Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

DNA-based molecular classifiers for the profiling of gene expression signatures.

Although gene expression signatures offer tremendous potential in diseases diagnostic and prognostic, but massive gene expression signatures caused challenges for experimental detection and computational analysis in clinical setting. Here, we introduce a universal DNA-based molecular classifier for profiling gene expression signatures and generating immediate diagnostic outcomes. The molecular classifier begins with feature transformation, a modular and programmable strategy was used to capture relative relationships of low-concentration RNAs and convert them to general coding inputs. Then, competitive inhibition of the DNA catalytic reaction enables strict weight assignment for different inputs according to their importance, followed by summation, annihilation and reporting to accurately implement the mathematical model of the classifier. We validated the entire workflow by utilizing miRNA expression levels for the diagnosis of hepatocellular carcinoma (HCC) in clinical samples with an accuracy 85.7%. The results demonstrate the molecular classifier provides a universal solution to explore the correlation between gene expression patterns and disease diagnostics, monitoring, and prognosis, and supports personalized healthcare in primary care.

Temperature-boosted PAM-less activation of CRISPR-Cas12a combined with selective inhibitors enhances detection of SNVs with VAFs below 0.01.

The precise identification of rare single nucleotide variations (SNVs) concomitant with excess wild-type DNA is a valuable method for minimally invasive disease diagnosis and early prediction of drug responsiveness. Selective enrichment of mutant variants via strand displacement reaction offers an ideal approach of SNVs analysis but fails to differentiate wildtype from mutants with variant allele fraction (VAF) < 0.01%. Here, we demonstrate that integration of PAM-less CRISPR-Cas12a and adjacent mutation-enhanced inhibition of wild-type alleles enables highly sensitive measurement of SNVs well below the 0.01% VAF threshold. Raising the reaction temperature to the upper limit of LbaCas12a helps to boost PAM-less activation of collateral DNase activity, which can be further enhanced using PCR additives, leading to ideal discriminative performance for single point mutations. Along with selective inhibitors bearing additional adjacent mutation, it allowed detection of model EGFR L858R mutants down to 0.001% with high sensitivity and specificity. Preliminary investigation on adulterated genomic samples prepared in two different ways also suggests that it can accurately measure ultralow-abundance SNVs extracted directly from clinical samples. We believe that our design, which combines the superior SNV enrichment capability of strand displacement reaction and unparalleled programmability of CRISPR-Cas12a, has the potential to significantly advance current SNV profiling technologies.

Detection of rare CTCs by electrochemical biosensor built on quaternary PdPtCuRu nanospheres with mesoporous architectures.

Circulating tumor cells (CTCs) are promising liquid biopsy biomarkers for early cancer detection and anti-cancer therapy evaluation. The ultra-low abundance of CTCs in blood samples requires highly sensitive and accurate detection ways. In this study, we propose the design of a dual-recognition electrochemical biosensor to improve both the specificity and signal response. PdPtCuRu mesoporous nanospheres (PdPtCuRu MNSs) with excellent three dimensions (3D) nanopore structures were synthesized by one-pot method and connected to mucin 1 (MUC1) aptamer to serve as signal amplification probe. Besides, superconductive carbon black, Ketjen Black (KB), and gold nanoparticles (AuNPs) modified organometallic frame (CeMOF-Au) were combined to work as signal transducer. The characteristic branching structure of KB provides abundant contact points to load CeMOF-Au to heighten the interface electron transfer rate. In addition, AuNPs were reduced on the surface of CeMOF, which could effectively bind the capture antibody and further enhance the conductivity. Under the optimized condition, the limit of detection (LOD) of the as-constructed biosensor was less than 10 cells mL-1 for model A549 cells, and showed good specificity and accuracy in spiked serum samples. We envision the as-proposed electrochemical biosensor would alternate as a useful tool for the clinical detection of CTCs for cancer diagnosis.

A PAM-free CRISPR/Cas12a ultra-specific activation mode based on toehold-mediated strand displacement and branch migration.

CRISPR (clustered regularly interspaced short palindromic repeats) technology has achieved great breakthroughs in terms of convenience and sensitivity; it is becoming the most promising molecular tool. However, only two CRISPR activation modes (single and double stranded) are available, and they have specificity and universality bottlenecks that limit the application of CRISPR technology in high-precision molecular recognition. Herein, we proposed a novel CRISPR/Cas12a unrestricted activation mode to greatly improve its performance. The new mode totally eliminates the need for a protospacer adjacent motif and accurately activates Cas12a through toehold-mediated strand displacement and branch migration, which is highly universal and ultra-specific. With this mode, we discriminated all mismatch types and detected the EGFR T790M and L858R mutations in very low abundance. Taken together, our activation mode is deeply incorporated with DNA nanotechnology and extensively broadens the application boundaries of CRISPR technology in biomedical and molecular reaction networks.

A novel fluorescence biosensor based on double-stranded DNA branch migration-induced HCR and DNAzyme feedback circuit for sensitive detection of Pseudomonas aeruginosa (clean version).

Pseudomonas aeruginosa (P. aeruginosa) is one of the most common bacteria in nosocomial infection. Here, a novel fluorescence biosensor based on double-stranded DNA branch migration-induced hybridization chain reaction (HCR) and DNAzyme feedback circuit was constructed for sensitive detection of P. aeruginosa. The binding of P. aeruginosa with its aptamer on a DNA three-way junction structure initiated the double-stranded DNA branch migration to form two DNA "Y" junction structures. One DNA "Y" junction structure opened the fluorescence-labelled DNA hairpins and triggered the HCR. The other DNA "Y" junction structure formed a double-stranded DNAzyme and cleaved the specific ribonucleotide site, producing new triggering probes to start the next cycle of the double-stranded DNA branch migration. Ultimately, a large number of DNA "Y" junction structures were produced, which greatly promoted signal amplification. Under optimized conditions, the proposed biosensor detected a wide linearity range of 102-107 CFU mL-1, and the limit of detection was 37 CFU mL-1 (S/N = 3). The recovery test results indicated that the biosensor has promising clinical application potential. Because of the simultaneous initiation of the HCR and the DNAzyme feedback circuit through the double-stranded DNA branch migration, the constructed biosensor provided an ideal platform for pathogenic bacteria detection without protein enzymes and complex signal amplification procedures.

Cooperative strand displacement circuit with dual-toehold and bulge-loop structure for single-nucleotide variations discrimination.

Nucleic acid nanotechnologies based on toehold-mediated strand displacement are ideally suited for single-nucleotide variations (SNVs) detection. But only a limited number of means could be used to construct selective hybridization probes via finely designed toehold and regulation of branching migration. Herein, we present a cooperative hybridization strategy relying on a dual-toehold and bulge-loop (DT&BL) probe, coupled with the strand displacement catalytic (SDC) cycle to identify SNVs. The dual-toehold can simultaneously hybridize the 5' and 3' ends of the target, so that it possessed the mutual correction function for improving the specificity in comparison with the single target-binding domain. Insertion of BLs into the dual-toehold probe allows tuning of Gibbs free energy change (ΔG) and control of the reaction rate during branching migration. Using the SDC cycle, the reactivity and selectivity of the DT&BL probe were increased drastically without elaborate competitive sequences. The feasibilities of this platform were demonstrated by the identification of three cancer-related genes. Moreover, the applicability of this biosensor to detect clinical samples showed satisfactory accuracy and reliability. We envision it would offer a new perspective for the construction of highly specific probes based on dynamic DNA nanotechnology, and serves as a promising tool for clinical diagnostics.

Site-specific insertion of endonuclease recognition sites into amplicons to improve post-PCR analysis sensitivity of gene mutation.

Precise detection of low-frequency gene mutations surrounded by excess wild-type DNA is important in many aspects of medical fields. Most hybridization-based methods for high-resolution mutant allele analysis are hindered by competition of the complementary strand with single-strand probes for the target strand. Here, we demonstrate that site-specific insertion of endonuclease recognition sites into amplicons allows post-PCR generation of short dsDNA or ssDNA, whereby improves the sensitivity of both melting temperature analysis (MTA) and end-point detection following up. Using a three-staged PCR protocol, enrichment of target gene and incorporation of specific restriction sites in amplicons were ensued with hardly any loss in amplification efficiency and specificity. It enables simultaneous discrimination among a panel of totally 11 EGFR 19 exon deletion mutations via MTA after post-PCR digestion by either FokI only or cooperated with CRISPR-Cas12a, using SYBR green I. By replacement of one double-strand cleavage site with a nickase binding domain post-PCR generation of ssDNA of interest via strand displacement amplification (termed as iSDA) is realized. Our preliminary investigation shows that iSDA permits analysis of single nucleotide variants down to 0.1% allelic-frequency using end-point detection. Given the good compatibility with the majority of mutant-enrich PCR methods, we envision it would advance the current gene profiling technologies to a large extent.

Proximity ligation assay mediated rolling circle amplification strategy for in situ amplified imaging of glycosylated PD-L1.

Glycosylated PD-L1 is a more reliable biomarker for immune checkpoint therapy and plays important roles in tumor immunity. Glycosylation of PD-L1 hinders antibody-based detection, which is partially responsible for the inconsistency between PD-L1 immunohistochemical results and therapeutic treatment response. Herein, we present a proximity ligation assay mediated rolling circle amplification (PLA-RCA) strategy for amplified imaging of glycosylated PD-L1 in situ. The strategy relies on a pair of DNA probes: an aptamer probe to specifically recognize cellular surface protein PD-L1 and a glycan conversion (GC) probe for metabolic glycan labeling. Upon proximity ligation of sequence binding to the two probes, the proximity ligation-triggered RCA occurs. The feasibility of the as-proposed strategy has been validated as it realized the visualization of PD-L1 glycosylation in different cancer cells and the monitoring of the variation of PD-L1 glycosylation during drug treatment. Thus, we envision the present work offers a useful alternative to track protein-specific glycosylation and potentially advances the investigation of the dynamic glycan state associated with the disease process.

A new method for anti-negative interference of calcium dobesilate in serum creatinine enzymatic analysis.

BACKGROUND: Serum creatinine is a widely used biomarker for evaluating renal function. Sarcosine oxidase enzymatic (SOE) analysis is currently the most widely used method for the detection of creatinine. This method was negatively interfered with by calcium dobesilate, causing pseudo-reduced results. The aim of this study was to explore a new method to alleviate the negative interference of this drug on creatinine detection. METHOD: We formulated eight drug concentrations and 12 creatinine concentrations from serum. The SOE method, the new method, and the Jaffe method were used for detection in five systems. Creatinine biases were analyzed under the conditions with or without the interference of calcium dobesilate, at consistent or inconsistent creatinine concentrations. Creatinine concentrations were also analyzed at three medical decision levels (MDLs). RESULTS: Calcium dobesilate had negative interference in creatinine SOE analysis. With the increase in calcium dobesilate concentrations, the negative bias increases. The new BG method showed an anti-negative interference effect. In the Roche system, the BG method reduced the negative bias from -71.11% to -16.7%. In the Abbott system, bias was reduced from -45.15% to -2.74%. In the Beckman system, the bias was reduced from -65.36% to -7.58%. In the Siemens system, the bias was reduced from -58.62% to -7.58%. In the Mindray system, the bias was reduced from -36.29% to -6.84%. CONCLUSION: The new method alleviated the negative interference of calcium dobesilate in creatinine SOE detection. The negative bias could be reduced from -60% or -70% to less than -20%.

An All-Freeze-Casting Strategy to Design Typographical Supercapacitors with Integrated Architectures.

The emergence of flexible and wearable electronics has raised the demand for flexible supercapacitors with accurate sizes and aesthetic shapes. Here, a strategy is developed to prepare flexible all-in-one integrated supercapacitors by combining all-freeze-casting with typography technique. The continuous seamless connection of all-in-one supercapacitor devices enhances the load and/or electron transfer capacity and avoids displacing and detaching between their neighboring components at bending status. Therefore, such a unique structure of all-in-one integrated devices is beneficial for retaining stable electrochemical performance at different bending levels. More importantly, the sizes and aesthetic shapes of integrated supercapacitors could be controlled by the designed molds, like type matrices of typography. The molds could be assembled together and typeset randomly, achieving the controllable construction and series and/or parallel connection of several supercapacitor devices. The preparation of flexible integrated supercapacitors will pave the way for assembling programmable all-in-one energy storage devices into highly flexible electronics.

Gastroenteritis Outbreaks Associated with the Emergence of the New GII.4 Sydney Norovirus Variant during the Epidemic of 2012/13 in Shenzhen City, China.

Noroviruses (NoVs) are the leading cause of gastroenteritis outbreaks in humans worldwide. Since late 2012, a new GII.4 variant Sydney 2012 has caused a significant increase in NoV epidemics in several countries. From November of 2012 to January of 2013, three gastroenteritis outbreaks occurred in two social welfare homes (Outbreaks A and B) and a factory (Outbreak C) in Shenzhen city of China. Feces and swabs were collected for laboratory tests for causative agents. While no bacterial pathogen was identified, all three outbreaks were caused by NoVs with detection rates of 26.2% (16/61) at Outbreak A, 35.2% (38/108) at Outbreak B), and 59.3% (16/27) at Outbreaks C. For Outbreak B, 25 of the 29 symptomatic individuals (86.2%) and 13 of the 79 asymptomatic individuals (16.5%) were found NoV-positive. For Outbreak C, an asymptomatic food handler was NoV-positive. All thirteen NoV sequences from the three outbreaks were classified into genogroup II and genotype 4 (GII.4), which we identified to be the GII.4 Sydney 2012 variant. The genome of two isolates from Outbreaks A and B were recombinant with the opening reading frame (ORF) 1 of GII.4 Osaka 2007 and ORF2 and 3 of the GII.4 New Orleans. Our study indicated that the GII.4 Sydney 2012 variant emerged and caused the outbreaks in China.

[Analyses of Binding Profiles of the GII. 12 Norovirus with Human Histo-blood Group Antigens].

Interactions between noroviruses (NoVs) and the receptors of histo-blood group antigens (HB-GAs) affect the infectivity and host susceptibility of NoVs. We elucidated the binding profile of a GII. 12 NoV to HBGAs. First, we synthesized the P domain sequence of the GII. 12 NoV strain Pune (GenBank accession number EU921353). Protein of the P domain was expressed in a prokaryotic system. Formation of the P particle was monitored by gel-filtration chromatography. Antiserum was prepared by immunization of mice with GII. 12 P particles. The binding profile of the GII. 12 NoV Pune strain was determined by binding of the P particle with a panel of saliva samples with various known HBGAs phenotypes. The GII. 12 NoV was bound strongly to saliva samples of subjects with B and AB types and weakly to A, O secretor, and non-secretor saliva samples, suggesting higher affinity with B antigen by GII. 12 NoV. These results were consistent with those determined by a previous crystallography study of GII. 12 NoV. These data suggested that individuals with B and AB blood types may be more susceptible to infection by GII. 12 NoV compared with those with other blood types. Our findings may provide a basis for the prevention and control of an epidemic of GII. 12 NoV.

[Full sequence analysis and characterization of the Shenzhen Norovirus strain SZ2010422].

OBJECTIVE: To obtain information on viral molecular structural and evolutionary characteristics, we conducted the SZ2010422 full-length genomic analysis. METHODS: Primers were designed by New Orleans full sequence, SZ2010422 full genome was amplified by RT-PCR, the whole genome sequence and the capsid domain amino acid sites was analysised after cloned and sequenced. RESULTS: The genome of G II-4 Norovirus SZ2010422 strain was consist of 7559 bp, it revealed three ORFs composites of the whole genome, ORF1 (5100 bp), ORF2 (1623 bp), ORF3 (807 bp) respectively, ORF1 and ORF2 had 19 nucleotide overlap. By evolutionary comparative analysis found SZ2010422 genomic nucleotide sequences with reference strains of G II-4 New Orleans1805 strains the highest homology with a total length of homology was 99.3%, of ORF1 (99.5%), ORF2 (99.2%), ORF3 (98.6%). Phylogenetic analyses showed SZ2010422 belonging to G II-4 New Orleans variant. Date of 541 amino acid analyses showed: New Orleans variant strains of popular sites: aa310N or K, --> S aa341D --> of N, aa359T--> S, aa396H --> P, aa460H --> Y. CONCLUSION: Norovirus SZ2010422 belonged to the G II-4 New Orleans variant. In This study, SZ2010422 full sequence can be used not only as a full-length NoV variant sequence standard for future comparison studies, but also as useful material for the public health field by enabling the diagnosis, vaccine development, and prediction of new emerging variants. Noroviruses; Genes; Sequence analysis

[Genetic characterization of G II.12 norovirus in Beijing from 2008 to 2009].

OBJECTIVE: To reveal the genetic characteristics of GII.12 Norovirus strains isolating from stool samples of adults with diarrhea in Beijing during 2008-2009. METHODS: RdRp, ORF2, ORF3 and ORF1/ORF2 overlap region were respectively amplified by primers using RT-PCR. The products were purified, cloned, sequenced and then aligned, phylogenetic and recombinant analyzed by softwares of DNAStar, MEGA and SimPlot. RESULTS: According to the phylogenetic analysis, 11 strains belonged to G II.g in the RdRp region,while GII.12 in the ORF2 and ORF3. SimPlot analysis further confirmed the 11 strains were recombinant strains ( G II.g [RdRp]/G II.12 [capsid]). CONCLUSION: G II.12 Norovirus prevailing in Beijing and other regions of the world belonged to the same strain, and we identified the genetic characteristics of G II.12 Norovirus in Beijing.

[Optimizing expression of the capsid protein VP2 from human Bocavirus and establish it's seroepidemiology assying methord].

OBJECTIVE: To obtain sufficient recombinant VP2 protein of human Bocavirus and establish it's seroepidemiology assying metbord. METHORD: Tbe capsid protein VP2 DNA genes of HBoV1 and 2 were optimized in accordance with tbe usage of the favorite codons in K coil so as to enhance its protein expression in prokaryotic expressing system. The protein was purified by Ni-NTA column, and its antigenicity was determined by Western Blot. Then establish ELISA to detect the specific anti-VP2 IgG antibodies against HBoV1 and 2 in healthy children aged 3-6 years in Nanjing, China. RESULTS: The recombinant protein 6 x His-VP2 was produced in a larger quantity at 25 degrees C induced by IPTG (1 mmol/L) over night and purified by Ni-NTA column. Seropositive rates of HBoV1 and 2 were 62.2% and 55.5% and their mixed seropositivity was 37%. CONCLUSION: The optimizing expression of the capsid protein VP2 from human Bocavirus constructed successfully and get a high yield under certain conditions. The established ELISA could be used to further analyze seroepidemiology of HBoV in China.