High Amplification Research Articles

De Novo Transcriptome Assembly and EST-SSR Marker Development and Application in Chrysosplenium macrophyllum.

Chrysosplenium macrophyllum Oliv., belonging to the family Saxifragaceae, is a traditional and unique Chinese herbal medicine. However, the lack of adequate molecular markers has hampered the progress regarding population genetics and evolution within this species. In this research, we used the DNBSEQ-T7 Sequencer (MGI) sequencing assay to analyze the transcriptome profiles of C. macrophyllum. SSR markers were developed on the basis of transcriptomic sequences and further validated on C. macrophyllum and other Chrysosplenium species. The genetic diversity and structure of the 12 populations were analyzed by using polymorphic expressed sequence tag simple sequence repeat (EST-SSR) markers. A potential pool of 3127 non-redundant EST-SSR markers were identified for C. macrophyllum in this study. The developed EST-SSR markers had high amplification rates and cross-species transferability in Chrysosplenium. Our results also showed that the natural populations of C. macrophyllum had a high level of genetic diversity. Genetic distance, principal component analysis, and popular structure analysis revealed that all 60 samples clustered into two major groups that were consistent with their geographical origins. This study provided a batch of highly polymorphic EST-SSR molecular markers that were developed via transcriptome sequencing. These markers will be of great significance for the study of the genetic diversity and evolutionary history of C. macrophyllum and other Chrysosplenium species.

Enzyme-Assisted Nucleic Acid Amplification in Molecular Diagnosis: A Review.

Infectious diseases and tumors have become the biggest medical challenges in the 21st century. They are driven by multiple factors such as population growth, aging, climate change, genetic predispositions and more. Nucleic acid amplification technologies (NAATs) are used for rapid and accurate diagnostic testing, providing critical information in order to facilitate better follow-up treatment and prognosis. NAATs are widely used due their high sensitivity, specificity, rapid amplification and detection. It should be noted that different NAATs can be selected according to different environments and research fields; for example, isothermal amplification with a simple operation can be preferred in developing countries or resource-poor areas. In the field of translational medicine, CRISPR has shown great prospects. The core component of NAAT lies in the activity of different enzymes. As the most critical material of nucleic acid amplification, the key role of the enzyme is self-evident, playing the upmost important role in molecular diagnosis. In this review, several common enzymes used in NAATs are compared and described in detail. Furthermore, we summarize both the advances and common issues of NAATs in clinical application.

VFG-Chip: A high-throughput qPCR microarray for profiling virulence factor genes from the environment

As zoonotic pathogens are threatening public health globally, the virulence factor genes (VFGs) they carry underlie latent risk in the environment. However, profiling VFGs in the environment is still in its infancy due to lack of efficient and reliable quantification tools. Here, we developed a novel high-throughput qPCR (HT-qPCR) chip, termed as VFG-Chip, to comprehensively quantify the abundances of targeted VFGs in the environment. A total of 96 VFGs from four bacterial pathogens including Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Salmonella enterica were targeted by 120 primer pairs, which were involved in encoding five types of virulence factors (VFs) like toxin, adherence, secretion system, immune evasion/invasion, and iron uptake. The specificity of VFG-Chip was both verified computationally and experimentally, with high identity of amplicon sequencing and melting curves analysis proving its robust capability. The VFG-Chip also displayed high sensitivity (by plasmid serial dilution test) and amplification efficiency averaging 97.7%. We successfully applied the VFG-Chip to profile the distribution of VFGs along a wastewater treatment system with 69 VFGs detected in total. Overall, the VFG-Chip provides a robust tool for comprehensively quantifying VFGs in the environment, and thus provides novel information in assessing the health risks of zoonotic pathogens in the environment.

The Interplay between RNA Editing Regulator ADAR1 and Immune Environment in Colorectal Cancer.

An abnormality in the regulation of adenosine deaminase acting on RNA (ADAR) enzymes, which catalyzed adenosine-to-inosine (A-to-I) RNA editing, was closely associated with the highly aggressive biologic behavior and poor prognosis in many malignancies. In the present study, we aimed to investigate the relationship among transcript factors-microRNAs regulatory network, immune environment, and ADAR gene in colorectal carcinoma (CRC). The association among the expression levels of ADAR mRNA and copy number variation, methylation, and mutation status were comprehensively analyzed using cBioPortal, Wanderer, and UALCAN databases in CRC datasets. ADAR-transcript factors (TFs) and ADAR-miRNA regulation networks were constructed by Cistrome Cancer and miRWalk2.0, respectively. The full network and subnetworks for ADAR coexpression genes were constructed using the STRING database and visualized by the MCODE module of the Cytoscape app. The relationship between ADAR mRNA expression and the abundance of infiltrating immune cells in CRC patients was explored by the Tumor Immune Estimation Resource, CIBERSORT, and single-gene gene set enrichment analysis (GSEA). ADAR mRNA was elevated and was a cancer essential gene in CRC. ADAR mRNA and transcripts P110 were significantly elevated in CRC compared to normal controls. Low-level methylation in the promoter region and high copy number amplification of ADAR were responsible for high levels of ADAR mRNA expression. ADAR coexpression genes were mainly involved in immunoregulation, especially T-lymphocyte activation. Hub genes, including CD2, CD274, and FASLG, were also significantly upregulated in the ADAR-high group compared to the control group. Besides, M1 macrophages were enriched in the ADAR-high group compared to the control group. This study demonstrated that ADAR, a new essential gene, was involved in the immune regulator and was a novel immune treatment target in CRC.

Nonlinear finite element analysis for seismic site amplification assessment of urban slopes showing surface geology and topography irregularities

In urban areas, buildings close to the crest of a slope can undergo serious seismic disturbances due to seismic site amplifications, often induced by local site conditions. In this context, this paper aims to assess how combined surface geology and topography irregularities can amplify the site response to the ground surface of the Ciloc slope, subject to the reference earthquake that occurred in Constantine province on October 27, 1985. Therefore, a two-dimensional nonlinear dynamic finite element analysis was conducted on various geometric models, in which both slope angle and lithology were simultaneously varied. To describe properly the earthquake frequency content, an analytical solution based on the interpolation method of the general equation of the dynamic motion was programmed under MATLAB language. Fast Fourier Transform (FFT) was also programmed to compute the Fourier spectral amplitude of a selected accelerogramme. Likewise, the computer software SeismoSignal has been used to compare analytical outcomes. Four slope configurations were simulated namely, an actual slope, a layered slope, a homogeneous marl slope and a homogeneous alluvial slope with various slope angles. It was found that maximum site amplifications had occurred behind the crest and not at the exact crest of the slope as commonly expected. Due to the dilatancy phenomenon, alluvial homogenous formations as cohesionless soils, show high spectral parameter amplifications than the other cases. It was also concluded that a seismic response analysis of these sedimentary deposits is strongly recommended as a preliminary study for the seismic analysis of the neighbouring buildings, to avoid serious seismic destructions.

Sensitive electrochemical immunosensor via amide hydrolysis by DT-diaphorase combined with five redox-cycling reactions

Amide hydrolysis using enzyme labels, such as proteases, occurs at a slower rate than phosphoester and carboxyl ester hydrolysis. Thus, it is not very useful for obtaining high signal amplification in biosensors. However, amide hydrolysis is less sensitive to nonenzymatic spontaneous hydrolysis, allowing for lower background levels. Herein, we report that amide hydrolysis by DT-diaphorase (DT-D) occurs rapidly and that its combination with five redox-cycling reactions allows for the development of a highly sensitive electrochemical immunosensor. DT-D rapidly generates ortho-aminohydroxy-naphthalene (oAN) from its amide substrate via amide hydrolysis, which not even trypsin, a highly catalytic protease, can achieve. NADH, which is required for amide hydrolysis, advantageously acts as a reducing agent for rapid electrooxidation-based redox-cycling reactions. In the presence of oAN, DT-D, and NADH, two redox-cycling reactions rapidly occur. In the additional presence of an electron mediator, Ru(NH3)63+ [Ru(III)], three more redox-cycling reactions occur because Ru(III) reacts rapidly with oAN and DT-D. Although the O2-related redox-cycling reactions and redox reaction decrease electrochemical signals, this signal-decreasing effect is not significant in air-saturated solutions. The slow electrooxidation of NADH at an indium tin oxide electrode and sluggish reaction between NADH and Ru(III) allow for low electrochemical backgrounds. When the developed signal amplification scheme is tested for the sandwich-type electrochemical detection of parathyroid hormone (PTH), a detection limit of ∼1 pg/mL is obtained. The detection method is highly sensitive and can accurately measure PTH in serum samples.

Switch-Mode MOSFETs for Class A Radio Frequency Amplification

Vertical double diffused MOSFETs (“VDMOSFETs”) developed for switching power supplies were successfully used in high voltage, high frequency linear amplifier circuits. These high voltage power switches are surprisingly useful for linear RF power amplifiers and transmitters. In most of their intended applications such as in switching power supplies or switched digital amplifiers, VDMOSFETs are pulsed by a high voltage and the pulsed output is heavily filtered. This avoids the linear region of the VDMOSFET, which normally limits the efficiency to a theoretical maximum of 25%. However, in a search of MOSFETs for high voltage linear amplification using Class A bias without heavy use of filtering, these low-cost switching MOSFETs were found to work best, if limited to radio frequencies above 1.5 MHz. Conditions, and in particular, very low impedance driving of the MOSFET gate, were discovered that gave efficiencies of 70%, rivaling that of non-linear pulse techniques such as Class C or Class D operation. The beneficial effect of linear operation combined with high efficiency at a high voltage is suggested to arise from Miller capacitance. This was only seen at radio frequencies and with the larger current, higher gate capacitance devices. Switching MOSFETs as described here should be considered alongside dedicated RF MOSFETs such as the more expensive LD (lateral diffused) MOSFET when working with RF power.

Rational design of nonlinear hybridization immunosensor chain reactions for simultaneous ultrasensitive detection of two tumor marker proteins.

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. The nonlinear hybridization chain reaction (NHCR) is widely used as an output signal amplification technique for biosensor platforms. A novel hairpin-free NHCR was developed in this study as a flow cytometric immunoassay to detect alpha-fetoprotein (AFP) and prostate specific antigen (PSA). First, the target AFP is captured on magnetic beads (MBs) that are modified with capture antibodies. Then, the prepared biotin-streptavidin-biotin (B-S-B) system, which links biotinylated detection antibodies and biotinylated trigger DNA together through the high affinity between biotin-streptavidin interaction, is added to label the target AFP, forming a sandwich complex with three trigger DNA chains. Each trigger DNA chain grows a dendritic DNA nanostructure following a nonlinear hybridization chain reaction. As the substrate flue chains are labeled with fluorophores, the self-assembly process of dendritic DNA is accompanied by the continuous release of fluorophores. Dendrites with strong fluorescence then form on the surface of MBs. Finally, the target AFP is quantified by analyzing the fluorescent MBs using flow cytometry. The proposed immunoassay has a high selectivity along with isothermal, enzyme-free, and exponential amplification efficiency. It shows a limit of detection (LOD) of 1.74 pg mL-1. The proposed biosensor was also successfully used to quantitatively detect AFP in serum samples. It may be utilized to detect multiple tumor markers simultaneously by changing the size of MBs and antibody-antigen pairs. Most tumor markers are only related to tumor diagnosis but without specificity, so the combined detection of multiple tumor markers can improve the accuracy of early tumor diagnoses.

Trace detection of chiral J-aggregated molecules adsorbed on single Au nanorods.

Trace detection of chiral molecules, which is of great significance in chemical, biological, medical and pharmaceutical sciences, requires microscopic techniques at the single-particle or single-molecule level. Although ensemble experiments show that the circular dichroism of chiral molecules can be amplified by plasmonic nanocrystals, trace detection of small chiral molecules remains challenging due to weak signals that are far below the detection limit. Herein, we demonstrate trace detection of chiral J-aggregated molecules adsorbed on individual Au nanorods (NRs) using single-particle circular differential scattering (CDS) spectroscopy. Through measuring the single-particle CDS spectra, we identified dip-peak bisignatures and further determined the chirality by matching them with calculations modelled with chiral media. We therefore find that plasmonic nanocrystals can dramatically amplify the circular dichroism of strongly coupled molecules to a detectable level so that the detection limit is as low as 3.9 × 103 molecules on an individual plasmonic nanoparticle, whereas 2.5 × 1012 molecules free in solution are barely detectable using a commercial circular dichroism instrument, suggesting a significant amplification factor of 108. Our method provides a promising strategy with a high amplification factor, shedding light on the trace detection of chiral molecules using optical microscopic methods.

Parahydrogen-Induced Hyperpolarization of Unsaturated Phosphoric Acid Derivatives

Parahydrogen-induced nuclear polarization offers a significant increase in the sensitivity of NMR spectroscopy to create new probes for medical diagnostics by magnetic resonance imaging. As precursors of the biocompatible hyperpolarized probes, unsaturated derivatives of phosphoric acid, propargyl and allyl phosphates, are proposed. The polarization transfer to 1H and 31P nuclei of the products of their hydrogenation by parahydrogen under the ALTADENA and PASADENA conditions, and by the PH-ECHO-INEPT+ pulse sequence of NMR spectroscopy, resulted in a very high signal amplification, which is among the largest for parahydrogen-induced nuclear polarization transfer to the 31P nucleus.

Entropy-Driven Self-Assembly of Single Quantum Dot Sensor for Catalytic Imaging of Telomerase in Living Cells

Telomerase is a highly valuable cancer diagnosis biomarker and a promising cancer therapy target. So far, most telomerase assays are limited by the involvement of tedious procedures, multiple enzymes, and complicated reaction schemes. Sensitive monitoring of low-abundant telomerase in living cells remains a challenge. Herein, we demonstrate an entropy-driven catalytic assembly of quantum dot (QD) sensors for accurate detection and imaging of telomerase activity in living cells. In this sensor, target telomerase specifically catalyzes extension of telomerase primer, and the extended primer subsequently acts as a catalyst to continuously initiate entropy-driven catalytic reaction, generating a large number of fluorophore- and biotin-labeled DNAs that can be self-assembled on the QD surface to induce an efficient Föster resonance energy transfer signal. The proposed sensor requires a single step for both recognition and amplification of the telomerase signal, eliminating the use of either protein enzymes or laborious procedures. Taking advantage of the inherent superiority of single-molecule fluorescence detection and high amplification efficiency of the entropy-driven reaction, this sensor demonstrates single-cell sensitivity for the in vitro assay. Moreover, it is capable of screening the telomerase inhibitor, discriminating different tumor cells from normal ones, and even real-time imaging telomerase in living cells, providing a novel platform for telomerase-associated cancer diagnosis and drug screening.

Conservation Genetics of the Asian Giant Soft-Shelled Turtle (Pelochelys cantorii) with Novel Microsatellite Multiplexes.

To understand the genetic structure of the protected turtle species Pelochelys cantorii we used transcriptome data to design more than 30,000 tri- and tetranucleotide repeat microsatellite primer pairs, of which 230 pairs were used for laboratory experiments. After two screenings, only 10 microsatellite markers with good specificity, high amplification efficiency, and polymorphisms were obtained. Using the selected primers, two multiplex PCR systems were established to compare and analyze the genetic diversity of artificially assisted breeding generations from four parents (two females and two males) continuously bred over two years. A total of 25 alleles were detected among the 10 microsatellite loci of the offspring. The polymorphism information content (PIC) was 0.313-0.674, with an average of 0.401, among which two loci were highly polymorphic (PIC ≥ 0.5). The number of alleles was 2-5 and the number of effective alleles was 1.635-3.614. The observed heterozygosity was 0.341-0.813, with an average of 0.582, whereas the average expected heterozygosity was 0.389-0.725, with an average of 0.493. Moreover, on the basis of Nei's genetic distance and the Bayesian clustering algorithm, the 182 offspring were divided into two subgroups, and the results corresponded to the two maternal lines. This is the first study to investigate the molecular markers of P. cantorii, and the results obtained can be used to protect genetic resources and provide a genetic basis for the design of population recovery plans.

Mid-IR pulse amplification to ∼millijoule energies in a single transverse mode using large core Er:ZBLAN fibers operating at 2.8µm.

We demonstrate single transverse mode and high energy nanosecond pulse amplification at ∼2.8-µm using large core Er:ZBLAN fibers. The highest energies achieved are 0.75mJ from a 50 µm core, and 420µJ from a 30 µm core fibers respectively, seeded with 95 ns long pulses generated by a ring-cavity Q-switched Er:ZBLAN fiber laser. Nearly diffraction-limited beams with M2 = 1.2-1.3 were obtained using a single-mode excitation technique of multi-mode core fibers. Achieved pulse energies exceed by approximately an order of magnitude the previously reported highest pulse energies in a single transverse mode from a fiber laser or amplifier at these mid-IR wavelengths.

Efficient Tumor Eradication at Ultralow Drug Concentration via Externally Controlled and Boosted Metallic Iron Magnetoplasmonic Nanocapsules.

With the aim to locally enhance the efficacy of cancer nanotherapies, here we present metal iron based magnetoplasmonic drug-loaded nanocapsules (MAPSULES), merging powerful external magnetic concentration in the tumor and efficient photothermal actuation to locally boost the drug therapeutic action at ultralow drug concentrations. The MAPSULES are composed of paclitaxel-loaded polylactic-co-glycolic acid (PLGA) nanoparticles partially coated by a nanodome shape iron/silica semishell. The iron semishell has been designed to present a ferromagnetic vortex for incorporating a large quantity of ferromagnetic material while maintaining high colloidal stability. The large iron semishell provides very strong magnetic manipulation via magnetophoretic forces, enabling over 10-fold higher trapping efficiency in microfluidic channels than typical superparamagnetic iron oxide nanoparticles. Moreover, the iron semishell exhibits highly damped plasmonic behavior, yielding intense broadband absorbance in the near-infrared biological windows and photothermal efficiency similar to the best plasmonic nanoheaters. The in vivo therapeutic assays in a mouse xenograft tumor model show a high amplification of the therapeutic effects by combining magnetic concentration and photothermal actuation in the tumor, leading to a complete eradication of the tumors at ultralow nanoparticle and drug concentration (equivalent to only 1 mg/kg PLGA nanoparticles containing 8 μg/kg of paclitaxel, i.e., 100-500-fold lower than the therapeutic window of the free and PLGA encapsulated drug and 13-3000-fold lower than current nanotherapies combining paclitaxel and light actuation). These results highlight the strength of this externally controlled and amplified therapeutic approach, which could be applied to locally boost a wide variety of drugs for different diseases.

Persistent high latitude amplification of the Pacific Ocean over the past 10 million years

While high latitude amplification is seen in modern observations, paleoclimate records, and climate modeling, better constraints on the magnitude and pattern of amplification would provide insights into the mechanisms that drive it, which remain actively debated. Here we present multi-proxy multi-site paleotemperature records over the last 10 million years from the Western Pacific Warm Pool (WPWP) – the warmest endmember of the global ocean that is uniquely important in the global radiative feedback change. These sea surface temperature records, based on lipid biomarkers and seawater Mg/Ca-adjusted foraminiferal Mg/Ca, unequivocally show warmer WPWP in the past, and a secular cooling over the last 10 million years. Compiling these data with existing records reveals a persistent, nearly stationary, extratropical response pattern in the Pacific in which high latitude (~50°N) temperatures increase by ~2.4° for each degree of WPWP warming. This relative warming pattern is also evident in model outputs of millennium-long climate simulations with quadrupling atmospheric CO2, therefore providing a strong constraint on the future equilibrium response of the Earth System.

Radiomics of dynamic contrast-enhanced magnetic resonance imaging parametric maps and apparent diffusion coefficient maps to predict Ki-67 status in breast cancer.

This study was aimed at evaluating whether a radiomics model based on the entire tumor region from breast dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) parametric maps and apparent diffusion coefficient (ADC) maps could indicate the Ki-67 status of patients with breast cancer. This retrospective study enrolled 205 women with breast cancer who underwent clinicopathological examination. Among them, 93 (45%) had a low Ki-67 amplification index (Ki-67 positivity< 14%), and 112 (55%) had a high Ki-67 amplification index (Ki-67 positivity ≥ 14%). Radiomics features were extracted from three DCE-MRI parametric maps and ADC maps calculated from two different b values of diffusion-weighted imaging sequences. The patients were randomly divided into a training set (70% of patients) and a validation set (30% of patients). After feature selection, we trained six support vector machine classifiers by combining different parameter maps and used 10-fold cross-validation to predict the expression level of Ki-67. The performance of six classifiers was evaluated with receiver operating characteristic (ROC) analysis, sensitivity, and specificity in both cohorts. Among the six classifiers constructed, a radiomics feature set combining three DCE-MRI parametric maps and ADC maps yielded an area under the ROC curve (AUC) of 0.839 (95% confidence interval [CI], 0.768-0.895) within the training set and 0.795 (95% CI, 0.674-0.887) within the independent validation set. Additionally, the AUC value, compared with that for a single parameter map, was moderately increased by combining features from the three parametric maps. Radiomics features derived from the DCE-MRI parametric maps and ADC maps have the potential to serve as imaging biomarkers to determine Ki-67 status in patients with breast cancer.

Submicron Vertical Channel Organic Electrochemical Transistors with Ultrahigh Transconductance

AbstractOrganic electrochemical transistors (OECTs) belong to the class of electrolyte gated organic transistors (EGOTs) that offer a smooth interface with biology in combination with high transconductance values of typically a few mS. Fabrication‐wise, though, most of the up‐to‐date reported devices are limited to two dimensional structures, where the transistor channel is patterned on the same plane as the source and drain electrodes. Here, a method is introduced for the fabrication of integrated vertical channel OECTs (vOECTs) with submicron channel length. By employing electrodeposition, a vertical channel sandwiched between the source and drain electrodes is created. Channel lengths down to 60 nm are demonstrated, giving rise to ultrahigh transconductance of up to 275 mS. Accounting for the voltage loss on the device connection tracks, an intrinsic transconductance of vOECTs of 500 mS is found. The vOECTs are three‐dimensional transistors finding relevant application in “organ‐on‐a‐chip” and implantable devices, where high amplification and small footprint are demanded.

Nanochip Technology for Monitoring Chemotherapy Response Using Extracellular RNAs and Proteins in Patient Plasma with Diffuse Large B-Cell Lymphoma As Biomarkers

Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Assessment of therapeutic response and monitoring for relapse are important components of clinical care in this entity. Although circulating tumor DNA (ctDNA) has shown to be a potential biomarker, it is affected by gene dysregulation and limited by cost, as it requires high amplification, especially in early stage DLBCL since only ~100 ng free DNA fragments are detected in 1 mL blood. Extracellular vesicles (EVs), on the other hand, are abundant in the blood (1011 - 1012 in 1 mL blood), making quantitative analysis easier and less expensive. This OSU based technology has been extensively studied in lung and pancreatic cancer as well as chronic lymphocytic leukemia and multiple myeloma with promising results. Hence, we sought to evaluate whether Immune Lipoplex Nanoparticle (ILN) biochip-based testing (the first of its kind to monitor patients with lymphomas) can serve as a useful means to follow select biomarkers for monitoring therapeutic response. Methods This is a single-center prospective study of de-novo DLBCL patients wherein the patient samples were longitudinally collected before, during, and after chemotherapy upto 2 years every 3 months. The patients were classified as responders if they achieved complete remission (CR) to chemotherapy following cycle 6 (C6), and non-responders/primary progressors if they had progression. In ILN biochip assay, tethered antibodies on the chip surface can capture and sort an EV subpopulation rich with specific membrane proteins after washing. The fluorescence labeled antibodies or cationic lipoplex nanoparticles (CLNs) containing molecular beacons (MBs) are then added to fuse with captured EVs for the detection of membrane protein or mRNA targets respectively. Well known B-cell markers (CD10, CD19, CD20, MUM1, BCL2, BCL6, and cMYC) and a relatively new lymphoma biomarker, CD47 was measured in plasma samples from DLBCL patients before and after chemotherapy with ILN biochips. EV subpopulations rich in CD20 and/or CD19 (DLBCL markers) or CD63/CD9/CD81 (exosome markers) were captured by antibodies tethered on the biochip surface. Subsequently, ILN biomarker measurements were obtained for CD10, CD19 and CD20 protein expressions by fluorescence-labeled antibodies on anti-CD19/CD20 captured B-cell dominated EV subpopulation; MUM1, BCL2, BCL6, cMYC, and PRMT5 mRNA expressions by MBs encapsulated in CLNs in anti-CD63/CD9/CD81 captured exosome-dominated EV subpopulation; and CD47 protein expression by fluorescence-labeled antibodies on the anti-CD47 captured/ CD47-rich EV subpopulation. Results For plasma samples taken before treatment (C0), the exosomal cMYC, BCL2, and BCL6 mRNA expressions could discriminate between healthy donors (HDs, n=17) and patients (n=27), while other B-cell markers did not perform well (Figure 1A). Since BCL2 and BCL6 detection demonstrated similar trends with BCL2 performing slightly better, exosomal cMYC and BCL2 mRNA expressions were selected as the biomarker classifiers. CD47 protein expression by CD47-rich EVs also discriminated between HDs and patients well in pre-treatment (C0) samples (Figure 1A). During chemotherapy, the EV CD47 expression demonstrated a greater decrease in responder samples (n=20) than in non-responder samples (n=7) obtained after C3 showing a statistically significant difference (Figure 1B). Longitudinal CD47 expression (i.e., C1 and C3) on CD47+ EVs could serve as a potential biomarker to discriminate between responders and non-responders of DLBCL patients undergoing chemotherapy with AUC~0.80 (Figure 2). The results of the longitudinal tri-biomarkers, i.e., CD47(C3)/cMYC(C0)/BCL2(C0) expressions will be presented at the meeting. Conclusions We discovered a new longitudinal tri-biomarker classifier based on CD47/cMYC/BCL2 expression after early chemotherapy treatment (C0 and C3) for monitoring chemotherapy response in DCBCL. However, the sample size, particularly in the non-responder cohort (n=7) is small to make any statistically meaningful conclusions. Additional patient samples are being collected to validate these findings. Lastly, to improve the biomarker classifier performance for chemotherapy monitoring (and maybe relapse surveillance), we are conducting an EV mRNA profiling study to identify novel biomarker candidates. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inducible positive amplification regulation coupled with the double-strand specific nuclease for FzD5 mRNA assay

Frizzled receptor 5 (FzD5) is a significant protein receptor in the Wingless/Int-1 (WNT) signaling pathway, which plays a crucial role in the genesis and development of breast cancer. Here, we constructed a biosensor based on Inducible positive amplification regulation (IPAR) amplification mode coupled with the Double-strand Specific Nuclease (DSN) for sensitive detection of FzD5 mRNA. IPAR as a mode of gene expression regulation in prokaryotes, organically integrated the Rolling circle amplification (RCA) and strand displacement amplification (SDA) for improving amplification efficiency. By clever design, the RCA and SDA cycles were linked to each other to produce large amounts of single-stranded DNA (ssDNA). And DSN as a terminal signal output sheared perfectly paired double-stranded DNA (dsDNA) of probes and ssDNA, greatly improving the specificity of the biosensor. Taking full advantage of the IPAR mode with high amplification efficiency, the detection limit of the biosensor was as low as 99 fM. Therefore, the biosensor was a promising tool in the detection of cancer markers. And it provided new ideas for RCA and SDA applications and new application value for IPAR mode.

A bent fiber optic sensor based on gold nanoparticle-decorated silver film for integration of cytosensor and plasmonic photothermal therapy: A simulation study

The fiber optic surface plasmonic resonance (SPR), with the unique characteristic of miniaturized devices, high refractive index sensitivity, and intense plasmonic photothermal effect, is a promising technique for advanced biosensors in clinical medicine. Studies on the synergistic influence factors of the dual performance of refractive index sensitivity and surface heat field are benefitted for integration of cytosensor and phototherapy. Herein, a gold nanoparticle-decorated silver film (AuNPs-AgFM) was simulated as the coating to construct a bent fiber optic surface plasmonic resonance (SPR) through a Finite Element Method (FEM). Bending leads core mode to shift away from the center of curvature into the fiber cladding, which further reacted with the AgFM and AuNPs to generate the short wavelength peaks for cytosensor and long wavelength peaks for plasmonic photothermal therapy, respectively. Furthermore, the parameters of bent radius, AuNPs’ size, and thickness of AgFM were optimized considering both refractive index sensitivity and surface temperature. Under the optimal condition, the designed SPR biosensor exhibit high amplification sensitivity (AS) of − 2301.6 (1/RIU), wavelength sensitivity (WS) of 3120 (nm/RIU), and surface temperature of 54.9 ℃ under irradiation of near-infrared ray (NIR) laser. The proposed AuNPs-AgFM-based fiber optic SPR biosensor provides an alternative scheme to integrate cytosensor and phototherapy, further pushing fiber optics to clinical application.