Lysosomes Initiating and DNAzyme-Assisted Intracellular Signal Amplification Strategy for Quantification of Alpha-Fetoprotein in a Single Cell.

Cellular trace proteins are critical for maintaining normal cell functions, with their quantitative analysis in individual cells aiding our understanding of the role of cell proteins in biological processes. This study proposes a strategy for the quantitative analysis of alpha-fetoprotein in single cells, utilizing a lysosome microenvironment initiation and a DNAzyme-assisted intracellular signal amplification technique based on electrophoretic separation. A nanoprobe targeting lysosomes was prepared, facilitating the intracellular signal amplification of alpha-fetoprotein. Following intracellular signal amplification, the levels of alpha-fetoprotein (AFP) in 20 HepG2 hepatoma cells and 20 normal HL-7702 hepatocytes were individually evaluated using microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF). Results demonstrated overexpression of alpha-fetoprotein in hepatocellular carcinoma cells. This strategy represents a novel technique for single-cell protein analysis and holds significant potential as a powerful tool for such analyses.

Vitamin B-6 Prevents Heart Failure with Preserved Ejection Fraction Through Downstream of Kinase 3 in a Mouse Model.

BACKGROUND: There is an urgent need to develop an efficient therapeutic strategy for heart failure with preserved ejection fraction (HFpEF), which is mediated by phenotypic changes in cardiac macrophages. We previously reported that vitamin B-6 inhibits macrophage-mediated inflammasome activation. OBJECTIVES: We sought to examine whether the prophylactic use of vitamin B-6 prevents HFpEF. METHODS: HFpEF model was elicited by a combination of high-fat diet and Nω-nitro-l-arginine methyl ester supplement in mice. Cardiac function was assessed using conventional echocardiography and Doppler imaging. Immunohistochemistry and immunoblotting were used to detect changes in the macrophage phenotype and myocardial remodeling-related molecules. RESULTS: Co-administration of vitamin B-6 with HFpEF mice mitigated HFpEF phenotypes, including diastolic dysfunction, cardiac macrophage phenotypic shifts, fibrosis, and hypertrophy. Echocardiographic improvements were observed, with the E/E' ratio decreasing from 42.0 to 21.6 and the E/A ratio improving from 2.13 to 1.17. The exercise capacity also increased from 295.3 to 657.7 min. However, these beneficial effects were negated in downstream of kinase (DOK) 3-deficient mice. Mechanistically, vitamin B-6 increased DOK3 protein concentrations and inhibited macrophage phenotypic changes, which were abrogated by an AMP-activated protein kinase inhibitor. CONCLUSIONS: Vitamin B-6 increases DOK3 signaling to lower risk of HFpEF by inhibiting phenotypic changes in cardiac macrophages.

Enhancing intracellular mRNA precise imaging-guided photothermal therapy with a nucleic acid-based polydopamine nanoprobe.

Despite significant advancements in cancer research, real-time monitoring and effective treatment of cancer through non-invasive techniques remain a challenge. Herein, a novel polydopamine (PDA) nucleic acid nanoprobe has been developed for imaging signal amplification of intracellular mRNA and precise photothermal therapy guidance in cancer cells. The PDA nucleic acid nanoprobe (PDA@DNA) is constructed by assembling an aptamer hairpin (H1) labeled with the Cy5 fluorophore and another nucleic acid recognition hairpin (H2) onto PDA nanoparticles (PDA NPs), which have exceptionally high fluorescence quenching ability and excellent photothermal conversion properties. The nanoprobe could facilitate cellular uptake of DNA molecules and their protection from nuclease degradation. Upon recognition and binding to the intracellular mRNA target, a catalytic hairpin assembly (CHA) reaction occurs. The stem of H1 unfolds upon binding, allowing the exposed H1 to hybridize with H2, forming a flat and sturdy DNA double-stranded structure that detaches from the surface of PDA NPs. At the same time, the target mRNA is displaced and engages in a new cyclic reaction, resulting in the recovery and significant amplification of Cy5 fluorescence. Using thymidine kinase1 (TK1) mRNA as a model mRNA, this nanoprobe enables the analysis of TK1 mRNA with a detection limit of 9.34 pM, which is at least two orders of magnitude lower than that of a non-amplifying imaging nucleic acid probe. Moreover, with its outstanding performance for in vitro detection, this nanoprobe excels in precisely imaging tumor cells. Through live-cell TK1 mRNA imaging, it can accurately distinguish between tumor cells and normal cells. Furthermore, when exposed to 808-nm laser irradiation, the nanoprobe fully harnesses exceptional photothermal conversion properties of PDA NPs. This results in a localized temperature increase within tumor cells, which ultimately triggers apoptosis in these tumor cells. The integration of PDA@DNA presents innovative prospects for tumor diagnosis and image-guided tumor therapy, offering the potential for high-precision diagnosis and treatment of tumors.

Design and synthesis of a novel chiral photoacoustic probe and accurate imaging detection of hydrogen peroxide in vivo.

Nanocatalytic medicine, which aims to accurately target and effectively treat tumors through intratumoral in situ catalytic reactions triggered by tumor-specific environments or markers, is an emerging technology. However, the relative lack of catalytic activity of nanoenzymes in the tumor microenvironment (TME) has hampered their use in biomedical applications. Therefore, it is crucial to develop a highly sensitive probe that specifically responds to the TME or disease markers in the TME for precision diagnosis and treatment of diseases. In this work, a chiral photoacoustic (PA) nanoprobe (D/L-Ce@MoO3) based on the H2O2-catalyzed TME activation reaction was constructed in a one-step method using D-cysteine (D-Cys) or L-cysteine (L-Cys), polymolybdate, and cerium nitrate as raw materials. The designed and synthesized D/L-Ce@MoO3 chiral nanoprobe can perform in situ, non-invasive, and precise imaging of pharmacological acute liver injury. In vivo and in vitro experiments have shown that the D/L-Ce@MoO3 probe had chiral properties, the CD signal decreased upon reaction with H2O2, and the absorption and PA signals increased with increasing H2O2 concentration. This is because of the catalytic reaction between Ce ions doped in the nanoenzyme and the high expression of H2O2 caused by drug-induced liver injury to produce ·OH, which has a strong oxidizing property to kill tumor cells and destroy the Mo-S bond in the probe, thus converting the chiral probe into an achiral polyoxometalate (POM) with PA signal.

Rational design of a near-infrared dual-emission fluorescent probe for ratiometric imaging of glutathione in cells.

Sensors for which the output signal is an intensity change for a single-emission peak are easily disturbed by many factors, such as the stability of the instrument, intensity of the excitation light, and biological background. However, for ratiometric fluorescence sensors, the output signal is a change in the intensity ratio of two or more emission peaks. The fluorescence intensity of these emission peaks is similarly affected by external factors; thus, these sensors have the ability to self-correct, which can greatly improve the accuracy and reliability of the detection results. To accurately image glutathione (GSH) in cells, gold nanoclusters (AuNCs) with intrinsic double emission at wavelengths of 606 nm and 794 nm were synthesized from chloroauric acid. With the emission peak at 606 nm as the recognition signal and the emission peak at 794 nm as the reference signal, a near-infrared dual-emission ratio fluorescence sensing platform was constructed to accurately detect changes in the GSH concentration in cells. In vitro and in vivo analyses showed that the ratiometric fluorescent probe specifically detects GSH and enables ultrasensitive imaging, providing a new platform for the accurate detection of active small molecules.

Development and evaluation of a culturally adapted digital-platform integrated multifaceted intervention to promote the utilization of maternal healthcare services: a single-arm pilot study.

BACKGROUND: The utilization of hospital delivery and antenatal care (ANC) is essential for improving maternal and newborn outcomes. However, social and cultural barriers in underdeveloped rural areas hindered maternal care utilization. This study aims to design and evaluate the effectiveness of a culturally adapted digital-platform intervention to promote maternal care utilization among women in ethnic minority communities in China. METHODS: From January 1st, 2020, to December 31st, 2021, all pregnant women in Mianshan town, Liangshan Autonomous Prefecture, were invited to participate in the intervention. The multifaceted intervention included participatory and cultural-tailored health education on a popular social media platform, transportation subsidies, and capacity building and economic incentives for healthcare providers. The effectiveness of the intervention was evaluated by comparing two groups: mothers who gave live birth before the intervention (January 1st to December 31st, 2019) and mothers whose entire pregnancy period was covered by the intervention. The primary outcomes were the rate of hospital delivery and ANC utilization. Data on pregnant women were retrospectively collected through telephone surveys and the maternal and newborn's health monitoring system. RESULTS: A total of 237 intervention sample and 138 pre-intervention sample were included. The intervention group demonstrated significantly higher rates of hospital delivery (97.5% vs. 87.7%, p < 0.001), timely initiation of ANC (73.0% vs. 62.3%, p = 0.031), and timely completion of five-time ANC visits (37.1% vs.4.3%, p < 0.001) compared to the pre-intervention group. The intervention group was more likely to utilize hospital delivery (OR = 9.26, 95%CI [2.83-30.24], p < 0.001) and ANC, including timely initiation of ANC (OR = 2.18, 95%CI [1.31-3.62], p = 0.003), completion of five ANC visits (OR = 1.72, 95%CI [1.05-2.83], p = 0.032), and timely completion of five ANC visits (OR = 15.12, 95%CI [6.24-36.64], p < 0.001). CONCLUSIONS: The culturally adapted digital-platform integrated multifaceted intervention effectively promoted the utilization of hospital delivery, timely initiation of ANC, and completion of ANC visits in the Yi ethnic community in China. This study provides valuable insights for future interventions targeting maternal healthcare services in underdeveloped ethnic minority communities worldwide. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2300073219. Registered 4 July 2023 - Retrospectively registered, https://www.chictr.org.cn/showproj.html?proj=199202 .

A Novel Fluorescent Aptamer Sensor with DNAzyme Signal Amplification for the Detection of CEA in Blood.

Carcinoembryonic antigen (CEA) is a tumor-specific biomarker; however, its low levels in the early stages of cancer make it difficult to detect. To address the need for analysis of ultra-low-level substances, we designed and synthesized a fluorescent aptamer sensor with DNAzyme signal amplification and used it for the detection of CEA in blood. In the presence of the target protein, the aptamer sequence in the recognition probe binds to the target protein and opens the hairpin structure, hybridizes with the primer and triggers a polymerization reaction in the presence of polymerase to generate double-stranded DNA with two restriction endonuclease Nb.BbvCl cleavage sites. At the same time, the target protein is displaced and continues to bind to another recognition probe, triggering a new round of polymerization reaction, forming a cyclic signal amplification triggered by the target. The experimental results show that the blood detection with CEA has a high sensitivity and a wide detection range. The detection range: 10 fg/mL~10 ng/mL, with a detection limit of 5.2 fg/mL. In addition, the sensor can be used for the analysis of complex biological samples such as blood.

Fabrication of Ag-CaCO3 Nanocomposites for SERS Detection of Forchlorfenuron.

In this study, Ag-CaCO3 nanocomposites were synthesized using silver nitrate as the precursor solution based on calcium carbonate nanoparticles (CaCO3 NPs). The synthesis involved the reaction of calcium lignosulphonate and sodium bicarbonate. The properties of Ag-CaCO3 nanocomposites were studied by various technologies, including an ultraviolet-visible spectrophotometer, a transmission electron microscope, and a Raman spectrometer. The results showed that Ag-CaCO3 nanocomposites exhibited a maximum UV absorption peak at 430 nm, the surface-enhanced Raman spectroscopy (SERS) activity of Ag-CaCO3 nanocomposites was evaluated using mercaptobenzoic acid (MBA) as the marker molecule, resulting in an enhancement factor of 6.5 × 104. Additionally, Ag-CaCO3 nanocomposites were utilized for the detection of forchlorfenuron. The results demonstrated a linear relationship in the concentration range of 0.01 mg/mL to 2 mg/mL, described by the equation y = 290.02x + 1598.8. The correlation coefficient was calculated to be 0.9772, and the limit of detection (LOD) was determined to be 0.001 mg/mL. These findings highlight the relatively high SERS activity of Ag-CaCO3 nanocomposites, making them suitable for analyzing pesticide residues and detecting toxic and harmful molecules, thereby contributing to environmental protection.

MicroRNA-23a-3p promotes macrophage M1 polarization and aggravates lipopolysaccharide-induced acute lung injury by regulating PLK1/STAT1/STAT3 signalling.

Macrophage polarization is an important effector process in acute lung injury (ALI) induced by sepsis. MicroRNAs (miRNAs) have emerged as important players in regulating ALI process. Here, we showed that elevated microRNA-23a-3p (miR-23a-3p) promoted LPS-induced macrophage polarization and ALI in mice, while inhibition of miR-23a-3p led to reduced macrophage response and ameliorated ALI inflammation. Mechanically, miR-23a-3p regulated macrophage M1 polarization through targeting polo-like kinase 1 (PLK1). PLK1 was downregulated in LPS-treated macrophages and ALI mouse lung tissues. Knockdown of PLK1 increased macrophage M1 polarization through promoting STAT1/STAT3 activation, while overexpression of PLK1 reduced macrophage immune response. Collectively, our results reveal a key miRNA regulon that regulates macrophage polarization for LPS-induced immune response.

A colorimetric aptasensor based on a hemin/EpCAM aptamer DNAzyme for sensitive exosome detection.

Exosomes are considered as potential biomarkers that can reflect information from their parent cell-associated cancer microenvironment. Recently, aptasensors have been widely used for cancer and tumor exosome detection. Aptamers related to exosome surface proteins are usually used to introduce a sequence; the aptamer is used for exosome recognition, and the introduced sequence is used to form G-quadruplexes and for signal amplification. In this paper, we found that the EpCAM aptamer is rich in guanine and unimolecular G-quadruplex with a two-layer G-tetrad under acidic conditions, and we investigated its topology, thermal stability and dissociation constant with hemin. Based on this, our proposed colorimetric aptamer sensor combines the unmodified EpCAM aptamer with hemin to construct a hemin/G-quadruplex DNAzyme and catalyze the TMB-H2O2 system to generate a strong colorimetric signal. Therefore, colorimetric signal changes were negatively correlated with the exosome concentration. The linear range of the 1 h assay was 106-108 particles per mL, and the detection limit was 3.94 × 105 particles per mL. In addition, this method can detect exosomes in complex fetal bovine serum samples with good specificity and high sensitivity toward exosomes from breast, liver, and lung cancers with abnormal EpCAM protein expression.

Adsorption and leaching of ß-blockers in fluvo-aquic and black soil: Behavior characteristic and enantiomer selectivity.

ß-blockers are widely used chiral pharmaceuticals to treat hypertension and cardiovascular diseases, which are ubiquitously detected in the water-soil environment. However, little is known about their biogeochemical behaviors and enantiomer selectivity during soil migration and transformation. In this study, the adsorption and leaching behaviors of ß-blockers in fluvo-aquic soil and black soil were investigated. The adsorption of ß-blockers was fit well by the Freundlich adsorption isotherm (R2 > 0.913) and the adsorption affinity of ß-blockers decreased in the following order: propranolol (logarithm of Freundlich adsorption coefficient log Kf = 1.46-2.55) > atenolol (log Kf = 0.53-1.04) > sotalol (log Kf = 0.32-1.01). An increase in ionic strength and dissolved organic matter (DOM) inhibited their soil adsorption. Ionic change is the main driving force for adsorption. Besides, hydrophobic partitioning and hydrogen bonding played key roles in the adsorption of propranolol and atenolol, respectively. The leaching behaviors of ß-blockers are related to their hydrophobicity. An increase in ionic strength enhanced the migration of ß-blockers to deeper soil layers, and the presence of DOM accelerated the migration of sotalol and propranolol. The migration potential of ß-blockers in black soil is lower than that in fluvo-aquic soil, which could be ascribed to the higher organic matter content and strong ion exchange ability of black soil. Further, more significant enantiomer selectivity of ß-blockers was found in black soil (e.g. enantiomer fraction of atenolol = 0.61) than in fluvo-aquic soil (e.g. enantiomer fraction of atenolol = 0.53) during the leaching process. The microbial activity might influence the enantiomer selectivity of studied ß-blockers during soil leaching.

Changes in residents' hygiene awareness and behaviors in public toilets before and during the COVID-19 pandemic in Hangzhou, China: a two-round cross-sectional study.

BACKGROUND: Hygiene behaviors in public toilets are important to prevent the transmission of infectious diseases, especially during the pandemic. All through the novel coronavirus (COVID-19) pandemic, governments in many countries published guidance on personal hygiene for the general population to prevent disease transmission. This study aimed to investigate improvements in residents' hygiene awareness and behaviors in public toilets before and during the pandemic. METHODS: We recruited 316 residents between November and December 2018 before the pandemic, and 314 residents between December 2020 and January 2021 during the pandemic in the same study sites in Hangzhou, a well-developed city in China. Residents' hygiene behaviors in public toilets, hygiene awareness, risk perception, and sociodemographic factors were collected. Bivariate analysis and multivariable logistic regressions were used to test the differences between the two rounds. We conducted an observational study to record the provision of hygiene amenities at toilets during the pandemic. RESULTS: After controlling for sociodemographic factors (gender, marital status, age, education level, and monthly household income), compared with respondents recruited before the pandemic, respondents recruited during the pandemic were more likely to perceive the risks of infection when using public toilets (aOR = 1.77, 95%CI [1.20, 2.60]), and were more likely to be aware of the risks of touching contaminated toilet facilities (aOR = 1.72, 95%CI [1.17, 2.54]) and the risks of not using soap to wash one's hands after using the toilet (aOR = 1.93, 95%CI [1.38, 2.72]). They were more likely to always clean their toilet seat with alcohol (aOR = 1.88, 95%CI [1.01, 3.51]), wash hands with soap (aOR = 1.52, 95%CI [1.09, 2.10]) and dry their hands with a dryer (aOR = 1.78, 95%CI [1.16, 2.71]), but they were less likely to always wash their hands after using the toilets (aOR = 0.57, 95%CI [0.32, 1.00]). Among 70 public toilets observed, 9 provided alcohol for toilet seat disinfection, 52 provided soap, 33 provided paper towels, and 41 had working hand dryers. CONCLUSIONS: Despite the overall improvement, residents' hygiene behaviors in public toilets and the supply of hygiene amenities were still suboptimal during the pandemic. Further hygiene education and an adequate supply of hygiene amenities in public toilets are needed to promote residents' hygiene behaviors.

Expectant parents' vaccine decisions influenced by the 2018 Chinese vaccine crisis: A cross-sectional study.

Immunization programs have been challenged by vaccine crises. Between 2013 and 2018, China has experienced three major vaccine scandals and crises, which has partly impaired Chinese public trust in domestically produced vaccines. This study aims to explore the associations between parental trust toward CDC-released crisis communication information, parents' critical understanding of crisis information, parental confidence in vaccine efficacy, and parental vaccine decisions. A cross-sectional survey was conducted among 1065 expectant parents two weeks after the 2018 vaccine crisis was revealed. The proportion of parental hesitancy toward domestically produced vaccines and overall vaccination increased from 30.6% to 82.7% and 8.3% to 52.1%, respectively, after the crisis. Parents with higher levels of trust toward crisis communication information were less likely to report vaccine hesitancy toward both domestically produced vaccines and vaccines overall after the crisis. Parents with better critical understanding of crisis information were less likely to report a vaccine hesitancy toward overall vaccine and more likely to maintain a vaccine intention. Additionally, parents with lower levels of confidence in vaccine efficacy were more likely to became vaccine hesitant but were also more likely to maintain their vaccine intentions after the crisis. It is crucial to guarantee the safety of vaccines, maintain parental confidence in vaccine efficacy, and eliminate the potential risks that result in parental vaccine hesitancy. Future crisis communication strategies are encouraged to ensure timely responses to sustain public confidence.

A DNA-functionalized biomass nanoprobe for the targeted photodynamic therapy of tumor and ratiometric fluorescence imaging-based visual cancer cell identification/antitumor drug screening.

Survivin is widely expressed in tumor tissue, in which the in situ ratiometric fluorescence imaging of intracellular survivin mRNA can provide accurate information for the diagnosis and treatment of cancers, as well as the screening of antitumor drugs. However, the development of a nanoprobe that can be used simultaneously in the diagnosis and treatment of tumors and the screening of antitumor drugs remains a challenge. In an effort to address these requirements, a multifunctional biomass nanoprobe was developed for the photodynamic therapy (PDT) of tumors as well as cancer cell identification and antitumor drug screening based on the ratiometric fluorescence imaging of intracellular survivin mRNA. This nanoprobe was assembled from near-infrared (NIR) biomass quantum dots (BQDs), single-stranded DNA and NIR dye (dylight680) labeled single-stranded DNA. The BQDs contain a large number of chlorophyll molecules, meaning that they can produce a large amount of singlet oxygen under NIR light irradiation, thus realizing the PDT of a tumor. However, the specific binding of the nanoprobe to intracellular survivin mRNA causes the release of dylight680 and reduces the fluorescence resonance energy transfer (FRET) efficiency between the BQDs and dylight680 in the probe, thereby achieving the ratiometric fluorescence imaging of survivin mRNA. Therefore, the prepared nanoprobe can not only be used in the diagnosis of cancers, but also in the targeted PDT of tumors.

Molecular characterization and functional analysis of multidrug resistance-associated genes of Pinewood nematode (Bursaphelenchus xylophilus) for nematicides.

Bursaphelenchus xylophilus (Pinewood nematode, PWN) is the causative agent of pine wilt disease (PWD) which caused serious threat to pine forests in the world, especially in East Asia and Western Europe. At present, the control of PWD mainly rely on the massive use of pesticide despite the damage to human health and environmental safety. Developing novel drug targets is the optimized strategy for developing new method to control PWN. In this study, four multidrug resistance-associated protein (MRP) genes containing highly conserved MRP-associated domains were cloned from PWN. The expression patterns of the four Bx-mrps under three different nematicides treatments were studied by quantitative reverse transcription PCR (qRT-PCR) and the function of the four genes in multidrug resistance were also validated by RNA interference (RNAi). Results showed that the expression of Bx-mrp1, Bx-mrp2, Bx-mrp3, and Bx-mrp4 were significantly increased when exposed to different nematicides, wherein, Bx-mrp4 exposed by 4.0 mg/mL of matrine own the highest expression level. The mortality rates of Bx-mrps silenced nematodes revealed significant increase(P < 0.05)under matrine, avermectin, and emamectin benzoate exposure. Specially, Bx-mrp4 exposed with 4.0 mg/mL matrine for 24 h own the highest mortality increase by 18.34%. After RNAi of Bx-mrps, feeding ability of the nematodes were also significantly decreased. These results demonstrate that Bx-mrps were linked to the detoxification process and feeding behavior of PWN. Silencing of Bx-mrps can lead to increased sensitivity of PWN to nematicides and decrease its feeding ability. Bx-mrps are potential new PWN control targets in the future.

An aptamer-based four-color fluorometic method for simultaneous determination and imaging of alpha-fetoprotein, vascular endothelial growth factor-165, carcinoembryonic antigen and human epidermal growth factor receptor 2 in living cells.

The extraordinary fluorescence quenching capability of graphene oxide (GO) was coupled to the specific recognition capability of aptamers to design a four-color fluorescent nanoprobe for multiplexed detection and imaging of tumor-associated proteins in living cells. Specifically, alpha-fetoprotein (AFP), vascular endothelial growth factor-165 (VEGF165), carcinoembryonic antigen (CEA), and human epidermal growth factor receptor 2 (HER2) were detected. Due to strong π interaction, the fluorescence of labeled aptamers is quenched by GO. Four fluorophore-labeled aptamers that bind the tumor-associated proteins were adsorbed on GO to form the four-color nanoprobe with quenched fluorescence. The nanoprobes were internalized into cells via endocytosis, where the aptamer/GO nanoprobes bind the intracellular tumor-associated proteins. The aptamer-protein complexes thus formed detach from GO, and fluorescence recovers. Each analyte has its typical color (AFP: blue; VEGF165: green; CEA: yellow; HER2: red). As a result, simultaneous detection and imaging of multiple tumor-associated proteins in living cells were achieved. This nanoprobe has a fast response and is highly specific and biocompatible. The linear ranges for AFP, VEGF165, CEA, and HER2 are 0.8 nM-160 nM, 0.5 nM-100 nM, 1.0 nM-200 nM, and 1.2 nM-240 nM, respectively. Detection limits were 0.45 nM for AFP, 0.30 nM for VEGF165, 0.62 nM for CEA, and 0.96 nM for HER2. The probe allows for a fast distinction between tumor cells and normal cells via imaging. Graphical abstract Schematic presentation of the development of a four-color fluorometic method based on aptamer and graphene oxide for simultaneous detection and imaging of alpha-fetoprotein, vascular endothelial growth factor-165, carcinoembryonic antigen and human epidermal growth factor receptor 2 in living cells.

Transcriptome-Based Analysis Reveals a Crucial Role of BxGPCR17454 in Low Temperature Response of Pine Wood Nematode (Bursaphelenchus xylophilus).

BACKGROUND: The causal agent of pine wilt disease is the pine wood nematode (PWN) (Bursaphelenchus xylophilus), whose ability to adapt different ecological niches is a crucial determinant of their invasion to colder regions. To discover the molecular mechanism of low temperature response mechanism, we attempted to study the molecular response patterns under low temperature from B. xylophilus with a comprehensive RNA sequencing analysis and validated the differentially expressed genes (DEGs) with quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatic software was utilized to isolate and identify the low-temperature-related BxGPCR genes. Transcript abundance of six low-temperature-related BxGPCR genes and function of one of the BxGPCR genes are studied by qRT-PCR and RNA interference. RESULTS: The results showed that we detected 432 DEGs through RNA sequencing between low-temperature-treated and ambient-temperature-treated groups nematodes. The transcript level of 6 low-temperature-related BxGPCR genes increased at low temperature. And, the survival rates of BxGPCR17454 silenced B. xylophilus revealed a significant decrease at low temperature. CONCLUSION: in conclusion, this transcriptome-based study revealed a crucial role of BxGPCR17454 in low temperature response process of pine wood nematode. These discoveries would assist the development of management and methods for efficient control of this devastating pine tree pest.

A fluorescent aptasensor based on single oligonucleotide-mediated isothermal quadratic amplification and graphene oxide fluorescence quenching for ultrasensitive protein detection.

In this work, we have developed a novel fluorescent aptasensor based on single oligonucleotide-mediated isothermal quadratic amplification (SOIQA) and graphene oxide (GO)-mediated fluorescence quenching for the ultrasensitive detection of proteins in a homogeneous solution. The SOIQA consists of a fluorophore-labeled aptamer hairpin probe containing T7 exonuclease (T7 Exo)-resistant 5'-protruding termini and a mismatch base at its 3'-end, DNA polymerase, T7 Exo and GO. The target analyte binds with the aptamer sequences and unfolds the fluorophore-labeled aptamer hairpin probe to form a new DNA hairpin, inducing the catalytic recycling of the target analyte (assisted by DNA polymerase) and DNA sequences (aided by T7 Exo) to achieve SOIQA, which results in the digestion of numerous fluorophore-labeled aptamer hairpin probes and the generation of a large amount of mononucleotides carrying the fluorophore. These mononucleotide products cannot be adsorbed onto the GO, leading to a dramatic increase in the fluorescence intensity for the amplified detection of the target molecules. In the absence of the target analyte, however, the SOIQA reaction is inhibited and the fluorophore-labeled aptamer hairpin probe is adsorbed onto the GO, leading to an extremely low fluorescence background signal. To test the feasibility of the SOIQA systems, a protein cancer marker, carcinoembryonic antigen (CEA) was used as the model analyte. The developed aptasensor could detect CEA with a detection limit of 28.5 fg mL-1 (∼142 aM), high specificity and a broad detection range of 6 orders of magnitude. And this one-step incubation can be completed in 60 min. In addition, the approach uses only one oligonucleotide strand, and is simple. Moreover, this SOIQA sensing method is suitable for rapid and direct quantification of proteins in complex biological samples such as clinical serum. Considering the simplicity and superior sensitivity/specificity, the developed sensing method provides a promising platform for the analysis of a variety of low-abundance biomolecules.

Low Temperature Extends the Lifespan of Bursaphelenchus xylophilus through the cGMP Pathway.

The causal agent of pine wilt disease, pine wood nematode (PWN) (Bursaphelenchus xylophilus), revealed extended lifespan at low temperature. To discover the molecular mechanism of this phenomenon, we attempted to study the molecular characterization, transcript abundance, and functions of three genes of the cyclic guanosine monophosphate (cGMP) pathway from B. xylophilus. Three cGMP pathway genes were identified from B. xylophilus. Bioinformatic software was utilized to analyze the characteristics of the three putative proteins. Function of the three genes in cold tolerance was studied with RNA interference (RNAi). The results showed that the deduced protein of Bx-DAF-11 has an adenylate and guanylate cyclase catalytic domain, indicating an ability to bind to extracellular ligands and synthesizing cGMP. Both Bx-TAX-2 and Bx-TAX-4 have cyclic nucleotide-binding domains and ion transport protein domains, illustrating that they are cGMP-gated ion channels. The transcript level of Bx-daf-11, Bx-tax-2, and Bx-tax-4 increased at low temperature. The survival rates of three gene silenced B. xylophilus revealed a significant decrease at low temperature. This study illustrated that the cGMP pathway plays a key role in low-temperature-induced lifespan extension in B. xylophilus.

Uptake Pathway, Translocation, and Isomerization of Hexabromocyclododecane Diastereoisomers by Wheat in Closed Chambers.

To study the uptake pathways of 3 main hexabromocyclododecane diastereoisomers (α-, ß-, and γ-HBCDs) in wheat, four closed chambers were designed to expose wheat to HBCDs via air and/or soil for 4 weeks. The results showed that HBCDs could be absorbed by wheat both via root from soil and via leaf from air. The Rt values (ratio of HBCDs from root-to-leaf translocation to the total accumulation in leaves) ranging from 14.4 to 29.8% suggested that acropetal translocation within wheat was limited. A negative linear relationship was found between log Rt and log Kow of the HBCD diastereoisomers (p < 0.05). The bioconcentration factors (BCFs, (µg/g wheat tissues)/(µg/g soil)) were in the order α- > ß- > γ-HBCD in wheat roots and stems, being negatively related to their Kow values. No such correlation was found in leaves, where the HBCDs came mainly from air distribution. The results of enantiomeric fractions indicated that the (-)-enantiomer of α- and γ-HBCDs and the (+)-ß-enantiomer were selectively accumulated. Furthermore, ß- and γ-HBCDs were transformed to α-HBCD in the wheat, with 0.309-4.80% and 0.920-8.40% bioisomerization efficiencies at the end of the experiment, respectively, being the highest in leaves. Additionally, no isomerization product from α-HBCD was found.