Thermus thermophilus Argonaute-Based Isothermal Amplification Assay for Ultrasensitive and Specific RNA Detection.

Recent advances in prokaryotic Argonaute proteins (pAgos) as potential genome-editing tools have provided new insights into the development of pAgos-based nucleic acid detection platforms. However, pAgos-based isothermal detection remains challenging. Here, we report a true isothermal amplification strategy, termed Thermus thermophilus Argonaute-based thermostable exponential amplification reaction (TtAgoEAR), to detect RNA with ultrasensitivity and single-nucleotide resolution at a constant temperature of 66 °C. We demonstrate the reliable detection of lncRNA, mRNA, and virus RNA with attomolar sensitivity and that TtAgoEAR can be applied to detect RNA targets in in cell lines, saliva, and tissues. We utilize this assay to distinguish pancreatic cancer cells carrying the mutation from wild-type cells with as little as 2 ng of RNA material. We also show that TtAgoEAR is easily adaptable to a lateral-flow-based readout. These results demonstrate that TtAgoEAR has great potential to facilitate reliable and easy RNA detection in point-of-care diagnosis and field analysis.

Graphene-enhanced hybrid terahertz metasurface sensor for ultrasensitive nortriptyline sensing and detection.

Graphene is a two-dimensional material with unique physical and chemical properties, whose excellent biocompatibility has also attracted widespread attention in the field of biosensing and medical detection. Graphene provides a novel solution for dramatically improving the sensitivity of terahertz metasurface sensors, since the electrical conductivity can be modified by contact with biomolecules. In this paper, a metal-graphene hybrid metasurface is proposed and demonstrated for high-sensitive nortriptyline sensing based on the plasmon-induced transparency (PIT) resonances. The π-π stacks between nortriptyline and graphene lead to an increase in the Fermi level of graphene and a decrease in the conductivity, thus enhancing the PIT resonance. Experimental results show that the peak-to-peak amplitude magnitude of the PIT window is enhanced up to 3.4-fold with 1 ng nortriptyline analyte, and the minimum detection limit is extended down to 0.1 ng. But no significant change is observed from the samples without graphene as a comparative experiment, which demonstrates that the presence of graphene greatly enhances the bonding to the drug molecules and improves the sensing sensitivity. This metasurface sensor has the advantages of high sensitivity, fast detection speed, label-free and steady properties, which has potential applications in the fields of trace molecular sensing and disease diagnosis.

Aptamer-Cholesterol-Mediated Proximity Ligation Assay for Accurate Identification of Exosomes.

Accurate identification of exosomes plays an essential role in facilitating disease diagnosis and therapies. Herein, we proposed an Aptamer-cholesterol-mediated Proximity Ligation Assay (AcmPLA) for accurate identification of exosomes in a dual-probe strategy, one aptamer probe for recognition of exosomal innate surface protein CD63 and another cholesterol probe for biolipid layer targeting. By integrating a proximity ligation of probes bound with exosomal biomarkers for specific recognition and a rolling circle amplification (RCA) strategy for signal amplification, we have successfully developed an exosomes-surface approach that can perform "AND" logic analysis of dual biomarkers, which not only could be used for exosomes quantification, but also for exosomes tracing. Besides RCA-initiated signal amplification, CD9 antibody-labeled magnetic beads were used to capture exosomes for isolation and secondary signal enrichment. Our approach can achieve specific exosomes isolation and accurate identification and thus could be exploited for broad applications in biological science, biomedical engineering, and personalized medicine.

Rapid and sensitive exosome detection with CRISPR/Cas12a.

Numerous studies have shown that exosomes are closely related to the pathogenesis of various diseases, especially cancers. Therefore, a rapid and sensitive method for exosome detection will be of great importance for the diagnosis and prognosis of diseases. We report here a method for exosome detection based on the CD63 aptamer and clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system. This method consists mainly of exosomal membrane protein recognition based on the CD63 aptamer and signal amplification based on CRISPR/Cas12a. The CD63 aptamer, as an easily adaptable nucleic acid strand, is responsible for the conversion of the amounts of exosomes into nucleic acid detection, whereas CRISPR/Cas12a is responsible for highly specific nucleic acid signal amplification. The detection range of the method was determined as 3 × 103-6 × 107 particles per microliter. Additionally, we successfully applied this method to detect exosomes in clinical samples from both healthy individuals and patients with lung cancer, and the results were highly consistent with those obtained by nanoparticle tracking analysis. In general, this method provides a highly sensitive and specific method for the detection of exosomes and offers an avenue toward future exosome-based diagnosis of diseases.

Target-triggered "signal-off" electrochemical aptasensor assisted by Au nanoparticle-modified sensing platform for high-sensitivity determination of circulating tumor cells.

In this study, we fabricated a high-sensitivity "signal-off" electrochemical aptasensing platform for quantifying circulating tumor cells (CTCs) based on target-triggered signal readout of methylene blue (MB). Au nanoparticles (AuNPs) were introduced to enlarge the specific surface area of the gold electrode (GE), which would immobilize homogeneous and more MB-aptamers. MB-modified and stem-loop-like aptamers were assigned as a recognition element with K562 cells. Thiolated complementary strands hybridized with MB-aptamers to form double-stranded DNA (dsDNA) conformation which were further self-assembled on the surface of AuNP-modified GE, leading to a marked current peak of MB signal. In the presence of K562 cells, the MB-aptamers preferred to recognize and bind with the cells, causing the disassembly of MB-aptamers from the GE surface. Therefore, the reduced value of MB signal was related to the number of K562 cells. With the proposed aptasensor, a dynamic linear range from 1 × 102 to 1 × 106 cells mL-1 was obtained with a detection limit of 23 cells mL-1. Moreover, the aptasensor showed good selectivity, stability, and reproducibility as well as potential use in the clinical setting. Meanwhile, characterization techniques such as field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, atomic force microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy were performed to analyze the evolution of the morphology and each fabricated step of the constructed aptasensor. Our proposed aptasensor could be designed as a universal platform for CTC determination by replacing tumor cell-specific aptamers, which is a promising strategy for basic research and clinical applications. Graphical abstract.

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).

Indirect surface-enhanced Raman scattering assay of insulin-like growth factor 2 receptor protein by combining the aptamer modified gold substrate and silver nanoprobes.

An indirect aptamer-based SERS assay for insulin-like growth factor 2 receptor (IGF-IIR) protein was developed. The gold substrate and silver nanoparticles (AgNPs) were employed simultaneously to achieve double enhancement for SERS signals. Firstly, the five commercial SERS substrates including Enspectr, Ocean-Au, Ocean-AG, Ocean-SP and Q-SERS substrates were evaluated using 4-mercaptobenzoic acid (4-MBA). The Q-SERS substrate was selected based on low relative standard deviation (RSD, 8.6%) and high enhancement factor (EF, 8.7*105), using a 785 nm laser. The aptamer for IGF-IIR protein was designed to include two sequences: one grafted on gold substrate to specifically capture the IGF-IIR protein and a second one forming a 3' sticky bridge to capture SERS nanotags. The SERS nanotag was composed by AgNPs (20 nm), 4-MBA and DNA probes that can hybridize with the aptamer. Due to the steric-hindrance effect, when the aptamer doesn't combine with IGF-IIR protein, it only can capture the SERS nanotags. Therefore, there was a negative correlation between the concentration of IGF-IIR protein and the intensity of 4-MBA at 1076 cm-1. The detection limit reached to 141.2 fM and linear range was from 10 pM to 1 µM. The SERS aptasensor also exhibits a high reproducibility with an average RSD of 4.5%. The interference test was conducted with other four proteins to verify the accuracy of measuring. The study provides an approach to quantitative determination of proteins based on specific recognition and nucleic acid hybridization of aptamers, to establish sandwich structure for SERS enhancement. Graphical abstractSchematic representation of surface-enhanced Raman scattering (SERS) assay on insulin-like growth factor 2 receptor (IGF-IIR) protein by combining the aptamer modified gold substrate and 4-mercaptobenzoic acid (4-MBA) and DNA probe modified silver nanoparticles.

THz Spectroscopy for a Rapid and Label-Free Cell Viability Assay in a Microfluidic Chip Based on an Optical Clearing Agent.

Simple, rapid, and efficient cell viability assays play a fundamental role in much of biomedical research, including cell toxicology investigations and antitumor drug screening. Here, we demonstrate for the first time a rapid and label-free cell viability assay using THz spectroscopy in combination with a new optical clearing agent (OCA) and microfluidic technology. This strategy uses a considerably less absorptive OCA to replace the highly absorptive water molecules around the living cells and thus to decrease the background signal interference. Three low-viscosity oils were screened as potential OCA candidates, among which fluorinated oil was selected because of its lower absorption and lowest cytotoxicity. After the liquid medium was replaced with fluorinated oil in a microfluidic chip, an obvious THz spectral difference was observed between the fluorinated oils with and without living cells. This change in THz response was preliminarily attributed to the distinguishable signals between the cells and the fluorinated oil. In addition, we applied this method to cell viability assays of human breast cancer cells (MDA-MB-231) after treatment with different antitumor drugs. The results indicated that THz spectroscopy with the aid of the proposed water-replacement strategy presented excellent quantification of cell viability with the advantages of a rapid, label-free, nondestructive microassay, which offers significant potential to developing a convenient and practical cell analysis platform.

All-fiber seawater salinity sensor based on fiber laser intracavity loss modulation with low detection limit.

An all-fiber seawater salinity sensor based on intracavity loss-modulated sensing in a fiber ring laser is proposed and experimentally demonstrated. An optical fiber multimode interferometer, which is based on single-mode-no-core-single-mode fiber structure, is cascaded with a fiber reflector and used as a reflected sensing head to enhance loss-modulated depth. It is inserted in a fiber ring laser and the intracavity loss-modulated salinity sensing is induced for the fiber laser's output intensity. The salinity sensitivity is measured to be 0.1 W/‰ with a high signal-to-noise ratio more than 49 dB and narrow full width at half maximum less than 40 pm. The temperature cross-sensitivity characteristic and stability are also analyzed. Considering the errors from cross-sensitivity, stability and resolution of the photodetector, the detection limit of the sensor system is 0.0023 ‰ (0.0002 S/m), which is comparable to the most advanced commercial electronic salinity sensor.

Development of duplex-crossed allele-specific PCR targeting of TPMT*3B and *3C using crossed allele-specific blockers to eliminate non-specific amplification.

Prospective testing for variants in the thiopurine S-methyltransferase (TPMT) is considered a key process in the development of thiopurine therapy. This testing is done to avoid toxicity and side effects in the management of diverse immunological and malignant conditions. Real-time fluorescent PCR techniques using duplex-crossed allele-specific primers in a single tube (DCAS-PCR) were developed in this study to genotype the common loss-of-function TPMT*3B c.460G > A (rs1800460) and TPMT*3C c.719A > G (rs1142345) usually occurring in individuals of Chinese ethnicity. In this method, several integrated strategies were used to completely eliminate the non-specific amplification that is commonly presented in traditional allele-specific (AS) PCR. These strategies include using AS-primers (ASP) that both are artificially mismatched in the penultimate positions and phosphorothioate modifications in the 5'-termini positions. In the assay, an AS-blocker was used, locus-specific TaqMan (LST) probes were used and we used at least two fragments were simultaneously amplified in a single tube which satisfy the thermodynamic characteristics of DNA polymerase to eliminate non-specific amplification. In a group of 200 unselected subjects, the results showed that 8 samples were heterozygous of TPMT*3C, and all samples possessed wild-type TPMT*3B. There was no non-specific amplification, and the genotypes were 100% consistent with Sanger sequencing.

Rapid screening of colistin-resistant Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa by the use of Raman spectroscopy and hierarchical cluster analysis.

Colistin is recognized as the last therapeutic option for multidrug-resistant Gram-negative bacteria infection. In addition, bacterial resistance to colistin could be transmitted between different species through plasmid-mediated mcr-1 gene transfer. Therefore, rapid screening of colistin-resistant isolates will play a key role in controlling the spread of resistance and improving patient outcomes. We developed a rapid method for the detection of colistin-resistance in Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa bacteria based on Raman spectroscopy and hierarchical cluster analysis. Bacteria were incubated with and without colistin using CAMHB as the liquid culture medium. They were then centrifuged and dried on a glass slide. Five Raman spectra of each of the samples were recorded and analyzed by the hierarchical cluster analysis method to determine whether the bacteria were resistant. To evaluate this method, 123 clinical bacterial isolates (42 isolates of E. coli, 41 isolates of A. baumannii and 40 isolates of P. aeruginosa) were tested. The detection sensitivity and specificity were 90.9% and 91.1%, respectively, compared with the reference broth microdilution method. The screening is easy to perform and can be completed in 1.5 h, suggesting that it holds great potential to be an initial screening method in countries and areas where colistin becomes the last resort antibiotic.

Surface-enhanced Raman scattering method for the identification of methicillin-resistant Staphylococcus aureus using positively charged silver nanoparticles.

The article describes a SERS-based method for diagnosis of bacterial infections. Positively charged silver nanoparticles (AgNPs+) were employed for identification of methicillin-resistant Staphylococcus aureus (MRSA). It is found that AgNPs+ undergo self-assembly on the surface of bacteria via electrostatic aggregation. The assembled AgNPs+ are excellent SERS substrates. To prove the capability of SERS to differentiate between S. aureus and other microorganisms, six standard strains including S. aureus 29213, S. aureus 25923, C. albicans, B. cereus, E. coli, and P. aeruginosa were tested. To further demonstrate its applicability for the identification of MRSA in clinical samples, 52 methicillin-sensitive S. aureus (MSSA) isolates and 215 MRSA isolates were detected by SERS. The total measurement time (include incubation) is 45 min when using a 3 µL sample. The method gives a strongly enhanced Raman signal (at 730 cm-1 and 1325 cm-1) with good reproducibility and repeatability. It was successfully applied to the discrimination of the six strain microorganisms. The typical Raman peaks of S. aureus at 730, 1154, 1325, and 1457 cm-1 were observed, which were assigned to the bacterial cell wall components (730 cm-1- adenine, glycosidic ring mode, 1154 cm-1- unsaturated fatty acid, 1325 cm-1- adenine, polyadenine, and 1457 cm-1 for -COO- stretching). S. aureus was completely separated from other species by partial least squares discriminant analysis (PLS-DA). Moreover, 52 MSSA isolates and 215 MRSA isolates from clinical samples were identified by PLS-DA. The accuracy was almost 100% when compared to the standard broth microdilution method. A classification based on latent structure discriminant analysis provided spectral variability directly. Conceivably, the method offers a potent tool for the identification of bacteria and antibiotics resistance, and for studies on antibiotic-resistance in general. Graphical abstract Schematic of the surface-enhanced Raman scattering (SERS) measurements on Staphylococcus aureus (S. aureus) using positively charged silver nanoparticles (AgNPs+). AgNPs+ are adsorbed on the bacterial cell wall by electrostatic attraction. SERS spectra were analyzed by PLS-DA for the identification of Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus aureus (MSSA). MRSA isolates were divided into four groups, including R1, R2, R3, and R4. MSSA just includes group S.

Terahertz spectroscopy for the isothermal detection of bacterial DNA by magnetic bead-based rolling circle amplification.

The demand for rapid and sensitive bacterial detection is continuously increasing due to the significant requirements of various applications. In this study, a terahertz (THz) biosensor based on rolling circle amplification (RCA) was developed for the isothermal detection of bacterial DNA. The synthetic bacterium-specific sequence of 16S rDNA hybridized with a padlock probe (PLP) that contains a sequence fully complementary to the target sequence at the 5' and 3' ends. The linear PLP was circularized by ligation to form a circular PLP upon recognition of the target sequence; then the capture probe (CP) immobilized on magnetic beads (MBs) acted as a primer to initialize RCA. As DNA molecules are much less absorptive than water molecules in the THz range, the RCA products on the surface of the MBs cause a significant decrease in THz absorption, which can be sensitively probed by THz spectroscopy. Our results showed that 0.12 fmol of synthetic bacterial DNA and 0.05 ng µL-1 of genomic DNA could be effectively detected using this assay. In addition, the specificity of this strategy was demonstrated by its low signal response to interfering bacteria. The proposed strategy not only represents a new method for the isothermal detection of the target bacterial DNA but also provides a general methodology for sensitive and specific DNA biosensing using THz spectroscopy.

Enhanced Specificity of BRAF V600E Genotyping Using Wild-Type Blocker Coupled with Internal Competitive Reference in a Single Tube.

BACKGROUND: Mutations in the BRAF gene have been strongly associated with failure in cancer treatment using epidermal growth factor receptor (EGFR) antibodies. To better diagnose and assess the prognosis of cancer patients, mutation screening of the BRAFV600E gene should be performed prior to clinical anti-tumor drug therapy to avoid ineffective treatment. METHODS: In our previous study, we developed a real-time wild-type blocking PCR (WTB-PCR), which can amplify the mutant allele at high efficiency while simultaneously inhibiting the amplification of wild-type alleles. In order to reduce base mismatch due to the high number of cycles, as well as to monitor the total quantity of DNA added to the reaction system, an internal reference gene was co-amplified together with the target gene on the basis of WTB-PCR. RESULTS: Our results showed that when 50 - 200 ng of the DNA templates was used, this current built method (realtime quantitative clamp-based PCR technology using wild-type blocker coupled with internal competitive reference to enhance amplification specificities, named wirePCR) completely blocked the amplification of the wild-type BRAFV600E gene with detection of the mutated allele at a mutant/wild-type ratio of 1:10,000, which was in line with the sensitivity requirement for the detection of trace amounts of the mutant gene. In the colorectal biopsies from 50 patients with suspected colorectal cancer, eight patients (16%) with BRAFV600E mutations were detected using wirePCR. The allele percentage of mutations can be obtained directly from the ΔCq between the targeted and reference genes, we demonstrated that among the V600E-positive patient samples, the percentage of BRAF DNA with the V600E mutation ranged from 24.99% to 54.31%. CONCLUSIONS: WirePCR is a rapid, simple, and low-cost quantitative analytical technique for the detection of trace amounts of mutant BRAFV600E genes in clinical samples.

Laboratory diagnosis of Ebola virus disease and corresponding biosafety considerations in the China Ebola Treatment Center.

Ebola virus disease (EVD), caused by Ebola virus (EBOV), is a potent acute infectious disease with a high case-fatality rate. Etiological and serological EBOV detection methods, including techniques that involve the detection of the viral genome, virus-specific antigens and anti-virus antibodies, are standard laboratory diagnostic tests that facilitate confirmation or exclusion of EBOV infection. In addition, routine blood tests, liver and kidney function tests, electrolytes and coagulation tests and other diagnostic examinations are important for the clinical diagnosis and treatment of EVD. Because of the viral load in body fluids and secretions from EVD patients, all body fluids are highly contagious. As a result, biosafety control measures during the collection, transport and testing of clinical specimens obtained from individuals scheduled to undergo EBOV infection testing (including suspected, probable and confirmed cases) are crucial. This report has been generated following extensive work experience in the China Ebola Treatment Center (ETC) in Liberia and incorporates important information pertaining to relevant diagnostic standards, clinical significance, operational procedures, safety controls and other issues related to laboratory testing of EVD. Relevant opinions and suggestions are presented in this report to provide contextual awareness associated with the development of standards and/or guidelines related to EVD laboratory testing.

Sensitive and specific detection of miRNA using an isothermal exponential amplification method using fluorescence-labeled LNA/DNA chimera primers.

MicroRNAs (miRNAs) are currently considered as potential biomarkers for various human diseases. In the present study, miRNA-triggered real-time fluorescent isothermal reaction with exponential amplification (ReFIRE) with or without Thermus aquaticus MutS (Taq MutS) was developed to analyze miRNAs using DNA polymerase, a nicking endonuclease, and fluorescently labeled primers. In the absence of Taq MutS, the ReFIRE system permitted the detection of 100 ymol of targeted miRNA in 80 min. However, this system enabled limited differentiation between homologous miRNA family members. Upon addition of Taq MutS to the ReFIRE system, non-specific amplification generated from the mishybridization between primers and primer dimers or primers and the template duplex was eliminated. The addition of Taq MutS enabled the ultrasensitive detection of as little as 10 ymol of targeted miRNAs in 50 min, which corresponds to less than 10 copies of miRNAs in a total volume of 20 µl. Additionally, the assay exhibited a dynamic range of up to 12 orders of magnitude. The ReFIRE system also showed high specificity, enabling differentiation between homologous miRNA family members exhibiting only single-base differences. The sensitivity, specificity, and dynamic range associated with this system were greater than most currently available miRNA isothermal amplification assays. Moreover, when target-specific primers were labeled with different fluorescent reporters, multiplex analysis was easily performed in a single tube, permitting accurate normalization of miRNA expression. This simple, fast, ultrasensitive, highly specific, and easy-to-multiplex method could significantly contribute to research investigations pertaining to the biological roles of miRNA, as well as clinical diagnosis of various diseases that involve miRNA disruptions. Graphical Abstract The principle of ReFIRE system.

Terahertz spectroscopy for bacterial detection: opportunities and challenges.

The demand for advanced bacterial detection tools is continuously increasing, promoted by its significant benefits in various applications. For instance, in the medical field, these tools would facilitate decision making about more tailored therapies once the infection source has been identified. In the past few years, terahertz (THz = 10(12) Hz) spectroscopy has also shown potential as a novel bacterial detection modality due to its unique advantages. Impressive breakthroughs have been achieved in this field related to bacterial component characterization, spore identification, and cell detection. However, some intrinsic limitations and technical bottlenecks have led to some debates about the practicability of its clinical adoption. In this review, we summarize the progress achieved in this field and discuss some challenges and strategies for future implementation of practical applications.

Activation of Dopamine D2 Receptor Suppresses Neuroinflammation Through αB-Crystalline by Inhibition of NF-κB Nuclear Translocation in Experimental ICH Mice Model.

BACKGROUND AND PURPOSE: Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced secondary brain injury. Recently, dopamine D2 receptor (DRD2) is identified as an important component controlling innate immunity and inflammatory response in central nervous system, and αB-crystallin (CRYAB) is a potent negative regulator on inflammatory pathways. Here, we sought to investigate the role of DRD2 on neuroinflammation after experimental ICH and the potential mechanism mediated by CRYAB. METHODS: Two hundred and twenty-four (224) male CD-1 mice were subjected to intrastriatal infusion of bacterial collagenase or autologous blood. Two DRD2 agonists quinpirole and ropinirole were administrated by daily intraperitoneal injection starting at 1 hour after ICH. DRD2 and CRYAB in vivo knockdown was performed 48 hours before ICH insult. Behavioral deficits and brain water content, Western blots, immunofluorescence staining, coimmunoprecipitation (Co-IP) assay, and proteome cytokine array were evaluated. RESULTS: Endogenous DRD2 and CRYAB expressions were increased after ICH. DRD2 knockdown aggravated the neurobehavioral deficits and the pronounced cytokine expressions. DRD2 activation by quinpirole and ropinirole ameliorated neurological outcome, brain edema, interleukin-1ß, and monocyte chemoattractant protein-1 expression, as well as microglia/macrophages activation, in the perihematomal region. These effects were abolished by pretreatment with CRYAB siRNAs. Quinpirole enhanced cytoplasmic binding activity between CRYAB and NF-κB and decreased nuclear NF-κB expression. Similar therapeutic benefits were observed using autologous blood injection model and intranasal delivery of quinpirole. CONCLUSIONS: DRD2 may have anti-inflammatory effects after ICH. DRD2 agonists inhibited neuroinflammation and attenuated brain injury after ICH, which is probably mediated by CRYAB and enhanced cytoplasmic binding activity with NF-κB.

Surface invasive cleavage assay on a maskless light-directed diamond DNA microarray for genome-wide human SNP mapping.

The surface invasive cleavage assay, because of its innate accuracy and ability for self-signal amplification, provides a potential route for the mapping of hundreds of thousands of human SNP sites. However, its performance on a high density DNA array has not yet been established, due to the unusual "hairpin" probe design on the microarray and the lack of chemical stability of commercially available substrates. Here we present an applicable method to implement a nanocrystalline diamond thin film as an alternative substrate for fabricating an addressable DNA array using maskless light-directed photochemistry, producing the most chemically stable and biocompatible system for genetic analysis and enzymatic reactions. The surface invasive cleavage reaction, followed by degenerated primer ligation and post-rolling circle amplification is consecutively performed on the addressable diamond DNA array, accurately mapping SNP sites from PCR-amplified human genomic target DNA. Furthermore, a specially-designed DNA array containing dual probes in the same pixel is fabricated by following a reverse light-directed DNA synthesis protocol. This essentially enables us to decipher thousands of SNP alleles in a single-pot reaction by the simple addition of enzyme, target and reaction buffers.

High-sensitivity PCR method for detecting BRAF V600E mutations in metastatic colorectal cancer using LNA/DNA chimeras to block wild-type alleles.

The response to epidermal growth factor receptor (EGFR)-targeted therapy in metastatic colorectal cancer (mCRC) is variable because of intra-tumor heterogeneity at the genetic level, and consequently, it is important to develop sensitive and selective assays to predict patient responses to therapy. Low-abundance BRAF V600E mutations are associated with poor response to treatment with EGFR inhibitors. We developed a method for the detection of BRAF V600E mutations in mCRC using real-time wild-type blocking PCR (WTB-PCR), in which a chimera composed of locked nucleic acids and DNA is incorporated to amplify the mutant allele at high efficiency while simultaneously inhibiting the amplification of wild-type alleles. Mixing experiments showed that this method is exquisitely sensitive, with detection of the mutated allele at a mutant/wild-type ratio of 1:10,000. To demonstrate the applicability of this approach for mCRC patients, we assessed the V600E mutations in 50 clinical cases of mCRC by real-time WTB-PCR. The percentage of patients with V600E mutation as determined by WTB-PCR (16%, 8/50) was higher than by traditional PCR (10%, 5/50), suggesting an increased sensitivity for WTB-PCR. By calculating the ΔC q for real-time traditional PCR, which amplifies all BRAF alleles, versus WTB-PCR, which selectively amplifies mutant BRAF, we demonstrated that among the V600E-positive mCRC patient samples, the percentage of BRAF DNA with the V600E mutation ranged from 0.05 to 52.32%. In conclusion, WTB-PCR provides a rapid, simple, and low-cost method to detect trace amounts of mutated BRAF V600E gene.