Colorimetric detection of circulating tumor cells in breast cancer based on ladder-branch hybridization chain reaction and DFs/AuNCs nanozyme.

Breast cancer is the most common malignant tumor in women, which accounts for 6.9% of all cancer-related deaths. Early diagnosis is crucial for making the best clinical decision and improving the prognosis of patients. Circulating tumor cells (CTCs) have been regarded as significant tumor biomarkers. Herein, we designed a colorimetric biosensor for breast cancer CTCs quantification based on ladder-branch hybridization chain reaction (HCR) and DNA flowers/gold nanoclusters (DFs/AuNCs) nanozyme. With the assistance of complementary DNA labeled on magnetic beads (MBs), the cleavage products of RNA-cleaving DNAzymes (RCDs) could be rapidly captured, subsequently triggering ladder-branch HCR. In addition, the DFs/AuNCs nanozyme was applied for colorimetric analysis, which further improved the sensitivity for the detection of target CTCs. Benefiting from specific RCDs, ladder-branch HCR and DFs/AuNCs, we achieved a superior detection limit of 3 cells/mL as well as a broad linear range of 10 cells/mL to 104 cells/mL. Conclusively, this colorimetric biosensor achieved sensitively and selectively detection of breast cancer CTCs without the participation of enzymes at room temperature, which might provide new insight into the early detection of breast cancer.

Effects of food-grade iron(III) oxide nanoparticles on cecal digesta- and mucosa-associated microbiota and short-chain fatty acids in rats.

Although iron(III) oxide nanoparticles (IONPs) are widely used in diverse applications ranging from food to biomedicine, the effects of IONPs on different locations of gut microbiota and short-chain fatty acids (SCFAs) are unclear. So, a subacute repeated oral toxicity study on Sprague Dawley (SD) rats was performed, administering low (50 mg/kg·bw), medium (100 mg/kg·bw), and high (200 mg/kg·bw) doses of IONPs. In this study, we found that a high dose of IONPs increased animal weight, and 16S rRNA sequencing revealed that IONPs caused intestinal flora disorders in both the cecal digesta- and mucosa-associated microbiota. However, only high-dose IONP exposure changed the abundance and composition of the mucosa-associated microbiota. IONPs increased the relative abundances of Firmicutes, Ruminococcaceae_UCG-014, Ruminiclostridium_9, Romboutsia, and Bilophila and decreased the relative abundance of Bifidobacterium, and many of these microorganisms are associated with weight gain, obesity, inflammation, diabetes, and mucosal damage. Functional analysis showed that changes in the gut microbiota induced by a high dose of IONPs were mainly related to metabolism, infection, immune, and endocrine disease functions. IONPs significantly elevated the levels of valeric, isobutyric, and isovaleric acid, promoting the absorption of iron. This is the first description of intestinal microbiota dysbiosis in SD rats caused by IONPs, and the effects and mechanisms of action of IONPs on intestinal and host health need to be further studied and confirmed.

Pretreatment with Daudi cells eliminates anti-CD47 monoclonal antibody interference in immunohematology testing.

BACKGROUND: Anti-CD47 monoclonal antibodies have increasing clinical applications in the treatment of cancer. However, anti-CD47 monoclonal antibodies interfere with immunohematology testing in patients who require blood transfusion. As the current approaches to removing any interferences have technical problems, new methods need to be developed to resolve anti-CD47 interference in immunohematology testing. MATERIALS AND METHODS: We evaluated the Daudi cell line for the adsorption of free anti-CD47 monoclonal antibodies from patients' plasma to facilitate immunohematology testing in patients treated with anti-CD47 monoclonal antibody. CD47 expression was identified on the Daudi cells using flow cytometry and confocal microscopy. Next, we tested the ability of intact Daudi cells mixed with simulating plasma and clinical samples to achieve efficient removal of interfering anti-CD47 monoclonal antibodies. The indirect antiglobulin test was used to verify whether interference from anti-CD47 monoclonal antibodies in plasma was eliminated and whether the detection of other irregular antibodies was affected. The effect of eliminating interference was also investigated in relation to the time that the Daudi cells were stored after having been fixed with paraformaldehyde. RESULTS: CD47 expression was higher on Daudi cells than on red blood cells. Analysis of the indirect antiglobulin test results revealed that anti-CD47 monoclonal antibody-treated patients' plasma absorbed by Daudi cells for 15 min at 37°C could completely prevent the interference of anti-CD47 monoclonal antibodies in immunohematology testing while the detection of the tested antibodies, including anti-D and anti-K, was unaffected. DISCUSSION: By decreasing the incubation time, we discovered that interferences in samples with agglutination strengths below 2+ could be eliminated after incubation for 5 min. Of importance, Daudi cells can be preserved with 4% paraformaldehyde for 14 days as short-term storage reagents. This is the first study in which Daudi cells were used to effectively resolve the interference of anti-CD47 monoclonal antibodies in pretransfusion tests.

Hepatic effect of subacute Fe2 O3 nanoparticles exposure in Sprague-Dawley rats by LC-MS/MS based lipidomics.

Fe2 O3 nanoparticles (Fe2 O3 NPs) are one of the components of food additives numbered E172 and have been widely used as food pigments to color sweets. Although a large number of studies have reported that Fe2 O3 NPs could induce hepatotoxicity, the pathogenesis is still unclear, especially the subacute effects on the metabolic network after oral exposure. Therefore, it is necessary to define a highly sensitive strategy to investigate the potential effects of Fe2 O3 NPs and the mechanism. In this study, an animal experiment showed that Fe2 O3 NPs had no obvious toxic effects on body weight, histopathology and oxide stress. In order to further investigate the potential effects of Fe2 O3 NPs in vivo, a more sensitive LC-MS/MS-based lipidomic study was performed. The results of multivariate statistical analysis and western blot analysis showed that Fe2 O3 NP exposure significantly affects the hepatic glycerophospholipid metabolism, decreasing triacylglycerol, diglyceride, lysophosphatidylethanolamine and free fatty acids, and increasing phosphatidylcholine, lysophosphatidylinositol and coenzyme Q9. These data provide further insight into the hepatic subacute effects of Fe2 O3 NPs obtained by conventional toxicology methods.

Application effect of sevoflurane in the cesarean section of pregnant women with pernicious placenta previa and its influence on maternal hemodynamics.

OBJECTIVE: To evaluate the application effect of sevoflurane in pregnant women with pernicious placenta previa who conduct the cesarean section and its influence on maternal hemodynamics. METHODS: A total of 94 women with pernicious placenta previa (PPP) admitted to our hospital were recruited in this study. They were randomly divided into two groups, with 47 each group. The control group was given ketamine, propofol and rocuronium while the observation group was given sevoflurane base on conventional general anesthesia. The available data, intraoperative indexes, coagulation function before and after operation, hemodynamics, umbilical arterial partial pressure of oxygen and carbon dioxide before the procedure (T0), 5 min after anesthesia (T1), 15 min after anesthesia (T2) and during fetal delivery (T4) were observed. The Apgar scores of 1 min, 5 min and 10 min after birth were recorded. RESULTS: No significant difference was seen in related indicators during operation and blood coagulation function before and after the operation between the two groups (P>0.05). The diastolic blood pressure and systolic blood pressure decreased at T1, T2 and T3 compared with T0 time (P<0.05). The decrease was more evident in the control group than in the observation group (P<0.001). The mean arterial pressure in the two groups at T1, T2 and T3 was higher than that at T0 (P<0.05). At T2, the increase in the control group was more obvious than that in the observation group (P<0.001). The heart rate at T1 and T2 was higher than that at T0 (P<0.05). Compared with the control group, the oxygen pressure increased and the carbon dioxide pressure decreased in the observation group (P<0.001). The Apgar score of the observation group was higher than that of the control group at 1 min and 5 min (P<0.001). CONCLUSION: Sevoflurane can stabilize hemodynamics, improve neonatal oxygen uptake rate and increase the safety of operation without affecting coagulation function, which is worthy of clinical application.

A study using LC-MS/MS-based metabolomics to investigate the effects of iron oxide nanoparticles on rat liver.

Iron oxide nanoparticles (IONPs) are widely used in food additives, but their metabolic mechanism in the body is still unclear. In this study, male Sprague-Dawley rats were orally administered with IONPs for 28 days to investigate the adverse effect and metabolic mechanism on liver by the combination of traditional toxicology technology and liquid chromatography tandem-mass spectrometry (LC-MS/MS)-based metabolomics. The results showed that IONPs could increase the concentration of blood glucose and the metabolites in the liver of the control and IONPs-treated group were significantly changed. A total of 32 different metabolites were found, including choline, Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), etc. Pathway analysis based on KEGG database demonstrated that the glycerophospholipid metabolism pathway would be affected. And the expression of the key enzymes of altered metabolomics pathway was further verified at the transcription level. In short, our study clarified oral exposure to IONPs would induce lipid metabolism disorders in the liver of rats, which provided useful information about their safety and potential risks.

Effects of All-Trans Retinoic Acid on the Optimization of Synovial Explant Induced by Tumor Necrosis Factor Alpha.

Current studies focused on the effects of all-trans-retinoic acid (ATRA) on synovial explants from rats with rheumatoid arthritis (RA) induced by lipopolysaccharides (LPS). In our study, synovial membranes were extracted aseptically from the quadriceps femoris of the knee joint of rats, and then incubated in medium containing 10% neonate bovine serum for 24 h adaptive culture. We first measured variations of correlation factors in synovium at 24, 48, 72, 96 and 120 h in control medium or in medium containing 20 ng/mL tumor necrosis factor alpha (TNF-α) (TNF-α-experiment). Then, we investigated the synovium exposed to three ATRA concentrations after 48 h incubation (ATRA-experiment). The effects of ATRA on synovitis were evaluated by observing the expression of inflammatory cytokines, angiogenic factors and the production of proteases in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and apoptosis and autophagy. In TNF-α-experiment, the secretion of nitric oxide (NO), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9) increased significantly after TNF-α stimulation without pathological damage to the synovium. Hence, we successfully obtained the synovial explants model, which had longer inflammatory response time. In the ATRA-experiment, ATRA suppressed the secretion of IL-6 and NO, downregulated the NF-κB P65 and Bcl-2, increased levels of autophagy marker protein LC3, but different doses of ATRA showed inconsistent regulatory effects on VEGF and MMP-9. In short, ATRA inhibited TNF-α induced synovitis by the regulation of inflammatory cytokines and inhibiting NF-κB signal transduction and potentially promoting autophagy, apoptosis and angiogenesis, displaying its role in alleviating synovial inflammation in patients with RA.

Capillary Electrophoresis with Laser Induced Fluorescence Detection for Study of the Association of HSP60 Gene Polymorphism with Gouty Arthritis.

Background: Gout arthritis is a common inflammatory arthritis and it poses a major threat to human health. Objective: A method of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for the detection of HSP60 gene polymorphism has been developed and applied to the exploration of correlation between gouty arthritis and HSP60 gene polymorphism. Methods: The genomic deoxyribonucleic acid from 59 patients with gouty arthritis and 64 control subjects was extracted and the conservative fragment of HSP60 was amplified. The products were digested with restriction endonuclease NlaIII and then separated and detected by the proposed method. Results: In the case group, there were 7 cases of TT genotype, 29 cases of CC genotype, and 23 cases of CT genotype. In the control group, there were 4 cases of TT genotype, 6 cases of CC genotype, and 54 cases of CT genotype. The detection results of the samples were statistically analyzed by binary logistic regression and Spearman correlation analysis. After adjusting gender, age, and other compounding factors, the TT genotype and CT genotype of the HSP60 gene were found to affect gouty arthritis. Conclusions: When used for gene polymorphism research, the proposed CE-LIF method has the advantages of high efficiency, rapidity, sensitivity, and low sample consumption. A moderate correlation between gouty arthritis and HSP60 genotype distribution was discovered for the first time. Highlights: A new method using CE-LIF for the detection of HSP60 gene polymorphism of 59 patients with gouty arthritis and 64 control subjects in China. The correlation between gouty arthritis and HSP60 gene polymorphism was explored for the first time.

Signal amplification strategies for DNA-based surface plasmon resonance biosensors.

DNA has well-defined ability to recognize a wide variety of targets, such as small biological molecules, proteins, inorganic ions and small organic molecules. As molecular recognition elements, DNA can be used to build simple, rapid and sensitive biosensors for detection of these targets. DNA-based SPR sensors are considered to be a real-time and label-free tool. We present a systematical and critical review on DNA-based SPR biosensors and their signal amplification via various strategies, focusing on recent advances in nanomaterials, novel DNA amplifications, redox reactions on surface, enzyme amplifications, as well as promising multiplex amplification strategies.

Fiber optic surface plasmon resonance sensor for detection of E. coli O157:H7 based on antimicrobial peptides and AgNPs-rGO.

A fiber optic surface plasmon resonance (FOSPR) sensor was developed for detection of Escherichia coli O157:H7 (E. coli O157:H7) in water and juice, based on antimicrobial peptides (AMP), Magainin I, as recognition elements and silver nanoparticles-reduced graphene oxide (AgNPs-rGO) nanocomposites assisted signal amplification. The uniform AgNPs-rGO was fixed on the surface of optical fiber and covered with gold film. Not only was the SPR response greatly enhanced, but also the AgNPs was prevented from being oxidized. The FOSPR showed a sensitivity of about 1.5 times higher than that fabricated only with gold film. In the assay, Magainin I, immobilized on the surface of gold film, could specifically capture E. coli O157:H7, resulting in the wavelength shift of the SPR absorption peak. Under the optimized conditions, the SPR resonance wavelength exhibited a good linear relationship with natural logarithm of the target bacteria concentration in the range of 1.0 × 103 to 5.0 × 107 cfu/mL with the detection limit of 5.0 × 102 cfu/mL (S/N = 3). The FOSPR sensor showed good specificity for E. coli O157:H7 detection compared to other bacteria similar to the target bacterial species. Furthermore, the FOSPR sensor was successfully applied to the detection of E. coli O157:H7 in water, fruit and vegetable juice with the satisfactory recoveries of 88-110%. This assay for E. coli O157:H7 detection possesses high sensitivity, good selectivity, reproducibility and stability. In addition, the AMP based SPR biosensing methodology could be extended to detect a wide variety of foodborne pathogens. Therefore, the versatile method might become a potential alternative tool in food analysis and early clinical diagnosis.

[The Detection of Micro RNA346 Gene Polymorphism by Capillary Electrophoresis].

OBJECTIVE: To develop a method for the detection of micro RNA346 gene polymorphism by capillary electrophoresis (CE). METHODS: The genome DNA was extracted with the kit of blood/cell/tissue genome DNA extraction,then micro RNA346 gene was amplified by PCR,digested by BciT130Ⅰrestriction enzyme and detected by CE. The conditions for CE separation were optimized. Samples from rheumatoid arthritis patients and healthy persons were detected under the optimal conditions. RESULTS: Under the optimized experimental conditions of CE (sieving medium mass concentration was 10 g/L and the separation voltage was 12 kV),the detection of the digested products of microRNA346 gene could be completed within 25 min. The intra-day relative standard deviation (RSD) of the method was 0.43%-0.63% and inter-day RSD was 1.49%-1.56%.Samples from 96 rheumatoid arthritis patients and 43 healthy persons were analyzed by the proposed method. The results showed that only micro RNA346Ⅰtype was detected but micro RNA346 Ⅱ type wasn't. CONCLUSION: This method is easy to operate,and has the advantages of high efficiency,fast speed,less sample consumption and high automation level. This method is suitable for the determination of RNA gene polymorphism of mirco RNA.

Determination of Pyrethroids in Tea Brew by GC-MS Combined with SPME with Multiwalled Carbon Nanotube Coated Fiber.

A new method has been developed to simultaneously determine 7 pyrethroid residues in tea brew using gas chromatography-mass spectrometry (GC-MS) combined with solid phase microextraction (SPME) with multiwalled carbon nanotubes (MWCNTs) coated fiber. The MWCNTs coated fiber of SPME was homemade by using stainless steel wire as coating carrier and polyacrylonitrile (PAN) solution as adhesive glue. Under the optimized conditions, a good linearity was shown for bifenthrin, fenpropathrin, permethrin, and cyfluthrin in 1-50 ng mL-1 and for cypermethrin, fenvalerate, and deltamethrin in 5-50 ng mL-1. The correlation coefficients were in the range of 0.9948-0.9999. The average recoveries of 7 pyrethroids were 94.2%-107.3% and the relative standard deviations (RSDs) were less than 15%. The detection limit of the method ranged from 0.12 to 1.65 ng mL-1. The tea brew samples made from some commercial tea samples were analyzed. Among them, bifenthrin, fenpropathrin, and permethrin were found. The results show that the method is rapid and sensitive and requires low organic reagent consumption, which can be well used for the detection of the pyrethroids in tea brew.

Inedible Azo Dyes and Their Analytical Methods in Foodstuffs and Beverages.

Edible colorants, as an important part of food additives, can not only enhance the sensorial attributes of foodstuffs, but can also increase one's appetite. They hold a very important position in food processing. In the past decades, the illegal addition of the inedible colorants has become one of the major issues of food safety. Industrial dyes, especially some azo dyes, are illegal additives frequently found in foodstuffs. They cannot provide any nutrients for the human body and even have toxicity, carcinogenic, or mutagenic effects, which may cause serious damage to consumers' health. There is an increasing demand to detect the inedible azo dyes in foodstuffs for health and safety reasons. In this review, the types and the physicochemical properties of the inedible azo dyes adulterated in foodstuffs and beverages are summarized, and the emphases are focused on the sample pretreatment methods and analytical techniques for monitoring of these dyes in foodstuffs and beverages.

Fluorescent aptasensor for detection of four tetracycline veterinary drugs in milk based on catalytic hairpin assembly reaction and displacement of G-quadruplex.

Based on a novel signal amplification strategy by catalytic hairpin assembly and displacement of G-quadruplex DNA, an enzyme-free, non-label fluorescent aptasensing approach was established for sensitive detection of four tetracycline veterinary drugs in milk. The network consisted of a pair of partially complementary DNA hairpins (HP1 and HP2). The DNA aptamer of four tetracycline veterinary drugs was located at the sticky end of the HP1. The ring region of HP1 rich in G and C could form a stable G-quadruplex structure, which could emit specific fluorescence signal after binding with the fluorescent dye and N-methylmesoporphyrin IX (NMM). When presented in the system, the target analytes would be repeatedly used to trigger a recycling procedure between the hairpins, generating numerous HP1-HP2 duplex complexes and displacing G-quadruplex DNA. Thus, the sensitive detection of target analytes was achieved in a wide linear range (0-1000 µg/L) with the detection limit of 4.6 µg/L. Moreover, this proposed method showed high discrimination efficiency towards target analytes against other common mismatched veterinary drugs, and could be successfully applied to the analysis of milk samples. Graphical abstract Schematic of target analyte detection based on catalytic hairpin assembly reaction and displacement of G-quadruplex.

[Rapid Detection of Adenovirus in Fecal Samples by Capillary Electrophoresis-laser Induced Fluorescence and Microchip Capillary Electrophoresis-laser Induced Fluorescence].

OBJECTIVE: To establish a rapid and sensitive method based on polymerase chain reaction (PCR) combined with capillary electrophoresis-laser induced fluorescence (CE-LIF) and microchip capillary electrophoresis-laser induced fluorescence (MCE-LIF) for detecting adenoviruses in fecal samples. METHODS: The DNA of adenovirus in fecal samples were extracted by the commercial kits and the conserved region of hexon gene was selected as the target gene and amplified by PCR reaction. After labeling highly sensitive nucleic acid fluorescent dye SYBR Gold and SYBR Orange respectively, PCR amplification products were separated by CE and MCE under the optimized condition and detected by LIF detector. RESULTS: PCR amplification products could be detected within 9 min by CE-LIF and 6 min by MCE-LIF under the optimized separation condition. The sequenced PCR product showed good specificity in comparison with the prototype sequences from NCBI. The intraday and inter-day relative standard deviation (RSD) of the size (bp) of the target DNA was in the range of 1.14%-1.34% and 1.27%- 2.76%, respectively, for CE-LIF, and 1.18%-1.48% and 2.85%-4.06%, respectively, for MCE-LIF. The detection limits was 2.33 x 10(2) copies/mL for CE-LIF and 2.33 x 10(3) copies/mL for MCE-LIF. The two proposed methods were applied to detect fecal samples, both showing high accuracy. CONCLUSION: The two proposed methods of PCR-CE-LIF and PCR-MCE-LIF can detect adenovirus in fecal samples rapidly, sensitively and specifically.