seen many applications in recent years. In this direction, we developed an MS2 platform (an integrated kit) for nucleic acid assay, which included a chitosan-modified minimalistic microfluidic chip (CM3-chip), a smartphone-based fluorescence detector (SF-detector), an APP for imaging and analysis, reagents, and accessories. Once the lysed sample was loaded into the CM3-chip modified by 1% concentration and 200-260 kDa molecular weight of chitosan, the following assay can be completed in approximately 1 h. The Mkit can detect 3 × 10° copies µL-1 of plasmid DNA and its polymerase chain reaction (PCR) efficiency was 96.8%. The CM3-chip equipped for the Mkit can enrich nucleic acid from the pH = 5 of lysis buffer, instead of using conventional adsorption mediums such as the magnetic beads and silica gel membranes, which could result in unexpected impurity residuals and tedious cleaning operations. In addition, the performance of the Mkit equipped with the pristine chip was demonstrated to perform poorer than that coupled with the CM3-chip in which the enriched nucleic acid can be all used for "in-situ PCR". The universality, selectivity, and user-friendliness of the Mkit were also validated. We finally demonstrated the feasibility of the Mkit for testing artificially prepared infected samples. H5N6 and IAV-infected saliva samples provided the limits of detection of 5 × 102 copies mL-1 and 3.24 × 102 copies mL-1 per chamber, respectively. The streamlined assay and compact device should enable the great potential of the Mkit in research and potential diagnostic uses.

Duplex real-time fluorescent recombinase polymerase amplification for the rapid and sensitive detection of two resistance genes in drug-resistant Staphylococcus aureus.

In the clinic, drug-resistant Staphylococcus aureus (S. aureus) is the most common suppurative infection pathogen in humans. It can cause local infections in humans and animals, such as pneumonia, mastitis, and other systemic illnesses. At present, the detection of drug-resistant S. aureus includes traditional isolation by culture and antimicrobial susceptibility tests. However, these methods are complicated in experimental design, specialized in operation and time consuming. Therefore, a rapid and accurate drug-resistant S. aureus detection technology is urgently needed. In this study, we combined duplex pairs of fluorescent probes with recombinase polymerase amplification (RPA) to realize the simultaneous detection of two resistance genes in drug-resistant S. aureus. The method shows low detection limit, detecting 20 copies within 10 min. The analytical specificity of this method was evaluated with several related drug-resistant bacterial strains (Non-resistant S. aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae), and the positive signal was only observed with drug-resistant S. aureus. In addition, the clinical suitability of this method was verified by 30 clinical isolates. Compared with qPCR, the coincidence rate of drug resistance genes were 100% (mecA) and 96.7% (ermA), respectively. These results show that the duplex real-time fluorescent RPA assay is a rapid, low detection limit and specific detection of mecA and ermA genes in isolates of drug-resistant S. aureus.

Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a severe health threat causing high-level morbidity and mortality in health care environments and in community settings. Though existing diagnostic methods, including PCR and culture-based methods, are routinely used in clinical practice, they are not appropriate for rapid point-of-care testing (POCT). Recently, since the development of the CRISPR/Cas technology, new possibilities for rapid point-of-care detection have emerged. In this study, we developed a rapid, accurate, and contamination-free platform for MRSA detection by integrating recombinase polymerase amplification (RPA) with the Cas12 system into one tube. Using this approach, visual MRSA detection could be achieved in 20 min. Based on the one-tube RPA-CRISPR/Cas12a platform, the assay results are visualized by lateral flow test strips (LFS) and fluorescent-based methods, including real-time and end-point fluorescence. This platform allows specific MRSA detection with a sensitivity of 10 copies for the fluorescence method and a range of 10-100 copies for the LFS. The results of 23 samples from clinical MRSA isolates showed that the coincidence rate was 100% and 95.7% of the fluorescence method and LFS, respectively, compared to qPCR. In conclusion, the one-tube RPA-CRISPR/Cas12a platform is an effective method for MRSA detection with significant potential in future practical POCT applications.

Exosomal miR-106a-5p accelerates the progression of nasopharyngeal carcinoma through FBXW7-mediated TRIM24 degradation.

Nasopharyngeal carcinoma (NPC) is prevalent in East Asia and causes increased health burden. Elucidating the regulatory mechanism of NPC progression is important for understanding the pathogenesis of NPC and developing novel therapeutic strategies. Nasopharyngeal carcinoma and normal tissues were collected. Nasopharyngeal carcinoma cell proliferation, migration, and invasion were evaluated using CCK-8, colony formation, wound healing, and transwell assays, respectively. A xenograft mouse model of NPC was established to analyze NPC cell growth and metastasis in vivo. The expression of miR-106a-5p, FBXW7, TRIM24, and SRGN was determined with RT-qPCR and Western blot. MiR-106a-5p, TRIM24, and SRGN were upregulated, and FBXW7 was downregulated in NPC tissues and cells. Exosomal miR-106a-5p could enter NPC cells, and its overexpression promoted the proliferation, migration, invasion, and metastasis of NPC cells, which were suppressed by knockdown of exosomal miR-106a-5p. MiR-106a-5p targeted FBXW7 to regulate FBXW7-mediated degradation of TRIM24. Furthermore, TRIM24 regulated SRGN expression by binding to its promoter in NPC cells. Suppression of exosomal miR-106a-5p attenuated NPC growth and metastasis through the FBXW7-TRIM24-SRGN axis in vivo. Exosomal miR-106a-5p accelerated the progression of NPC through the FBXW7-TRIM24-SRGN axis. Our study elucidates novel regulatory mechanisms of NPC progression and provides potential exosome-based therapeutic strategies for NPC.

Spatial frequency domain imaging technology based on Fourier single-pixel imaging.

SIGNIFICANCE: Optical properties (absorption coefficient and scattering coefficient) of tissue are the most critical parameters for disease diagnosis-based optical method. In recent years, researchers proposed spatial frequency domain imaging (SFDI) to quantitatively map tissue optical properties in a broad field of contactless imaging. To solve the limitations in wavebands unsuitable for silicon-based sensor technology, a compressed sensing (CS) algorithm is used to reproduce the original signal by a single-pixel detectors. Currently, the existing single-pixel SFDI method mainly uses a random sampling policy to extract and recover signals in the acquisition stage. However, these methods are memory-hungry and time-consuming, and they cannot generate discernible results under low sampling rate. Explorations on high performance and efficiency single-pixel SFDI are of great significance for clinical application. AIM: Fourier single-pixel imaging can reconstruct signals with less time and space costs and has fewer reconstruction errors. We focus on an SFDI algorithm based on Fourier single-pixel imaging and propose our Fourier single-pixel image-based spatial frequency domain imaging method (FSI-SFDI). APPROACH: First, we use Fourier single-pixel imaging algorithm to collect and compress signals and SFDI algorithm to generate optical parameters. Given the basis that the main energy of general image signals is concentrated in the range of low frequency of Fourier frequency domain, our FSI-SFDI uses a circular-sampling scheme to sample data points in the low-frequency region. Then, we reconstruct the image details from these points by optimization-based inverse-FFT method. RESULTS: Our algorithm is tested on simulated data. Results show that the root mean square error (RMSE) of optical parameters is lower than 5% when the data reduction is 92%, and it can generate discernible optical parameter image with low sampling rate. We can observe that our FSI-SFDI primarily recovers the optical properties while keeping the RMSE under the upper bound of 4.5% when we use an image with 512 × 512 resolution as the example for calculation and analysis. Not only that but also our algorithm consumes less space and time for an image with 256 × 256 resolution, the signal reconstruction takes only 1.65 ms, and requires less RAM memory. Compared to CS-SFDI method, our FSI-SFDI can reduce the required number of measurements through optimizing algorithm. CONCLUSIONS: Moreover, FSI-SFDI is capable of recovering high-quality resolvable images with lower sampling rate, higher-resolution images with less memory and time consumed than previous CS-SFDI method, which is very promising for clinical data collection and medical analysis.

A self-quenching fluorescence probe-mediated exponential isothermal amplification system for highly sensitive and specific detection of microRNAs.

We designed an efficient self-quenching fluorescence probe and constructed this probe-mediated exponential isothermal amplification system for miRNA detection. Owing to the significant improvement in the detective signal-to-background ratio, a wide dynamic range of 9 orders of magnitude and a limit of detection as low as 0.08 aM can be easily achieved in a single step. Furthermore, benefiting from the additional advantages of high specificity and biocompatibility, the proposed method has been demonstrated to be capable of accurately quantifying miRNA biomarkers in serum, which will provide promising perspectives for clinical diagnosis.

[Clinical characteristics and prognosis of sudden sensorineural hearing loss with rheumatoid arthritis].

Objective:To analyze the clinical features and prognosis of sudden sensorineural hearing loss（SSNHL） with rheumatoid arthritis（RA）, and evaluate the effect of the course of RA on the hearing recovery. Methods:We collected the clinical data of 43 SSNHL patients（46 ears） with RA（RA group） who were hospitalized in our hospital, and compared their clinical characteristics and prognosis with 386 SSNHL patients（400 ears） without RA（non-RA group）. 43 SSNHL patients with RA were further grouped into <5 years group, 5-10 years group and >10 years group, and the hearing recovery was compared among three groups. Results:In the RA group, the initial pure tone average（PTA） of SSNHL and non-SSNHL ears were （64.53±12.77） dB HL and （31.28±8.53） dB HL, which were higher than those in the non-RA group（54.31±13.45） dB HL and（24.83±6.06） dB HL（P<0.05）. After treatment, in the RA group, posttreatment PTA of SSNHL and non-SSNHL ears were （48.26±13.49） dB HL and （27.93±10.22） dB HL, which were higher than those in the non-RA group （33.65±9.22） dB HL and （21.86±6.88） dB HL（P<0.05）, and the hearing gains of SSNHL ear and the rate of overall recovery were （16.27±6.01） dB HL and 52.17%, which were lower than those in the non-RA group （20.66±6.21） dB HL and 75.00%. No statistic difference was observed in the hearing gains of non-SSNHL ear between the two groups（P>0.05）. The hearing gains in the <5 years group, 5-10 years group and >10 years group were （20.77±8.63） dB HL, （17.00±6.81） dB HL and （11.94±5.73） dB HL, statistic differences were observed among the three groups（P=0.010）, but no statistic difference was observed in the rate of complete recovery, marked recovery, recovery and no recovery（P>0.05）. Conclusion:SSNHL patients with RA often suffers a severe hearing loss, and the hearing recovery is poor. The longer the course of RA, the worse the prognosis.

Identification of miR-4644 as a suitable endogenous normalizer for circulating miRNA quantification in hepatocellular carcinoma.

Background: Circulating microRNAs (miRNAs) have proved to be promising biomarkers for early diagnosis and therapeutic monitoring in cancers. Particularly for hepatocellular carcinoma (HCC), detection of circulating miRNA biomarkers as a new diagnostic approach has been written into the latest Guidelines for Diagnosis and Treatment of Primary Liver Cancer in China (2019 edition). However, no general consensus on an ideal endogenous normalizer for circulating miRNAs quantification has been reached, so it will affect the accuracy of quantitative results. In this study, we aim to identify a stable endogenous normalizer for analyzing circulating miRNAs. Methods: Candidate miRNAs were selected by screening dataset GSE104310, as well as data statistics and analysis. Five commonly reference genes were chosen for further comparison and verification. Then, the expression levels of these genes in serum were analyzed by quantitative reverse transcription PCR (RT-qPCR) among four groups, including patients diagnosed with HCC, chronic hepatitis B (CHB), liver cirrhosis, and healthy subjects. Furthermore, the stability of target genes was evaluated using geNorm, NormFinder, comparative ΔCq programs, and validated by database. We also explored the availability of the miRNA combination, and compared the performance difference between combination and individuals, as well as the selectivity of miRNA references in the combinations. Results: 11 candidate miRNAs were obtained, and miR-4644 stood out among these miRNAs, and proved to be much more stable than other endogenous miRNAs. Further study showed that miR-4644 exhibited higher stability and expression abundance than other commonly miRNA reference controls. Finally, we discovered the combination of miR-4644 and miR-16 revealed high performance in stability when compared to miRNA individuals. Furthermore, the combination consisted of references with closer nature could give rise to amplification effects in stability. Conclusions: Our findings demonstrated that miR-4644 is an ideal endogenous normalizer for circulating microRNA quantification in hepatocellular carcinoma. Besides, combining miR-4644 with miR-16 into a whole as a reference control would greatly improve the accuracy of quantification.

A Novel Microfluidic Device Integrated with Chitosan-Modified Capillaries for Rapid ZIKV Detection.

The outbreak of Zika virus (ZIKV) has posed a great challenge to public health in recent years. To address the urgent need of ZIKV RNA assays, we integrate the microfluidic chip embedded with chitosan-modified silicon dioxide capillaries, smartphone-based detection unit to be a C3-system for the rapid extraction and detection of ZIKV RNA. The C3-system is characterized by: (1) four chitosan-modified silicon dioxide capillaries integrated in the microfluidic chip for target ZIKV RNA enrichment and "in situ PCR" (polymerase chain reaction) amplification; (2) smartphone-based point of care (POC) device consisting of a pneumatic subsystem for controlling the nucleic acid extraction processes in the microfluidic chip, a heating subsystem for sample lysis and PCR amplification, and an optical subsystem for signal acquisition. The entire detection processes including sample lysis, ZIKV RNA enrichment, and reverse-transcription polymerase chain reaction (RT-PCR) is achieved in the microfluidic chip. Moreover, PCR buffers can be directly loaded into the chitosan-modified silicon dioxide capillaries for "in situ PCR", in which the captured ZIKV RNA is directly used for downstream PCR without any loss. ZIKV RNA extracted by the C3-system can be successfully recovered at very low concentrations of 50 transducing units (TU)/mL from crude human saliva. This means that our method of detecting viremia in patients infected with ZIKV is reliable.

A Lab-on-a-Chip Device Integrated DNA Extraction and Solid Phase PCR Array for the Genotyping of High-Risk HPV in Clinical Samples.

Point-of-care (POC) molecular diagnostics play a crucial role in the prevention and treatment of infectious diseases. It is necessary to develop portable, easy-to-use, inexpensive and rapid molecular diagnostic tools. In this study, we proposed a lab-on-a-chip device that integrated DNA extraction, solid-phase PCR and genotyping detection. The ingenious design of the pneumatic microvalves enabled the fluid mixing and reagent storage to be organically combined, significantly reducing the size of the chip. The solid oligonucleotide array incorporated into the chip allowed the spatial separation of the primers and minimized undesirable interactions in multiplex amplification. As a proof-of-concept for POC molecular diagnostics on the device, five genotypes of high-risk human papillomavirus (HPV) (HPV16/HPV18/HPV31/HPV33/HPV58) were examined. Positive quality control samples and HPV patient cervical swab specimens were analyzed on the integrated microdevice. The platform was capable of detection approximately 50 copies of HPV virus per reaction during a single step, including DNA extraction, solid-phase PCR and genotype detection, in 1 h from samples being added to the chip. This simple and inexpensive microdevice provided great utility for the screening and monitoring of HPV genotypes. The sample-to-result platform will pave the way for wider application of POC molecular testing in the fields of clinical diagnostics, food safety, and environmental monitoring.

Fucosyltransferase 2 induced epithelial-mesenchymal transition via TGF-ß/Smad signaling pathway in lung adenocarcinaoma.

Fucosyltransferase 2 (FUT2), the enzyme catalyzing α-1,2-fucosylation in mammals, has been implicated in cancer. The up-regulation of FUT2 has been observed in lung adenocarcinoma (LUAD), and FUT2 can enhance the cell migration and invasion of LUAD cell lines. However, the underlying mechanism of FUT2 in LUAD remains largely unknown. Abundant studies have revealed that epithelial-mesenchymal transition (EMT) played a pivotal role during lung cancer metastasis and progression. In the present study, we showed that knocking down FUT2 in LUAD cell lines increased the expression of E-cadherin and reduced the expression of Vimentin, N-cadherin, TßRII, p-Smad2, p-Smad3 and Snail, which were the makers of EMT. Meanwhile, the expression of E-cadherin was decreased, and the expression of Vimentin was increased by restoring the expression of FUT2 in RNA interference FUT2 (RNAi-FUT2) cells, suggesting that FUT2 enhanced the EMT process in LUAD. Additionally, silencing FUT2 expression can up-regulate E-cadherin and down-regulate Vimentin, significantly attenuated EMT in vivo. Treated with the SIS3, a new-type inhibitor of p-Smad3 of TGF-ß signaling, the expression of E-cadherin, Vimentin and Snail were not affected by RNAi-FUT2 cells, indicating that the effect of FUT2 on EMT depended on TGF-ß/Smad signaling. Overall, the current results indicated that FUT2 might promote LUAD metastasis through the EMT initiated by TGF-ß/Smad signaling. Therefore, FUT2 might be a prognostic factor and therapeutic target for LUAD.

Clinical significance and biological function of fucosyltransferase 2 in lung adenocarcinoma.

Fucosylation, which is catalyzed by fucosyltransferases (FUTs), is one of the most important glycosylation events involved in cancer. Studies have shown that fucosyltransferase 8 (FUT8) is overexpressed in NSCLC and promotes lung cancer progression. However, there are no reports about the pathological role of fucosyltransferase 2 (FUT2) in lung cancer. To identify FUT2 associated with lung cancer, the expression and clinical significance of FUT2 in lung cancer was investigated by Real-Time PCR, Immunohistochemistry and Western Blot. In addition, we investigated the effect of knockdown FUT2 in lung adenocarcinoma cells. The results showed that the expression of FUT2 in lung adenocarcinoma is higher than that in adjacent noncancerous tissues. Knocking down FUT2 in A549 and H1299 cells decreased cell proliferation, migration and invasion, and increased cell apoptosis compared to corresponding control cells. Furthermore, Western Blot showed that knockdown FUT2 can impact the expression of migration-associated and apoptosis-associated proteins in A549 cells. Our results suggest that FUT2 may be associated with lung adenocarcinoma development and thus is a potential biomarker or/and therapeutic target in lung adenocarcinoma.

MoS2/Ag nanohybrid: A novel matrix with synergistic effect for small molecule drugs analysis by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

This paper reports a facile synthesis of molybdenum disulfide nanosheets/silver nanoparticles (MoS2/Ag) hybrid and its use as an effective matrix in negative ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The nanohybrid exerts a strong synergistic effect, leading to high performance detection of small molecule analytes including amino acids, peptides, fatty acids and drugs. The enhancement of laser desorption/ionization (LDI) efficiency is largely attributed to the high surface roughness and large surface area for analyte adsorption, better dispersibility, increased thermal conductivity and enhanced UV energy absorption as compared to pure MoS2. Moreover, both Ag nanoparticles and the edge of the MoS2 layers function as deprotonation sites for proton capture, facilitating the charging process in negative ion mode and promoting formation of negative ions. As a result, the MoS2/Ag nanohybrid proves to be a highly attractive matrix in MALDI-TOF MS, with desired features such as high desorption/ionization efficiency, low fragmentation interference, high salt tolerance, and no sweet-spots for mass signal. These characteristic properties allowed for simultaneous analysis of eight different drugs and quantification of acetylsalicylic acid in the spiked human serum. This work demonstrates for the first time the fabrication and application of a novel MoS2/Ag hybrid, and provides a new platform for use in the rapid and high throughput analysis of small molecules by mass spectrometry.

Microfiber-coupler-assisted control of wavelength tuning for Q-switched fiber laser with few-layer molybdenum disulfide nanoplates.

Based on the liquid exfoliated method, we obtained the few-layer molybdenum disulfide (MoS2) nanoplates solution. By thermal evaporation method, we directly deposited MoS2 thin film onto the facet of a fiber patch cord. The modulation depth of the film is as high as 29%, and a Q-switched fiber laser was achieved. We also provided a new method to continuously tune the output laser with a tuning sensitivity of ∼5.5 nm/(1% strain) by controlling the cavity loss with a strained microfiber coupler (MFC).