Cascade CRISPR/Cas12a and DSN for the electrochemical biosensing of miR-1246 in BC-derived exosomes.

MiR-1246 in breast cancer-derived exosomes was a promising biomarker for early diagnosis of breast cancer(BC). However, the low abundance, high homology and complex background interference make the accurate quantitative detection of miR-1246 facing great challenges. In this study, we developed an electrochemical biosensor based on the subtly combined of CRISPR/Cas12a, double-stranded specific nuclease(DSN) and magnetic nanoparticles(MNPs) for the detection of miR-1246 in BC-derived exosomes. Ascribed to the good synergistic effect of DSN, Cas12a and MNPs, the developed electrochemical biosensor exhibited excellent performance with the linear range from 500 aM to 5 pM, and the detection limit as low down to about 50 aM. The target-specific triggered enzyme-digest activity of DSN and Cas12a system, as well as the powerful separation ability of MNPs ensure the high specificity of developed electrochemical biosensor which can distinguish single base mismatches. In addition, the developed electrochemical biosensor has been successfully applied to detect miR-1246 in blood-derived exosomes and realize distinguishing the BC patients from the healthy individuals. It is expected that the well-designed biosensing platform will open up new avenues for clinical liquid biopsy and early screening of breast cancer, as well as provide deeper insights into clinical oncology treatment.

Nanozymes for the Therapeutic Treatment of Diabetic Foot Ulcers.

Diabetic foot ulcers (DFU) are chronic, refractory wounds caused by diabetic neuropathy, vascular disease, and bacterial infection, and have become one of the most serious and persistent complications of diabetes mellitus because of their high incidence and difficulty in healing. Its malignancy results from a complex microenvironment that includes a series of unfriendly physiological states secondary to hyperglycemia, such as recurrent infections, excessive oxidative stress, persistent inflammation, and ischemia and hypoxia. However, current common clinical treatments, such as antibiotic therapy, insulin therapy, surgical debridement, and conventional wound dressings all have drawbacks, and suboptimal outcomes exacerbate the financial and physical burdens of diabetic patients. Therefore, development of new, effective and affordable treatments for DFU represents a top priority to improve the quality of life of diabetic patients. In recent years, nanozymes-based diabetic wound therapy systems have been attracting extensive interest by integrating the unique advantages of nanomaterials and natural enzymes. Compared with natural enzymes, nanozymes possess more stable catalytic activity, lower production cost and greater maneuverability. Remarkably, many nanozymes possess multienzyme activities that can cascade multiple enzyme-catalyzed reactions simultaneously throughout the recovery process of DFU. Additionally, their favorable photothermal-acoustic properties can be exploited for further enhancement of the therapeutic effects. In this review we first describe the characteristic pathological microenvironment of DFU, then discuss the therapeutic mechanisms and applications of nanozymes in DFU healing, and finally, highlight the challenges and perspectives of nanozyme development for DFU treatment.

Ultrasensitive Exosomal MicroRNA Detection with a Supercharged DNA Framework Nanolabel.

DNA self-assembly has created various nanostructured probes deployed in biosensors, whereas their direct charge contribution to sensitive bioassays remains elusive. Here, we report a supercharged tetrahedral DNA nanolabel-based electrochemical (eTDN) sensor for ultrasensitive detection of exosomal microRNAs (Exo-miRs). By using an "assembly before testing" strategy, there is high-efficiency recognition between the target Exo-miR and self-assembled TDN probe in a homogenous solution relative to surface-based hybridization. The TDN-miR complex can be further bridged specifically to form a stable sandwich construct with the surface-confined capture sequence via the base-stacking effect. The intrinsic supercharges of the TDN can adsorb numerous electroactive molecules in stoichiometry, which largely enhances the detection sensitivity, particularly by using electroneutral peptide nucleic acid instead of DNA probes to minimize the background signal. Using this approach, the eTDN sensor achieves a high sensitivity (34 aM), high specificity (against the single mismatch), and high selectivity (in serum). Furthermore, this ultrasensitive sensor provides a conjugation-free, non-enzymatic Exo-miR detection in blood and accurately distinguishes the breast cancer patients from normal individuals, showing to be a promising tool in the early diagnosis of malignant tumors.

Bile acid profiles in bile and feces of obese mice by a high-performance liquid chromatography-tandem mass spectrometry.

Bile acids (BAs) play a pivotal role in manipulating the development of metabolic diseases. However, due to the compositional complexity and functional variation of BAs, it remains unclear about the changes in BA pool for individuals with obesity or metabolic syndrome. We established a high-performance liquid chromatography-mass spectrometer detection system for the simultaneous analysis of both unconjugated and conjugated BAs in the bile and feces of mice. Ten BAs were completely separated, identified, and quantified with low limit of detection (0.5 ng/mL) and inter/intraday precision (relative standard deviation < 12%). By using this method, these BAs in bile and feces of mice were quantified. The result showed that taurochenodeoxycholic acid, taurine-conjugated α-muricholic acids, and taurine-conjugated ß-muricholic acids were the dominated BAs in bile, whereas deoxycholic acid and chenodeoxycholic acid predominated in feces. Further, most of the BA levels were significantly elevated in either bile or fecal samples of high-fat diet-fed mice as compared with those in normal chow diet-fed mice, indicating that excessive production of BAs was closely associated with the occurrence of lipid metabolism disorders. In summary, the present method is practicable for analysis of BAs in bile and fecal samples of patients with obesity.

AuNP-Amplified Surface Acoustic Wave Sensor for the Quantification of Exosomes.

In this study, we report a gold nanoparticle (AuNP)-amplified surface acoustic wave (SAW) sensor for exosome detection with high sensitivity. The SAW chip was self-assembled with mercapto acetic acid to generate carboxylic groups via the Au-S bond. Anti-CD63 was then anchored onto the chip by pretreatment with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide,1-hydroxypyrrolidine-2,5-dione (NHS). Due to the existence of a membrane protein, CD63, on the exosome surface, exosomes could be bound onto the antibody-immobilized SAW chip. To amplify the detection signal, both the biotin-conjugated epithelial cell adhesion molecule (EpCAM) antibody as a secondary antibody and AuNP-labeled streptavidin were applied onto the exosome-bound SAW chip, resulting in AuNP assembly on the chip through biotin-avidin recognition. The sensor was capable of detecting 1.1 × 103 particles/mL exosomes, which was about 2 orders of magnitude higher than those detected by the strategy without using signal amplification. The sensor also achieved a satisfactory specificity and could detect the low-abundance exosomes directly in blood samples from cancer patients with minimal disturbance. This makes the SAW sensor useful for early diagnosis of cancer.

Resveratrol induces apoptosis in SGC-7901 gastric cancer cells.

The aim of the present study was to investigate the effect of resveratrol on apoptosis in SGC-7901 gastric cancer cells and its molecular mechanisms of action. Following resveratrol treatment, the inhibition rate of SGC-7901 cells was determined using an MTT assay. The morphological changes in apoptosis were observed by fluorescence microscopy based on acridine orange/ethidium bromide double staining. Furthermore, cell cycle and apoptosis were detected using flow cytometry, and the expression levels of nuclear factor κB (NF-κB) as well as apoptosis-associated proteins [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3 and cleaved caspase-8] were analyzed by western blotting. The results of the present study indicated that resveratrol was able to significantly inhibit the viability of SGC-7901 cells in a dose- and time-dependent manner. When treated with 200 µM resveratrol, the inhibition rate of SGC-7901 cells reached ~50%. In the presence of resveratrol, the proportion of apoptotic cells was also increased in a dose-dependent manner. Flow cytometry revealed that resveratrol induced S-phase arrest of SGC-7901 cells. When treated with 50, 200 and 400 µM resveratrol, the proportions of SGC-7901 cells in the S-phase were respectively increased to 33.8±2.42, 60.01±2.43 and 56.05±2.67%, compared with 25.62±3.29% for the control group cells in S-phase. Additionally, the levels of the pro-apoptotic proteins Bax, cleaved caspase-3 and cleaved caspase-8 were upregulated in a dose-dependent manner, whereas the level of the anti-apoptotic protein Bcl-2 was downregulated dose-dependently. Importantly, the activation of NF-κB (p65) was evidently decreased following treatment with resveratrol compared with in the control group. In conclusion, the results of the present study revealed that resveratrol was able to inhibit viability and induce apoptosis in SGC-7901 cells by suppressing NF-κB activation. Therefore, resveratrol may be considered as a potential drug candidate for the treatment of gastric cancer.

A MoS2 Nanosheet-Based Fluorescence Biosensor for Simple and Quantitative Analysis of DNA Methylation.

MoS2 nanomaterial has unique properties, including innate affinity with ss-DNA and quenching ability for fluorescence dyes. Here, we present the development of a simple fluorescence biosensor based on water-soluble MoS2 nanosheets and restriction endonuclease BstUI for methylation analysis of p16 promoter. The biosensing platform exhibited excellent sensitivity in detecting DNA with a linear range of 100 pM~20 nM and a detection limit of 140 pM. More importantly, our method could distinguish as low as 1% difference in methylation level. Compared with previous methylation analysis, our design is both time saving and simple to operate, avoiding the limitations of PCR-based assays without compromising performance.

[1 case of vocal cord plexiform schwannoma].

Summary A 36 years old patient with hoarseness for 2 years and got worsen for one month, electronic laryngoscopy showed a red smooth-faced wide based neoplasm on the posterior 2/3 of the right side of the vocal cords. The neoplasm was excised under suspension laryngoscope . The pathologic results showed:Cells were weave patterned, infiltrative growth, mitotic figure was rare. Immunohistochemical results showed CD34 (-), SMA (-), DM (-), S - 100 (+). The pathological diagnosis was plexiform schwannoma.

PNA-assembled graphene oxide for sensitive and selective detection of DNA.

DNA detection based on peptide nucleic acid (PNA)-DNA hybridization is emerging as an important method in the area of DNA microarrays and biosensors because PNA shows remarkable hybridization properties. In this work, we provide a novel, simple, sensitive, and selective strategy based on a PNA-graphene oxide (GO) assembled biosensor for fluorescence turn-on detection of DNA, in which the new nanomaterial GO was used as a scaffold for PNA and a quencher for the fluorophore. The PNA-GO assembled biosensor is capable of distinguishing sequence specificity including complementary, one-base mismatched and non-complementary targets. Moreover, the results show that the biosensor is able to detect target DNA down to hundreds of picomolar. This sensing platform has been demonstrated to be highly sensitive and specific, and we expect that it will find great applications in the field of biomedicine and disease diagnostics.

Label-free detection of carbohydrate-protein interactions using nanoscale field-effect transistor biosensors.

Carbohydrate-protein interactions play a significant role in cell communication, cell adhesion, cell trafficking, and immune responses. Many efforts have been made to demonstrate detection of carbohydrate-protein interactions. However, the existing methods are still tedious and expensive. Therefore, the detection of carbohydrate-protein interactions is of great significance, and new, efficient methods are required for fast and sensitive recognition testing. In this report, we, for the first time, developed the silicon nanowire (SiNW)-based biosensor capable of label-free electrical detection of carbohydrate-protein interactions with high selectivity and sensitivity by covalently immobilizing unmodified carbohydrates on the sensor surface. We fabricated new SiNW sensor chips with more SiNW arrays for potential detection of multiple analytes. In order to realize the immobilization of the unmodified carbohydrates on the SiNW surface, we used X-ray photoelectron spectra and fluorescence microscopy to verify the successful surface functionalization on the silicon surface. Furthermore, we demonstrated real-time detection of carbohydrate-protein interactions using the carbohydrate-modified SiNW sensor chips. The results show good specificity between galactose-lectin EC and mannose-Con A, which is in good agreement with that reported previously. Finally, the results also show that we are able to use the galactose-modified SiNW biosensor to detect lectin EC as low as 100 fg/mL, which is 4 orders of magnitude lower than that reported by other technologies. We believe that the developed SiNW biosensor paves a novel way for studying carbohydrate-protein interactions.

Programmed cell death 1 gene polymorphisms is associated with ankylosing spondylitis in Chinese Han population.

Programmed cell death 1 (PD-1) has been reported to have a genetic association in several autoimmune diseases. The aim of this study was to investigate the association of PD-1 polymorphisms and haplotypes with ankylosing spondylitis (AS) in Chinese Han population. In a case-control association study, three single-nucleotide polymorphisms (SNP), PD-1.3 G/A, PD-1.5 C/T and PD-1.9 T/C, were genotyped in 216 AS patients and 264 healthy controls using polymerase chain reaction-restriction fragment length polymorphism assay. All genotype distributions in the patients and in the controls were in Hardy-Weinberg equilibrium. The associations of genotypes and alleles with AS were analyzed. The genotype distributions of PD-1.9 were significantly different between the patients with AS and the controls (P = 0.025). The frequencies of TC genotype and T allele of PD-1.9 were higher in the patients than those in the controls (P = 0.026 and 0.004). No association for PD-1.5 in AS was found, and PD-1.3 was non-polymorphic in Chinese Han population. Moreover, the frequency of the CT haplotype (PD-1.5 C/T, PD-1.9 T/C) was significantly higher in AS patients than the controls (21.6 vs. 13.9%, P = 0.002). The CC haplotype was more common in the controls than in the patients (57.1 vs. 44.6%, P = 0.000). The results support a genetic association between the PD-1 polymorphism and susceptibility to AS in Chinese Han population.

High frequencies of HLA-B27 in Chinese patients with suspected of ankylosing spondylitis.

This study was performed to investigate the frequency of human leukocyte antigen (HLA)-B27 in Chinese patients with suspected of ankylosing spondylitis (AS) and to assess the clinical significance of HLA-B27 typing. A total of 1,016 patients suspected of AS were classified into six groups based on one major AS-related clinical manifestation. HLA-B27 was determined by polymerase chain reaction using sequence-specific primers. The frequency of B27 ranged between 24.3 and 46.7% among the patient groups, significantly higher than in healthy controls (2.4%). In the same group, the frequency of B27 in young (< or = 40 years) and in male patients was significantly higher than in the old and in female (P < 0.01). During a 1-year follow-up, 102 subjects were definitely diagnosed as AS, but only one B27(-) patient. Of the 102 definite patients, 69 (67.6%) definite patients were distributed in group 1 (low back pain and stiffness) with the higher incidence (28.5%) of AS. The incidence of AS in the same group was found with a similar pattern to the frequency of B27, in male and young patients significantly greater, except groups 4 and 6 (peripheral arthritis and alteration of skin). These findings confirm that HLA-B27 is one of sensitive diagnostic tools for early AS and suggest that there was a remarkable clinical significance of HLA-B27 typing in Chinese patients suspected of AS, particularly a young man who presents with low back pain and stiffness for > 3 months.

The association of HLA-B*27 subtypes with ankylosing spondylitis in Wuhan population of China.

The aim of this study was to investigate the association of the B27 subtypes with ankylosing spondylitis (AS) in the Wuhan population of China. We selected 317 HLA-B27-positive individuals (145 controls and 172 patients with ankylosing spondylitis). The B27 subtypes were characterized using a PCR-SSP method. Six B27 subtypes were determined: B*2702, 03, 04, 05, 06 and B*13. HLA-B*2704 and HLA-B*2705 were the two high frequency genotypes in controls and patients. Compared with the controls, the AS patients had high frequency of B*2704 (patients 69.2% vs. controls 53.8%) and low frequency of B*2705 (patients 23.8% vs. controls 33.1%). B*2703 was detected in 10 (5.8%) patients and in 13 (8.9%) controls. B*2702, 06 and B*2713 were relatively rare. Our results show that the allele conferring risk to AS in the Wuhan population of China was B*2704 and B*2705. B*2704 is strongly associated with AS.

A quantitative DNA methylation assay using mismatch hybridization and chemiluminescence.

OBJECTIVE: To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence. METHODS: Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes. RESULTS: The percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines. CONCLUSION: Compared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.

Bioluminescent method for detecting telomerase activity.

BACKGROUND: Telomerase is a promising biomarker in cancer diagnosis and therapy. The elongation of telomeric repeats catalyzed by telomerase is accompanied by release of six PP(i) for each TTAGGG repeat (1 pmol PP(i)/310 pg telomeric repeats). We developed a novel method to measure telomerase activity by use of an enzymatic luminometric PP(i) assay (ELIPA). METHODS: Extracts of cell lines and tissues were incubated with primer at 30 degrees C for 30 min. Released PP(i) was converted to ATP by sulfurylase, and ATP was detected by a luciferase bioluminescence system. The ELIPA results were compared with results obtained with the conventional telomeric repeat amplification (TRAP)-ELISA in 42 lung carcinoma tissues and 27 control tissues without malignancy. RESULTS: The lower detection limits of ELIPA and TRAP-ELISA were 5 and 10 cells, respectively. The within-run imprecision (CV) of ELIPA was < or =12%. When compared with TRAP-ELISA, the correlation coefficient (r) was 0.79. When we used the cutoff value from ROC analysis to distinguish malignant and nonmalignant tissues, the sensitivity and specificity of ELIPA were 83% and 96%, respectively, whereas the sensitivity and specificity of TRAP-ELISA were 71% and 96%, respectively. CONCLUSION: ELIPA is a simple and sensitive homogeneous method to quantify telomerase activity.