The impact of MIF deficiency on alterations of fecal microbiota in C57BL/6 mice induced by Trichinella spiralis infection.

Trichinellosis caused by Trichinella spiralis (T. spiralis) is a major food-borne parasitic zoonosis worldwide. Prevention of trichinellosis is an effective strategy to improve patient quality of life. Macrophage migration inhibitory factor (MIF) is closely related to the occurrence and development of several parasitic diseases. Studying the impact of MIF deficiency (Mif-/- ) on the alterations in host fecal microbiota due to T. spiralis infection may contribute to proposing a novel dual therapeutic approach for trichinellosis. To reveal the diversity and differences in fecal microbial composition, feces were collected from T. spiralis-uninfected and T. spiralis-infected wild-type (WT) and MIF knockout (KO) C57BL/6 mice at 0, 7, 14, and 35 days post-infection (dpi), and the samples were sent for 16S rRNA amplicon sequencing on the Illumina NovaSeq platform. Flow cytometry was used to determine the expression levels of IFN-γ and IL-4 in the CD4+ /CD8+ T-cell sets of mouse spleens. The results showed that operational taxonomic unit (OTU) clustering, relative abundance of microbial composition, alpha diversity, and beta diversity exhibited significant changes among the eight groups. The LEfSe analysis selected several potential biomarkers at the genus or species level, including Akkermansia muciniphila, Lactobacillus murinus, Coprococcus catus, Firmicutes bacterium M10_2, Parabacteroides sp. CT06, and Bacteroides between the KTs and WTs groups. The predicted bacterial functions of the fecal microbiota were mainly involved in metabolism, such as the metabolism of carbohydrates, amino acids, energy, cofactors, vitamins, nucleotides, glycans, and lipids. Flow cytometry revealed an increased CD3+ CD8- /CD3+ CD8+ T-cell ratio and increased IFN-γ and IL-4 levels in CD3+ CD8- T-cell sets from WT and MIF KO mice at 7 dpi. The results indicated that both MIF KO and infection time have a significant influence on the CD3+ CD8- IFN-γ+ and CD3+ CD8- IL-4+ response in mice after T. spiralis. In conclusion, this research showed alterations of the fecal microbiota and immune response in both WT and MIF KO mice before and after T. spiralis infection. These results revealed a potential role of MIF in regulating the pathogenesis of trichinellosis related to the intestinal microbiota. Importantly, the selected potential biomarkers combined with MIF will also offer a novel therapeutic approach to treat trichinellosis in the future.

Effect of particle sizes of biochar on CO2 emissions in a poplar plantation of ancient Yellow River channel, China.

Forest soil is a vital pool of organic carbon, which is sensitive to management. Biochar addition could change the CO2 emissions from soil, but its effects are still ambiguous. Moreover, the impacts of particle sizes of biochar on CO2 emissions are still unknown. In this study, a series of field experiments were conducted to investigate the effects of biochar addition on CO2 emissions in a poplar plantation (Populus nigra), China. Biochar with two application rates of (10 and 50 t/ha) and three particle sizes (3-1 mm, 1-0.1 mm, and <0.1 mm) was applied into the surface soil (0-10 cm), and the soil without biochar was set as control. The results showed that a high level of fine biochar addition (1-0.1 mm and <0.1 mm) had similar and positive effects on CO2 emissions by increasing the contents of soil ammonium, available phosphorus, easily oxidizable carbon, soil moisture, soil capillary pore, and the activity of ß-glucosidase. However, biochar addition (1-0.1 mm and <0.1 mm) reduced the bioavailability of dissolved organic carbon (DOC), producing a negative relationship between DOC content and CO2 emissions. This investigation highlights the importance of biochar with different particle sizes in adjusting CO2 emissions from temperate soils.

Oxymatrine hydrazone (OMTH) synthesis and its protective effect for rheumatoid arthritis through downregulation of MEK/NF-κB pathway.

Rheumatoid arthritis (RA) is one of the inflammatory diseases detected in more than 1% of the world population. In the present study, oxymatrine hydrazone (OMTH) was synthesized and investigated for treatment of RA in vitro in TNF-α induced fibroblast-like synoviocyte cell model. Cell viability and apoptosis were detected using MTT and flow cytometry assays, respectively. ELISA was used for determination of inflammatory cytokines and western blotting for evaluation of protein expression. Pretreatment of HFLS-RA cells with 0.5, 1.0, 1.5, 2.0, and 2.5 µM doses of OMTH suppressed TNF-α induced promotion of proliferative potential in dose-based manner. The OMTH pretreatment of TNF-α exposed HFLS-RA cells significantly increased apoptotic cell proportion. In TNF-α exposed HFLS-RA cells OMTH pretreatment elevated Bax and suppressed Bcl-2 expression. Treatment of HFLS-RA cells with OMTH prevented TNF-α mediated elevation of IL-1ß, IL-6 and IL-8. Moreover, OMTH treatment of HFLS-RA cells effectively suppressed TNF-α mediated elevated levels of MMP-1 and MMP-13. Pretreatment of HFLS-RA cells with OMTH reversed TNF-α mediated promotion of iNOS and COX-2 levels. The MEK/1/2 and p65 phosphorylation in TNF-α exposed HFLS-RA cells was reduced by OMTH pre-treatment in dose-based manner. Thus, OMTH successfully inhibited TNF-α-mediated increased viability of RA synovial cells and activated apoptosis. Pretreatment of TNF-α exposed synovial cells with OMTH targeted phosphorylation of MEK/NF-κB. Therefore, OMTH may act as potential therapeutic agent for RA treatment.

A novel quantification platform for point-of-care testing of circulating MicroRNAs based on allosteric spherical nanoprobe.

MiRNA-150, a gene regulator that has been revealed to be abnormal expression in non-small cell lung cancer (NSCLC), can be regarded as a serum indicator for diagnosis and monitoring of NSCLC. Herein, a new sort of nanoprobe, termed allosteric spherical nanoprobe, was first developed to sense miRNA-150. Compared with conventional hairpin, this new nanoprobe possesses more enrichment capacity and reaction cross section. Structurally, it consists of magnetic nanoparticles and dual-hairpin. In the absence of miRNA-150, the spherical nanoprobes form hairpin structure through DNA self-assembly, which could promote the Förster resonance energy transfer (FRET) of fluorophore (FAM) and quencher (BHQ1) nearby. However, in the presence of target, the target-probe hybridization can open the hairpin and form the active "Y" structure which separated fluorophore and quencher to yield "signal on" fluorescence. In the manner of multipoint fluorescence detection, the target-bound allosteric spherical nanoprobe could provide high detection sensitivity with a linear range of 100 fM to 10 nM and a detection limit of 38 fM. More importantly, the proposed method can distinguish the expression of serum miRNA-150 among NSCLC patients and healthy people. Finally, we hoped that the potential bioanalytical application of this nanoprobe strategy will pave the way for point-of-care testing (POCT).

Exogenous H2S Inhibits Autophagy in Unilateral Ureteral Obstruction Mouse Renal Tubule Cells by Regulating the ROS-AMPK Signaling Pathway.

BACKGROUND/AIMS: The induction of excessive autophagy by increased levels of oxidative stress is one of the main mechanisms underlying unilateral ureteral obstruction (UUO)-induced vascular endothelial cell dysfunction. Hydrogen sulfide (H2S) has been shown to have an anti-oxidative effect, but its mode of action on excessive autophagy in vascular endothelial cells is unclear. METHODS: Surgery was used to induce UUO in male C57BL/6 mice as an in vivo model. Human renal epithelial cells (HK-2) were treated with H2O2 as an in vitro model. NaHS was used as an exogenous H2S donor. Transmission electron microscopy was applied to observe the structure of renal autophagosomes. The expression of proteins related to autophagy and apoptosis was detected by western blot analysis in vivo and in vitro. Flow cytometry (DCFH-DA) was used to examine the levels of intracellular reactive oxygen species (ROS). The terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to detect cell apoptosis. Compound C was used to analyze the association of AMPK with autophagy. RESULTS: Compared with the sham group, in which the ureter was exposed but not ligated, the cell apoptosis index, number of autophagosomes, protein expression of microtubule-associated protein 1 light-chain 3 (LC3)-II/I, beclin-1, and p-AMPK/AMPK were significantly increased in the UUO group. On the other hand, p62, cystathionine ß-synthase, and cystathionine γ-lyase protein expression levels and H2S concentration were significantly decreased (p < 0.05). These alterations were ameliorated by the addition of NaHS (p < 0.05). Similar results were observed in vitro. By using the AMPK inhibitor compound C, it was indicated that AMPK was involved in ROS-induced autophagy. In addition, using tissue from patients with obstructive nephropathy, excessive autophagy was observed by an increased LC3-II/LC3-I ratio. CONCLUSION: NaHS-treatment may exert a protective effect on mouse kidney against UUO by suppressing the ROS-AMPK pathway. ROS-AMPK-mediated autophagy may represent a promising therapeutic target for obstructive nephropathy.

In vivo thrombolysis with targeted microbubbles loading tissue plasminogen activator in a rabbit femoral artery thrombus model.

The increasingly high incidence of ischemic stroke caused by thrombosis of the arterial vessels is one of the major factors that threaten people's health and lives in the world. The present treatments for thrombosis are unsatisfactory yet. We developed the microbubbles loading tissue plasminogen activator (tPA) and their in vitro thrombolysis efficacy under ultrasound exposure has been proved previously. We tried to investigate their thrombolysis effect in vivo in this present study. Thrombus model was made by clamping bilateral femoral arteries in 70 arteries of 40 rabbits. The targeted tPA-loaded microbubbles were made by lyophilization, taking arginine-glycine-aspartic acid-serine peptide as the targeting ligand. Its thrombolysis efficacy, calculated as count rate and efficiency rate of recanalization, was evaluated by Pearson's χ(2) and One-way ANOVA, respectively. The count rate of recanalization of the targeted tPA-loaded microbubbles under ultrasound exposure (70%) was similar to that of the combination of tPA, microbubbles and ultrasound exposure (80%) (P = 0.61), while its tPA dosage (0.06 mg/kg) was much less than that of latter (0.9 mg/kg). Its efficiency rate of recanalization was the highest among all groups (53.22 ± 40.39%) (P < 0.01). Ultrasound-induced targeted tPA-loaded microbubbles release is a promising thrombolytic method with satisfactory thrombolytic efficacy, lowered tPA dose and potentially decreased hemorrhagic risk.

A novel asymmetric-loop molecular beacon-based two-phase hybridization assay for accurate and high-throughput detection of multiple drug resistance-conferring point mutations in Mycobacterium tuberculosis.

The accurate and high-throughput detection of drug resistance-related multiple point mutations remains a challenge. Although the combination of molecular beacons with bio-immobilization technology, such as microarray, is promising, its application is difficult due to the ineffective immobilization of molecular beacons on the chip surface. Here, we propose a novel asymmetric-loop molecular beacon in which the loop consists of 2 parts. One is complementary to a target, while the other is complementary to an oligonucleotide probe immobilized on the chip surface. With this novel probe, a two-phase hybridization assay can be used for simultaneously detecting multiple point mutations. This assay will have advantages, such as easy probe availability, multiplex detection, low background, and high-efficiency hybridization, and may provide a new avenue for the immobilization of molecular beacons and high-throughput detection of point mutations.

Quantitative determination of fibrinogen of patients with coronary heart diseases through piezoelectric agglutination sensor.

Fibrinogen can transform fibrin through an agglutination reaction, finally forming fibrin polymer with grid structure. The density and viscosity of the reaction system changes drastically during the course of agglutination. In this research, we apply an independently-developed piezoelectric agglutination sensor to detect the fibrinogen agglutination reaction in patients with coronary heart diseases. The terminal judgment method of determining plasma agglutination reaction through piezoelectric agglutination sensor was established. In addition, the standard curve between plasma agglutination time and fibrinogen concentration was established to determinate fibrinogen content quantitatively. The results indicate the close correlation between the STAGO paramagnetic particle method and the method of piezoelectric agglutination sensor for the detection of Fibrinogen. The correlation coefficient was 0.91 (γ = 0.91). The determination can be completed within 10 minutes. The fibrinogen concentration in the coronary heart disease group was significantly higher than that of the healthy control group (P < 0.05). The results reveal that high fibrinogen concentration is closely correlated to the incurrence, development and prognosis of coronary heart diseases. Compared with other traditional methods, the method of piezoelectric agglutination sensor has some merits such as operation convenience, small size, low cost, quick detecting, good precision and the common reacting agents with paramagnetic particle method.

Management of difficult or failed biliary access in initial ERCP: A review of current literature.

Selective bile duct cannulation is the prerequisite for all endoscopic biliary therapeutic interventions, but this cannot always be achieved easily. Despite advances and new developments in endoscopic accessories, selective biliary access fails in 5%-15% of cases, even in expert high volume centers. Various techniques - such as double-guidewire induced cannulation, pre-cut papillotomy or transpancreatic sphincterotomy with or without placement of a pancreatic stent - have been used to improve cannulation success rates. Repeated and prolonged attempts at cannulation increase the risk of pancreatitis. Repeating the ERCP within a few days after initial failed pre-cut is a successful strategy and should be tried before contemplating more invasive, alternative interventions such as percutaneous-endoscopic or endoscopic ultrasound guided rendezvous procedure, percutaneous transhepatic or surgical intervention. However, standard guidelines or sequential protocol has not been existed up to now. In certain circumstances, there are unique clinical indications for which invasive, alternative interventions should be preferred. We present and discuss the methods that can be used in difficult or failed initial ERCP, therefore to provide practical advice for endoscopists, especially those who are inexperienced.

Hybridization assay of hepatitis B virus by QCM peptide nucleic acid biosensor.

Although the analyses of HBV genomic DNA have traditionally been performed with commercial techniques, the high cost and long time consumed have hindered their applications in routinely diagnosis and prognosis of infection. We construct peptide nucleic acid (PNA) piezoelectric biosensor for real-time monitoring of hybridization of hepatitis B virus (HBV) genomic DNA. The PNA probe can combine to target DNA sequences more effectively and specifically than a DNA probe. The PNA probe was designed and immobilized on the surface of the biosensor to substitute the conventional DNA probe for direct detection of HBV genomic DNA without previous amplification by PCR. The hybridization assay was completed in 50 min. The detection limit was 8.6 pg/L and the clinical specificity was 94.44% compared with real time-PCR (RT-PCR). The PNA probe was able to distinguish sequences that differ only in one base. Detection sensitivity can be improved and detection time can be decreased by adding RecA protein-coated complementary ssDNA which complement to HBV gene regions. The QCM system we designed has the advantages of being rapid, label-free and highly sensitive and can be a useful supplement to commercial assay methods in clinical chemistry.

Detection of Staphylococcus epidermidis by a Quartz Crystal Microbalance Nucleic Acid Biosensor Array Using Au Nanoparticle Signal Amplification.

Staphylococcus epidermidis is a critical pathogen of nosocomial blood infections, resulting in significant morbidity and mortality. A piezoelectric quartz crystal microbalance (QCM) nucleic acid biosensor array using Au nanoparticle signal amplification was developed to rapidly detect S. epidermidis in clinical samples. The synthesized thiolated probes specific targeting S. epidermidis 16S rRNA gene were immobilized on the surface of QCM nucleic acid biosensor arrays. Hybridization was induced by exposing the immobilized probes to the PCR amplified fragments of S. epidermidis, resulting in a mass change and a consequent frequency shift of the QCM biosensor. To further enhance frequency shift results from above described hybridizations, streptavidin coated Au nanoparticles were conjugated to the PCR amplified fragments. The results showed that the lowest detection limit of current QCM system was 1.3×10³ CFU/mL. A linear correlation was found when the concentration of S. epidermidis varied from 1.3×10³ to 1.3×107 CFU/mL. In addition, 55 clinical samples were detected with both current QCM biosensor system and conventional clinical microbiological method, and the sensitivity and specificity of current QCM biosensor system were 97.14% and 100%, respectively. In conclusion, the current QCM system is a rapid, low-cost and sensitive method that can be used to identify infection of S. epidermidis in clinical samples.

Nucleoside reverse transcriptase inhibitors induced hepatocellular mitochondrial DNA lesions and compensatory enhancement of mitochondrial function and DNA repair.

Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of combined antiretroviral therapy (cART) and are widely used in anti-human immunodeficiency virus (HIV) therapy. Long-term administration of NRTIs can result in mitochondrial dysfunction in certain HIV-1-infected patients. However, NRTI-associated liver mitochondrial toxicity is not well known. Herein, the liver autopsy of acquired immune deficiency syndrome (AIDS) patients and the liver tissues of mice with 12 months of NRTI exposure were used to identify NRTI-associated liver toxicity with immunofluorescence, quantitative real-time polymerase chain reaction (qPCR), Amplex red and horseradish peroxidase, and cloning and sequencing. Laser capture microdissection was used to capture hepatocytes from liver tissues. We observed DNA oxidative damage and mitochondrial DNA (mtDNA) loss in the livers of AIDS patients, and cART patients had higher DNA oxidative damage and lower DNA repair function in liver tissues than non-cART patients. We also observed liver oxidative damage, increased DNA repair and mtDNA loss in mice with exposure to four different NRTIs for 12 months, and hepatocytes had no more mtDNA loss than liver tissues. Although NRTIs could induce mitochondrial hydrogen peroxide production, increased mitochondrial oxygen consumption was found with a Clark-type electrode. The captured hepatocytes had greater diversity in their mtDNA D-loop, dehydrogenase subunit1 (ND1) and ND4 than the controls. Long-term NRTI exposure induced single nucleotide variation in hepatocellular mtDNA D-loop, ND1 and ND4. Our findings indicate that NRTIs can induce liver mtDNA lesions, but simultaneously enhance mitochondrial function and mtDNA repair.

A novel piezoelectric quartz micro-array immunosensor for detection of immunoglobulinE.

A novel multi-channel 2 x 5 model of piezoelectric (PZ) micro-array immunosensor has been developed for quantitative detection of human immunoglobulinE (IgE) in serum. Every crystal unit of the fabricated piezoelectric IgE micro-array immunosensor can oscillate without interfering each other. A multi-channel 2 x 5 model micro-array immunosensor as compared with the traditional one-channel immunosensor can provide eight times higher detection speeds for IgE assay. The anti-IgE antibody is deposited on the gold electrode's surface of 10 MHz AT-cut quartz crystals by SPA (staphylococcal protein A), and serves as an antibody recognizing layer. The highly ordered antibody monolayers ensure well-controlled surface structure and offer many advantages to the performance of the sensor. The uniform amount of antibody monolayer coated by the SPA is good, and non-specific reaction caused by other immunoglobulin in sample is found. The fabricated PZ immunosensor can be used for human IgE determination in the range of 5-300 IU/ml with high precision (CV is 4%). 50 human serum samples were detected by the micro-array immunosensor, and the results agreed well with those given by the commercially ELISA test kits. The correlation coefficient is 0.94 between ELISA and PZ immunosensor. After regeneration with NaOH the coated immunosensor can be reused 6 times without appreciable loss of activity.

Anti-tumor effect of ultrasound-induced Nordy-loaded microbubbles destruction.

BACKGROUND: Synthesized dl-Nordihydroguaiaretic acid (dl-NGDA or "Nordy") can inhibit the growth of malignant human tumors, especially the tumor angiogenesis. However, its liposoluble nature limits its in vivo efficacy in the hydrosoluble circulation of human. PURPOSE: We tried to use the ultrasonic microbubble as the carrier and the ultrasound-induced destruction for the targeted release of Nordy and evaluate its in vitro and in vivo anti-tumor effect. METHODS: Nordy-loaded lipid microbubbles were prepared by mechanical vibration. Effects of ultrasound-induced Nordy-loaded microbubbles destruction on proliferation of human umbilical vein endothelial cells (HUVECs), tumor derived endothelial cells (Td-ECs), and rabbit transplanted VX2 tumor models were evaluated. RESULTS: The ultrasound-induced Nordy-loaded microbubbles destruction inhibited the proliferations of HUVECs and Td-ECs in vitro, and inhibited the tumor growth and the microvasculature in vivo. Its efficacy was higher than those of Nordy used only and Nordy with ultrasound exposure. CONCLUSION: Ultrasonic microbubbles can be used as the carrier of Nordy and achieve its targeted release with improved anti-tumor efficacy in the condition of ultrasound-induced microbubbles destruction.

miR-451 suppresses bladder cancer cell migration and invasion via directly targeting c-Myc.

MicroRNA (miRNA) expression is shown dysregulated in tumors. It has been reported that miR-451 alters gene expression and regulates tumorigenesis in various cancer tissues. However, its underlying biological significance in bladder cancer remains to be clarified. In the present study, we investigated the function and molecular mechanism of miR-451 involved in bladder cancer cell migration and invasion. Our results showed that miR-451 was downregulated in clinical bladder carcinoma tissues compared with adjacent bladder tissues. Overexpression of miR-451 significantly retarded the proliferation, migration and invasion of bladder cancer T24 and 5637 cells in vitro. Moreover, the attenuated cell migration and invasion by miR-451 was correlated with increased apoptosis. However, our dual-luciferase reporter assay validated that c-Myc, an oncogene in many tumors, was a direct target gene of miR-451 in bladder cancer. The expression of c-Myc was repressed by miR-451 in bladder cancer cells, and knockdown of c-Myc mimicked the effects of miR-451 overexpression. This discovery suggested that miR-451 is a tumor suppressor modulating bladder cancer cell migration and invasion by directly targeting c-Myc. In addition, apoptosis promoted by miR-451 may participates in this biological behavior. Therefore, target miR-451 may be a novel therapeutic intervention for bladder cancer.

Construction of a novel peptide nucleic acid piezoelectric gene sensor microarray detection system.

A novel 2 x 5 clamped style piezoelectric gene sensor microarray has been successfully constructed. Every crystal unit of the fabricated gene sensor can oscillate independently without interfering with each other. The bis-peptide nucleic acid (bis-PNA) probe, which can combine with target DNA or RNA sequences more effectively and specifically than a DNA probe, was designed and immobilized on the surface of the gene sensor microarray to substitute the conventional DNA probe for direct detection of the hepatitis B virus (HBV) genomic DNA. Detection conditions were then explored and optimized. Results showed that PBS buffer of pH 6.8, an ion concentration of 20 mmol/liter, and a probe concentration of 1.5 micromol/liter were optimal for the detection system. Under such optimized experimental conditions, the specificity of bis-PNA was proved much higher than that of DNA probe. The relationship between quantity of target and decrease of frequency showed a typical saturation curve when concentrations of target HBV DNA varied from 10 pg/liter to 100 microg/liter, and 10 microg/liter was the watershed, with a statistic linear regression equation of I gC = -2.7455 + 0.0691 deltaF and the correlating coefficient of 0.9923. Fortunately, this is exactly the most ordinary variant range of the HBV virus concentration in clinical hepatitis samples. So, a good technical platform is successfully constructed and it will be applied to detect HBV quantitatively in clinical samples.

A review of 42 cases of intestinal pseudo-obstruction in patients with systemic lupus erythematosus based on case reports.

Intestinal pseudo-obstruction (IpsO) is considered a severe manifestation of systemic lupus erythematosus (SLE) characterized by clinical and radiological evidence of intestinal obstruction with no identifiable mechanical lesion. We performed a systematic review to document IpsO in SLE. Twenty-eight articles with 42 patients were included. The median age of onset of IpsO was 27.5 (10-57) years. The female to male ratio was 38:4. Twenty-two (52.4%) patients had IpsO as the initial presentation of their underlying lupus. Three (7.1%) patients manifested in inactive lupus. The duration of abdominal symptoms before admitted ranged from 3 days to 3 years, however most of the patients responded well to systemic corticosteroid or immunosuppressive treatment within 2 days to about 3 months. Concomitant ureterohydronephrosis was present in approximately three-fourths of the cases. More interestingly, 4 patients presented hepatobiliary dilatation without mechanical obstruction together with IPO and ureterohydronephrosis. In conclusion, IpsO is an uncommon but important manifestation of SLE. The finding of coexisting ureterohydronephrosis and hepatobiliary dilatation suggests that there may be generalized visceral muscle dysmotility. Early recognition of IpsO is necessary to institute appropriate medical treatment and to avoid inappropriate surgical intervention.

An aptamer-based biosensing platform for highly sensitive detection of platelet-derived growth factor via enzyme-mediated direct electrochemistry.

In this work, a new label-free electrochemical aptamer-based sensor (aptasensor) was constructed for detection of platelet-derived growth factor (PDGF) based on the direct electrochemistry of glucose oxidase (GOD). For this proposed aptasensor, poly(diallyldimethylammonium chloride) (PDDA)-protected graphene-gold nanoparticles (P-Gra-GNPs) composite was firstly coated on electrode surface to form the interface with biocompatibility and huge surface area for the adsorption of GOD layer. Subsequently, gold nanoclusters (GNCs) were deposited on the surface of GOD to capture PDGF binding aptamer (PBA). Finally, GOD as a blocking reagent was employed to block the remaining active sites of the GNCs and avoid the nonspecific adsorption. With the direct electron transfer of double layer GOD membranes, the aptasensor showed excellent electrochemical response and the peak current decreased linearly with increasing logarithm of PDGF concentration from 0.005 nM to 60 nM with a relatively low limit of detection of 1.7 pM. The proposed aptasensor exhibited high specificity, good reproducibility and long-term stability, which provided a new promising technique for aptamer-based protein detection.

Isothermal detection of multiple point mutations by a surface plasmon resonance biosensor with Au nanoparticles enhanced surface-anchored rolling circle amplification.

In this study, we developed a surface plasmon resonance (SPR) DNA biosensor method using surface-anchored rolling circle amplification (RCA) and Au nanoparticles modified probes (AuNPs) to isothermally detect multiple point mutations associated with drug-resistance in multidrug-resistant Mycobacterium Tuberculosis (MDRTB). A set of probes contains an allele-specific padlock probe (PLP), a capture probe and an AuNPs. The linear PLPs, circularized by ligation upon the recognition of the point mutation on DNA targets, hybridize to the capture probes via the specific tag/anti-tag recognition. Upon recognition each point mutation is identified by locating into the corresponding channel on the chip. Then the immobilized primer (capture probe)-template (circular PLP) complex are amplified isothermally as RCA and further amplified by AuNPs. The RCA products immobilized on the chip surface cause great SPR angle changes consequently. The 5 pM synthetic oligonucleotides and 8.2 pg uL(-1) of genomic DNA from clinical samples can be detected by the method. The positive mutation detection is achieved with a wild-type to mutant ratio of 5000:1. The method was demonstrated by targeting five clinically meaningful mutations in MDRTB. Thirty clinical samples were identified and they were in good agreement with the results from sequencing.

Ultrasound molecular imaging of acute cellular cardiac allograft rejection in rat with T-cell-specific nanobubbles.

BACKGROUND: Acute rejection (AR) is one of the main obstacles of cardiac transplantation; however, a noninvasive diagnostic method, which reflects its pathologic nature, has not been developed yet. In this study, we prepared a specific nanobubbles targeting to the activated T cells and applied it in the ultrasound molecular imaging of AR in heart transplantation by myocardial contrast echocardiography (MCE). METHODS: Nanobubbles loading anti-CD25 antibody (NB(specific)) or isotype control antibody (NB(nonspecific)) were prepared and then applied in the ultrasound molecular imaging by MCE in a rat model. MCE was performed in 24 allografts and 18 isografts that were divided into three groups, including days 2, 4, and 6 after transplantation. Confocal laser scanning microscopy was used to evaluate the binding of nanobubbles and T cells in four allografts and four isografts. RESULTS: MCE with NB(specific) in allograft showed a "delayed enhancement," and the time-intensity curve presented a second peak. The intensity and time of second peak were both positively correlated with the transplant time (P<0.01) and the pathologic grade of AR (P<0.01). Confocal laser scanning microscopy demonstrated the binding of nanobubbles and lymphocytes in myocardium post-MCE with NB(specific). CONCLUSIONS: Ultrasound molecular imaging of AR after heart transplantation can be achieved by using MCE with the nanobubbles targeted to T cells. The appearance of delayed enhancement indicates the occurrence of AR, and the intensity and time of the second peak in time-intensity curve provide potential quantitative indications for diagnosis and severity of AR.