A PAM-Free One-Step Asymmetric RPA and CRISPR/Cas12b Combined Assay (OAR-CRISPR) for Rapid and Ultrasensitive DNA Detection.

Current research endeavors have focused on the combination of various isothermal nucleic acid amplification methods with CRISPR/Cas systems, aiming to establish a more sensitive and reliable molecular diagnostic approach. Nevertheless, most assays adopt a two-step procedure, complicating manual operations and heightening the risk of contamination. Efforts to amalgamate both assays into a single-step procedure have faced challenges due to their inherent incompatibility. Furthermore, the presence of the protospacer adjacent motif (PAM) motif (e.g., TTN or TTTN) in the target double-strand DNA (dsDNA) is an essential prerequisite for the activation of the Cas12-based method. This requirement imposes constraints on crRNA selection. To overcome such limitations, we have developed a novel PAM-free one-step asymmetric recombinase polymerase amplification (RPA) coupled with a CRISPR/Cas12b assay (OAR-CRISPR). This method innovatively merges asymmetric RPA, generating single-stranded DNA (ssDNA) amenable to CRISPR RNA binding without the limitations of the PAM site. Importantly, the single-strand cleavage by PAM-free crRNA does not interfere with the RPA amplification process, significantly reducing the overall detection times. The OAR-CRISPR assay demonstrates sensitivity comparable to that of qPCR but achieves results in a quarter of the time required by the latter method. Additionally, our OAR-CRISPR assay allows the naked-eye detection of as few as 60 copies/µL DNA within 8 min. This innovation marks the first integration of an asymmetric RPA into one-step CRISPR-based assays. These advancements not only support the progression of one-step CRISPR/Cas12-based detection but also open new avenues for the development of detection methods capable of targeting a wide range of DNA targets.

Novel Oridonin Analog CYD0682 Inhibits Hepatic Stellate Cell Activation via the Heat Shock Protein 90-Dependent STAT3 Pathway.

INTRODUCTION: Activated hepatic stellate cells (HSCs) are the primary effector cells in hepatic fibrosis, over depositing extracellular matrix (ECM) proteins. Our previous work found oridonin analog CYD0682 attenuates proliferation, Transforming Growth Factor ß (TGFß)-induced signaling, and ECM production in immortalized HSCs. The underlying mechanism behind these reductions is unclear. The Signal Transduction and Activator of Transcription 3 (STAT3) pathway plays a central role in HSC activation and has been found to be overexpressed in models of hepatic injury. In this study, we will examine the effect of CYD0682 on STAT3 signaling. METHODS: Immortalized human (LX-2) and rat (HSC-T6) HSC lines were treated with CYD0682 or Tanespimycin (17-AAG) with or without TGF-ß. Nuclear and cytosolic proteins were extracted. Protein expression was analyzed with Western blot. DNA binding activity was assessed with STAT3 DNA Binding ELISA. Cell viability was assessed with Alamar blue assay. RESULTS: CYD0682 treatment inhibited STAT3 phosphorylation at tyrosine 705 in a dose-dependent manner in LX-2 and HSC-T6 cells. STAT3 DNA binding activity and STAT3 regulated protein c-myc were significantly decreased by CYD0682. Notably, TGFß-induced STAT3 phosphorylation and ECM protein expression were inhibited by CYD0682. STAT3 is reported to be a Heat Shock Protein 90 (HSP90) client protein. Notably, CYD0682 attenuated the expression of endogenous STAT3 and other HSP90 client proteins FAK, IKKα, AKT and CDK9. HSP90 specific inhibitor 17-AAG suppressed endogenous and TGFß-induced STAT3 phosphorylation and ECM protein production. CONCLUSIONS: CYD0682 attenuates endogenous and TGFß-induced STAT3 activation and ECM production via an HSP90 dependent pathway in HSCs. Further study of this pathway may present new targets for therapeutic intervention in hepatic fibrosis.

A new simplified sequence-dependent loop-mediated isothermal amplification (LAMP) detection method.

Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detection method, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the 5' end of the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10 min faster than several other probe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection.

Combined impacts of microplastics and cadmium on the liver function, immune response, and intestinal microbiota of crucian carp (Carassius carassius).

Microplastics (MPs) and the heavy metal cadmium (Cd) have attracted global attention for their toxicological interactions in aquatic organisms. The purpose of this investigation was evaluating the effect of MPs (1 mg L-1) and Cd (5 mg L-1) on the liver function, immune response of crucian carp (Carassius carassius) after 96 h exposure, and intestinal microbiota after 21 days, respectively. Co-exposure to MPs and Cd significantly enhanced MP accumulation in the liver of the crucian carp compared to the accumulation with exposure to MPs alone. Co-exposure to MPs and Cd triggered notable histopathological alterations accompanied by increased hepatic cell necrosis and inflammation, and was associated with higher aspartate aminotransferase and alanine aminotransferase levels, lower superoxide dismutase and catalase activity levels, but higher malondialdehyde content and total antioxidant capacity in the liver. Moreover, the combined treatment of MPs and Cd led to the up-regulated transcription of genes related to immune response, such as interleukin 8 (il-8), il-10, il-1ß, tumor necrosis factor-α, and heat shock protein 70, both in the liver and spleen. Co-exposure to MPs and Cd reduced the variety and abundance of the intestinal microbiota in the crucian carp. Our research indicates that the combined exposure to MPs and Cd may exert synergistic toxic effects on crucian carp, which could impede the sustainable growth of the aquaculture industry and pose potential risks to food safety.

Effects of non-lethal Cry1F toxin exposure on the growth, immune response, and intestinal microbiota of silkworm (Bombyx mori).

Bt (Bacillus thuringiensis) maize is expected to be commercial cultivated widely in China. When Bt maize is planted near mulberry trees, it renders silkworms (Bombyx mori) vulnerable, as they belong to the same class as the Lepidoptera insects targeted by Bt maize. Cry1F has been found to be highly toxic to silkworms, particularly in their early larval stages. In this study, we aimed to assess the effects of non-lethal Cry1F exposure on the growth, immune response, and intestinal microbiota in silkworms. The results showed that feeding silkworms with mulberry leaves soaked in 100 µg/mL Cry1F for 96 h had an impact on larval body weight acquisition, leading to a decrease in cocoon and pupae weight. Cry1F exposure disrupted the intestinal integrity of silkworms by affecting the columnar cells of the midgut. The activity of detoxification enzymes (CarE, AChE, and GST) as well as antioxidant enzymes (SOD, CAT, and POD) were also affected by Cry1F. After 96 h Cry1F exposure, the evenness of the bacterial community was disrupted, resulting in alterations in the structure of the intestinal microbiota. Additionally, Cry1F exposure affected the relative expression levels of the peritrophic membrane (PM) protein and the corresponding immune pathways genes of silkworms. Most of the immune-related gene expressions were inhibited after exposure to Cry1F toxin but increased with prolonged treatment. This study demonstrates that non-lethal Cry1F exposure can affect the growth, immune response, and intestinal microbiota of silkworm.

Altered Gene Expression of the Parasitoid Pteromalus puparum after Entomopathogenic Fungus Beauveria bassiana Infection.

Both parasitoids and entomopathogenic fungi are becoming increasingly crucial for managing pest populations. Therefore, it is essential to carefully consider the potential impact of entomopathogenic fungi on parasitoids due to their widespread pathogenicity and the possible overlap between these biological control tools during field applications. However, despite their importance, little research has been conducted on the pathogenicity of entomopathogenic fungi on parasitoids. In our study, we aimed to address this knowledge gap by investigating the interaction between the well-known entomopathogenic fungus Beauveria bassiana, and the pupal endoparasitoid Pteromalus puparum. Our results demonstrated that the presence of B. bassiana significantly affected the survival rates of P. puparum under laboratory conditions. The pathogenicity of B. bassiana on P. puparum was dose- and time-dependent, as determined via through surface spraying or oral ingestion. RNA-Seq analysis revealed that the immune system plays a primary and crucial role in defending against B. bassiana. Notably, several upregulated differentially expressed genes (DEGs) involved in the Toll and IMD pathways, which are key components of the insect immune system, and antimicrobial peptides were rapidly induced during both the early and late stages of infection. In contrast, a majority of genes involved in the activation of prophenoloxidase and antioxidant mechanisms were downregulated. Additionally, we identified downregulated DEGs related to cuticle formation, olfactory mechanisms, and detoxification processes. In summary, our study provides valuable insights into the interactions between P. puparum and B. bassiana, shedding light on the changes in gene expression during fungal infection. These findings have significant implications for the development of more effective and sustainable strategies for pest management in agriculture.

Toll and IMD Immune Pathways Are Important Antifungal Defense Components in a Pupal Parasitoid, Pteromalus puparum.

Insects employ multifaceted strategies to combat invading fungi, with immunity being a promising mechanism. Immune pathways function in signal transduction and amplification, ultimately leading to the activation of antimicrobial peptides (AMPs). Although several studies have shown that immune pathways are responsible for defending against fungi, the roles of parasitoid immune pathways involved in antifungal responses remain unknown. In this study, we evaluated the roles of the Toll and IMD pathways of a pupal parasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae), in fighting against Beauveria bassiana (Hypocreales: Cordycipitaceae). Successful colonization of B. bassiana on P. puparum adults was confirmed by scanning electron microscopy (SEM). AMPs were induced upon B. bassiana infection. The knockdown of key genes, PpTollA and PpIMD, in Toll and IMD signaling pathways, respectively, significantly compromised insect defense against fungal infection. The knockdown of either PpTollA or PpIMD in P. puparum dramatically promoted the proliferation of B. bassiana, resulting in a decreased survival rate and downregulated expression levels of AMPs against B. bassiana compared to controls. These data indicated that PpTollA and PpIMD participate in Toll and IMD-mediated activation of antifungal responses, respectively. In summary, this study has greatly broadened our knowledge of the parasitoid antifungal immunity against fungi.

Advances in amplification-free detection of nucleic acid: CRISPR/Cas system as a powerful tool.

Amplification technologies such as polymerase chain reaction (PCR) play an important role in nucleic acid detection. However, they require bulky and sophisticated thermal cycling instrument, as well as are prone to get false-positive results due to amplicon contamination. Currently, CRISPR/Cas system has become an increasingly popular diagnostic tool for nucleic acid with the discovery of its trans-cleavage activity which can degrade single-stranded DNA or RNA at a very high turnover rate. This inherent signal amplification capability allows CRISPR/Cas system to detect unamplified nucleic acids. Here, we reviewed the recent advances of CRISPR-based amplification-free methods for nucleic acid detection. With the assistance of various signal enhancement strategies, the detection sensitivity could be comparable to that of amplification-based methods. We then presented the pros and cons of these methods. And the subsistent challenges including sample preparation, off-target effect, sequences limit, quantitative and multiplex detection were further discussed in this review. It is probable for CRISPR-powered detection methods to pave the road for rapid, cheap, highly sensitive and specific on-site detection without amplification.

A CRISPR/Cas12a-Mediated Dual-Mode Electrochemical Biosensor for Polymerase Chain Reaction-Free Detection of Genetically Modified Soybean.

A clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-mediated dual-mode electrochemical biosensor without polymerase chain reaction (PCR) amplification was designed for sensitive and reliable detection of genetically modified soybean SHZD32-1. A functionalized composite bionanomaterial Fe3O4@AuNPs/DNA-Fc&Ru was synthesized as the signal unit, while a characteristic gene fragment of SHZD32-1 was chosen as the target DNA (tDNA). When Cas12a, crRNA, and tDNA were present simultaneously, a ternary complex Cas12a-crRNA-tDNA was formed, and the nonspecific cleavage ability of the CRISPR/Cas12a system toward single-stranded DNA was activated. Thus, the single-stranded DNA-Fc in the signal unit was cleaved, resulting in the decrease in the fast scan voltammetric (FSV) signal from ferrocene (Fc) and the increase in the electrochemiluminescence (ECL) signal from ruthenium complex (Ru) inhibited by Fc. The linear range was 1-107 fmol/L for ECL and 10-108 fmol/L for FSV, and the limit of detection (LOD) was 0.3 fmol/L for ECL and 3 fmol/L for FSV. Accuracy, precision, stability, selectivity, and reliability were all satisfied. In addition, PCR-free detection could be completed in an hour at room temperature without requiring complicated operation and sample processing, showing great potential in the field detection of genetically modified crops.

HJC0416 Attenuates Fibrogenesis in Activated Hepatic Stellate Cells via STAT3 and NF-κB Pathways.

BACKGROUND: Hepatic fibrosis is wound-healing response that is the result of hepatic stellate cell (HSC) activation and subsequent excess extracellular matrix deposition. HSCs can be activated by a variety of inflammatory stimuli as well as through the signal transducer and activator of transcription 3 (STAT3) pathway. HJC0416 is a novel, orally bioavailable small-molecule inhibitor of STAT3 that was developed by our team using a fragment-based drug design approach. Previously, our team has shown that HJC0416 has antifibrogenic effects in activated HSCs. Recently, increasing evidence suggests that nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays an important role in the activation of HSCs. In the present study, we examined the role of NF-κB inhibition of HSC activation by HJC0416. METHODS: LX-2 (human) and HSC-T6 (rat) cell lines were used. Expression levels of extracellular proteins, NF-κB and STAT3 expression and DNA binding, and inflammatory cytokine levels were determined using western blot, ELISA, and immunofluorescence assay. RESULTS: HJC0416 decreased cell viability in a dose-dependent manner in both cell lines and arrested the cell cycle at the S phase. Increased apoptosis was seen in LX-2 cells through Yo-Pro-1 and propidium iodide immunofluorescent stating. HJC0416 significantly decreased expression of fibronectin and collagen I as well as markedly decreased α-SMA and laminin. HJC0416 inhibited the STAT3 pathway by decreasing phosphorylation of STAT3, as well as signal transduction pathway activation. Notably, HJC0416 also inhibited the classic and alternative pathways of NF-κB activation. HJC0416 inhibited LPS-induced p65 nuclear translocation and DNA binding, as well as prevented phosphorylation and degradation of inhibitory protein IκBα. HJC0416 also prevented phosphorylation of serine residue 536 on p65. CONCLUSIONS: HJC0416, an inhibitor of STAT3, was found to have antifibrogenic properties in activated hepatic stellate cell lines. In addition, HJC0416 was found to inhibit the NF-κB pathway. Owing to this double effect, HJC0416 demonstrates promise for in vivo experimentation as an antifibrosis treatment.

[Establishment of a field visual detection method for genetically modified maize 'Shuangkang'12-5 by fluorescence RPA].

The genetically modified (GM) maize 'Shuangkang'12-5 has good insect resistance and herbicide tolerance, which is one of the first series of GM maizes obtained a safety certificate in China, and it has broad application prospect in the future. This study established an on-site rapid detection method for GM 'Shuangkang'12-5 based on recombinase polymerase amplification (RPA) technology, which primes and probe were designed according to the specific flank sequence. Then the best combination of primers and probe was obtained through a screeing process. The amplification results of fluorescence RPA can be directly visualized under blue light. The results showed that the visual detection system of GM 'Shuangkang'12-5 with high specificity, and the detection sensitivity of the method could reached 10 copies. Further research found that the RPA amplification system had a wide range of temperature (34℃-46℃). According to this property, the common self-heating warm pastes on the market were used replace the traditional heating instruments to stimulate the RPA.The results showed that the self-heating warm paste meets the temperature requirement of the RPA system. Finally, we combined the self-heating warm pastes with the RPA visual detection system to conduct on-site detection of GM 'Shuangkang'12-5, and compared the results with the detection results of qPCR. The detection showed that the results of on-site visual detection method established in this study were consistent with the detection results of the qPCR. Moreover, the visual detection method was more shorter in time and the final detection result was clear and easy to distinguish. The rapid on-site visual detection method for GM 'Shuangkang' 12-5 established in this study has high specificity, high sensitivity and convenience. It not only meets the needs of on-site rapid detection of GM 'Shuangkang'12-5, but also provides highlight for the development of other on-site rapid detection methods.

High-throughput detection and screening of plants modified by gene editing using quantitative real-time polymerase chain reaction.

Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene-edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high-throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high-throughput quantitative real-time (qPCR)-based method. The qPCR-based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild-type and a gene-edited mutant. We showed that the qPCR-based method can accurately distinguish CRISPR/Cas9-induced mutants from the wild-type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR-based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T0 transgenic plants. In a 384-well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post-polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T0 transgenic plants, which will be widely used in the area of plant gene editing.

Antifibrosis Effect of Novel Oridonin Analog CYD0618 Via Suppression of the NF-κB Pathway.

BACKGROUND: Liver fibrosis is characterized as excessive deposition of the extracellular matrix proteins, primarily by activated hepatic stellate cells (HSCs). NF-κB has been reported as one of the major mediators of HSC activation. Previously, our team reported that oridonin exhibited antihepatic fibrogenetic activity in vitro. In this study, we examined the effects of its novel derivative CYD0618 on HSC viability, apoptosis, and NF-κB signaling. METHODS: Cell proliferation of activated human and rat HSC lines LX-2 and HSC-T6 was measured using Alamar Blue Assay. Apoptosis was measured by a Cell Death Detection ELISA kit. Cellular proteins were determined by Western blots and immunofluorescence. RESULTS: CYD0618 significantly inhibited LX-2 and HSC-T6 cell proliferation in a dose-dependent manner. CYD0618 induced cell apoptosis in both cell lines. CYD0618 treatment increased cell cycle inhibitory protein p21, p27, and induced apoptosis marker cleaved poly (ADP-ribose) polymerase, while suppressing the expression of Collagen type 1. CYD0618 blocked lipopolysaccharide (LPS)-induced NF-κB p65 nuclear translocation and DNA binding activity and prevented LPS-induced NF-κB inhibitory protein IκBα phosphorylation and degradation. LPS-stimulated NF-κB downstream target cytokines IL-6 and MCP-1 were attenuated by CYD0618. Endogenous and LPS-stimulated NF-κB p65 S536 phosphorylation was inhibited by CYD0618 treatment. CONCLUSIONS: The potent antihepatic fibrogenetic effect of CYD0618 may be mediated via suppression of the NF-κB pathway.

Luteolin-Mediated Inhibition of Hepatic Stellate Cell Activation via Suppression of the STAT3 Pathway.

Hepatic stellate cell (HSC) activation is responsible for hepatic fibrogenesis and is associated with an overexpression of transcription 3 (STAT3). Luteolin, a common dietary flavonoid with potent anti-inflammatory properties, has previously demonstrated antifibrogenic properties in HSCs but the mechanism has not been fully elucidated. Activated human and rat hepatic stellate cell lines LX-2 and HSC-T6 were used to study the effects of luteolin on HSCs. Cellular proteins were determined by western blot and immunofluorescence. Cell proliferation was assessed with Alamar Blue assay. Luteolin significantly decreased LX-2 and HSC-T6 cell viability in a time-and-dose-dependent manner, as well as decreased HSC end-products α-smooth muscle actin (α-SMA), collagen I, and fibronectin. Luteolin decreased levels of total and phosphorylated STAT3, suppressed STAT3 nuclear translocation and transcriptional activity, and attenuated expression of STAT3-regulated proteins c-myc and cyclin D1. STAT3 specific inhibitors stattic and SH-4-54 demonstrated similar effects on HSC viability and α-SMA production. In LX-2 and HSC-T6 cells, luteolin demonstrates a potent ability to inhibit hepatic fibrogenesis via suppression of the STAT3 pathway. These results further elucidate the mechanism of luteolin as well as the effect of the STAT3 pathway on HSC activation.

Use of a novel metal indicator to judge loop-mediated isothermal amplification for detecting the 35S promoter.

Loop-mediated isothermal amplification (LAMP) is a widely used isothermal nucleic acid amplification method. Here we developed a new closed-tube colorimetric method for judging LAMP with a novel metal indicator. First, the metal indicator, acid chrome blue K (ACBK), was evaluated in the LAMP reaction with various combinations of reaction reagents, such as reaction buffer, dNTP mixtures, primer mixtures, or Mg2+. We found that the solution color of the LAMP reaction with ACBK changed from red to blue based on a decrease in the Mg2+ concentration in the reaction solution. We then optimized the LAMP with ACBK method for detecting the Cauliflower Mosaic Virus 35S promoter. Further, the specificity of the new colorimetric assay using ACBK in the LAMP reaction for detecting the 35S promoter was tested with diverse transgenic events in different crops, and the sensitivity threshold of the assay was ∼50 copies for transgenic rice genomic DNA and 100 ng of 0.1 % DNA from rice, soybean, rapeseed, and maize. Finally, the applicability of the LAMP assay was successfully validated using practical maize samples. All the detection results could be easily discerned either by UV-vis spectroscopy or the naked eye. Graphical Abstract The visual detect LAMP amplification by the addition of ACBK as a signal indicator. The color of the LAMP-ACBK solution turned from red to blue as the concentration of free Mg2+ decreases. The detection results could be easily discerned either by UV-vis spectroscopy or the naked eye.

Targeting the middle region of CP4-EPSPS protein for its traceability in highly processed soy-related products.

Transgenic components in genetically modified organisms consist not only of the transgenic genes, but also the transgenic protein. However, compared with transgenic DNA, less attention has been paid to the detection of expressed protein, especially those degraded from genetically modified soybean after food processing. In this study, the full length 5-enolpyruvyl-shikimate-3-phosphate synthase (CP4-EPSPS, 47.6 kD) protein was probed with the SC-16 (S19-R33) and the DC-16 (D219-K233) polyclonal antibodies in immunoblots. Both antibodies were able to detect the full length CP4-EPSPS and its residues in soy powder made from Roundup-Ready soybeans after heating and microwaving treatments which also reduced the molecular weight of the protein to 45.8 and 38.7 kD, respectively. Taken together the immunoblot results suggest that the middle region of the CP4-EPSPS protein possessed better stability than its N-terminal during thermal processing. This deduction was further validated by autoclave treatment, where a 37.4 kD residue of the protein was recognized by DC-16. A similar result was obtained in processed smoked sausage containing Roundup Ready soybean protein isolate (as an extender). The additional use of a further polyclonal antibody CK-17 (C372-K388), showed that compared with only the one signal for CP4-EPSPS detected by the SC-16 and CK-17 antibodies, the DC-16 middle region antibody detected four signals for CP4-EPSPS from five market sourced soy protein concentrates. Taken together, the study suggested that the middle region of CP4-EPSPS was more useful than the N- and C-terminal for tracing transgenic CP4-EPSPS protein and its remnants in highly processed soy-related products.

Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize.

This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.

A powerless on-the-spot detection protocol for transgenic crops within 30 min, from leaf sampling up to results.

The requirement of power-dependent instruments or excessive operation time usually restricts current nucleic acid amplification methods from being used for detection of transgenic crops in the field. In this paper, an easy and rapid detection method which requires no electricity supply has been developed. The time-consuming process of nucleic acid purification is omitted in this method. DNA solution obtained from leaves with 0.5 M sodium hydroxide (NaOH) can be used for loop-mediated isothermal amplification (LAMP) only after simple dilution. Traditional instruments like a polymerase chain reaction (PCR) amplifier and water bath used for DNA amplification are abandoned. Three kinds of dewar flasks were tested and it turned out that the common dewar flask was the best. Combined with visual detection of LAMP amplicons by phosphate (Pi)-induced coloration reaction, the whole process of detection of transgenic crops via genetically pure material (leaf material of one plant) could be accomplished within 30 min. The feasibility of this method was also verified by analysis of practical samples.

Enhanced anti-fibrogenic effects of novel oridonin derivative CYD0692 in hepatic stellate cells.

Oridonin, isolated from Rabdosia rubescens, has been proven to possess various anti-neoplastic and anti-inflammatory properties. Previously, we reported the anti-fibrogenic effects of oridonin for liver in vitro. In the present study, we investigated the effects of a newly designed analog CYD0692 in vitro. Cell viability was measured by Alamar Blue assay. Cell apoptosis was assessed by Cell Death ELISA and Yo-Pro-1 staining. Western blots were performed for cellular proteins. Flow cytometry was used to measure cell cycle regulation. CYD0692 significantly inhibited LX-2 cells proliferation in a dose- and time-dependent manner with an IC50 value of ~0.7 µM for 48 h, ~tenfold greater potency than oridonin. Similar results were observed in HSC-T6 cells. In contrast, on the human hepatocyte cell line C3A, only 12 % of the cell growth was inhibited with 5 µM of CYD0692 treatment for 48 h, while 30 % inhibited at 10 µM. After CYD0692 treatment on LX-2 cells, apoptosis and S-phase cell cycle arrest were induced; cleaved-PARP, p21, and p53 were activated while cyclin-B1 levels declined. In addition, α-smooth muscle actin, type I Collagen, and fibronectin (FN) were markedly down regulated. Transforming growth factor ß1 (TGF ß1) has been identified as a dominant stimulator for ECM production in HSC. Our results indicated that pretreatment with CYD0692 blocked TGF ß1-induced FN expression, thereby decreasing the downstream factors of TGF ß1 signaling, such as Phospho-Smad2/3 and phospho-ERK. In comparison with oridonin, its novel derivative CYD0692 has demonstrated to be a more potent and potentially safer anti-fibrogenic agent for the treatment of hepatic fibrosis.

Enhanced effects of novel oridonin analog CYD0682 for hepatic fibrosis.

BACKGROUND: Activated hepatic stellate cells (HSCs) are responsible for excess extracellular matrix (ECM) protein deposition in liver fibrosis. Previously, our group reported that the natural compound oridonin induces apoptosis, inhibits cell proliferation, and downregulates ECM proteins in activated HSC. In this study, the antifibrogenic effects of oridonin derivative CYD0682 on the activated human LX-2 and rat HSC-T6 stellate cell lines were investigated. METHODS: Cell proliferation was measured by alamarBlue assay. Apoptosis was detected by Cell Death ELISA and staining of Yo-Pro-1 and propidium iodide. Cell cycle was determined by flow cytometry. Immunoblot and immunofluorescence staining were performed for cellular protein expression. RESULTS: CYD0682 treatment significantly inhibited LX-2 cell proliferation in a dose- and time-dependent manner with an IC50 value of 0.49 µM for 48 h, ∼10-fold greater potency than oridonin. Similar results were observed in HSC-T6 cells. In contrast, 2.5 µM of CYD0682 treatment had no significant effects on proliferation of the human hepatocyte cell line C3A. CYD0682 treatment induced LX-2 cell apoptosis and S-phase cell cycle arrest and was associated with activation of p53, p21, and cleaved caspase-3. The myofibroblast marker protein α-smooth muscle actin and major ECM proteins type I collagen and fibronectin were markedly suppressed in a time- and dose-dependent fashion by CYD0682. Furthermore, pretreatment with CYD0682 blocked transforming growth factor-ß-induced type I collagen and fibronectin production. CONCLUSIONS: In comparison with oridonin, its novel derivative CYD0682 may act as a more potent antihepatic fibrosis agent.