Fluorescence Quantitative RT-PCR Research Articles

Attenuated Salmonella typhimurium L forms suppress tumor growth and promote apoptosis in murine ovarian tumors

To study the effects of attenuated Salmonella typhimurium L forms on the in vivo tumorigenicity and apoptosis of murine epithelial ovarian cancer cells, as well as the related mechanisms. Attenuated Salmonella typhimurium VNP20009 was induced into bacterial L forms by using antibiotic ceftriaxone. CCK-8 cell proliferation assay showed that attenuated S. typhimurium L forms can inhibit the proliferation of murine ovarian epithelial cancer ID8 cells. Attenuated ST L forms can induce apoptosis and inhibit invasion ability of epithelial ovarian cancer cells in vitro. TUNEL assay showed that attenuated ST L forms can induce apoptosis of ID8 cells in murine ovarian tumors. Meanwhile, attenuated ST L forms inhibit tumor growth in murine ovarian tumors. The tumorigenicity-related proteins of xenograft tumors detected by immunohistochemistry and fluorescence quantitative RT-PCR assays showed that attenuated ST L forms can reduce the expression of proteins that promote tumor growth and metastasis, such as Lgals9 and MMP9. This study confirmed that attenuated ST L forms can suppress tumor growth and promote apoptosis in murine ovarian tumors. Attenuated ST L forms may serve as a novel biological agent for bacterial-mediated tumor therapy in epithelial ovarian cancer.

Establishment and application of quadruple fluorescence quantitative RT-PCR method for the identification of waterfowl astrovirus.

Avian astrovirus can infect a variety of poultry species and cause viral diarrhea, with a wide epidemic range strong pathogenicity and a high incidence. Among them, Duck astrovirus 3(DAstV-3), Duck astrovirus 4(DAstV-4), Goose astrovirus 1(GoAstV-1) and Goose astrovirus 2(GoAstV-2) are four types of astroviruses newly discovered in waterfowl in recent years. In order to realize the rapid detection of these four kinds of waterfowl stellate viruses, specific primers and probes were engineered to target a highly conserved region of ORF1b gene of DAstV-3, GoAstV-1 and GoAstV-2 and the ORF2 gene of DAstV-4, and a quadruple fluorescence quantitative RT-PCR method was developed. The results indicate that the method established in this study has good specificity and no cross reactivity with other pathogens. This method can detect viruses with a minimum concentration of 1 × 101 copies/μL for DAstV-4, GoAstV-1 and GoAstV-2, respectively, while the minimum concentration for DAstV-3 is 1 × 102 copies/μL. Compared with the routinely used RT-PCR method, the limit of detection by the multiplex RT-PCR lower. Both intra- and inter-assay variability tests revealed excellent reproducibility. This method was then used to analyze 269 field samples, and the results were verified by genome sequencing. In conclusion, this study presents a sensitive, accurate, and specific method for detecting DAstV-3, DAstV-4, GoAstV-1, and GoAstV-2 in a single reaction, enabling the monitoring and differential diagnosis of these four types of waterfowl astroviruses.

The Physiological Response Mechanism of Peanut Leaves under Al Stress.

Aluminum (Al) toxicity in acidic soils can significantly reduce peanut yield. The physiological response of peanut leaves to Al poisoning stress still has not been fully explored. This research examined the influences of Al toxicity on peanut leaves by observing the leaf phenotype, scanning the leaf area and perimeter, and by measuring photosynthetic pigment content, physiological response indices, leaf hormone levels, and mineral element accumulation. Fluorescence quantitative RT-PCR (qPCR) was utilized to determine the relative transcript level of specific genes. The results indicated that Al toxicity hindered peanut leaf development, reducing their biomass, surface area, and perimeter, although the decrease in photosynthetic pigment content was minimal. Al toxicity notably affected the activity of antioxidative enzymes, proline content, and MDA (malondialdehyde) levels in the leaves. Additionally, Al poisoning resulted in the increased accumulation of iron (Fe), potassium (K), and Al in peanut leaves but reduced the levels of calcium (Ca), manganese (Mn), copper (Cu), zinc (Zn), and magnesium (Mg). There were significant changes in the content of hormones and the expression level of genes connected with hormones in peanut leaves. High Al concentrations may activate cellular defense mechanisms, enhancing antioxidative activity to mitigate excess reactive oxygen species (ROS) and affecting hormone-related gene expression, which may impede leaf biomass and development. This research aimed to elucidate the physiological response mechanisms of peanut leaves to Al poisoning stress, providing insights for breeding new varieties resistant to Al poisoning.

Differential expression of microRNAs in serum exosomes of obese and non-obese mice and analysis of their function

ObjectiveTo extract exosomes from obese and non-obese mice, screen specifically expressed microRNAs by high-throughput sequencing and explore their roles. MethodsAn animal obesity model was constructed, and the successful construction of the obesity model was verified by HE staining, Western Blot and RT-qPCR. In addition, exosomes were extracted and verified by Western Blot. High-throughput sequencing was performed on the extracted serum exosomes to screen for differentially expressed microRNAs. fluorescence quantitative RT-PCR (RT-qPCR) was used to validate the differentially expressed miRNAs and explore their functions. Results8 microRNAs were up-regulated and 11 microRNAs were down-regulated. mmu-miR-674-5p and X_28316 were significantly down-regulated and had the greatest impact on protein pathways. 8_13258 was significantly up-regulated and affected multiple protein pathways. GO enrichment analysis suggested that the differentially expressed microRNAs were mainly involved in the cleavage of microtubule activity, transferase activity/transferase pentameric acid. GO enrichment analysis suggested that differentially expressed microRNAs were mainly involved in the processes of cleavage microtubule activity, transferase activity/transfer pentamer, and threonine phosphatase/threonine kinase activity.KEGG pathway enrichment analysis showed that differentially expressed microRNAs were mainly involved in the processes of regulating the phosphorylation of TP53 activity, the G2/M DNA damage checkpoint, and the processing of the ends of DNA double-strand breaks. Protein interaction networks were enriched for Stat3, Fgr, Camk2b, Rac1, Asb6, and Ankfy1. Suggesting that they may be mediated by differential genes to participate in the process of insulin resistance. qRT-PCR results showed that the expression trend of mmu-miR-674-5p was consistent with the sequencing results. It suggests that it may be able to participate in the regulation of insulin resistance as a target gene. ConclusionmicroRNAs were differentially expressed in serum exosomes of obese and non-obese mice and might be involved in the specific regulation of insulin resistance. mmu-miR-674-5p was differentially expressed significantly and the validation trend was consistent with it, suggesting that it might be able to participate in the regulation of insulin resistance as a target gene.

Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus.

A sensitive and specific multiplex fluorescence rapid detection method was established for simultaneous detection of SARS-CoV-2, influenza A virus, and influenza B virus in a self-made device within 30 min, with a minimum detection limit of 200 copies/mL. Based on the genome sequences of SARS-CoV-2, influenza A virus (FluA), and influenza B virus (FluB) with reference to the Chinese Center for Disease Control and Prevention and related literature, specific primers were designed, and a multiplex fluorescent PCR system was established. The simultaneous and rapid detection of SARS-CoV-2, FluA, and FluB was achieved by optimizing the concentrations of Taq DNA polymerase as well as primers, probes, and Mg2+. The minimum detection limits of the nucleic acid rapid detection system for SARS-CoV-2, FluA, and FluB were evaluated. By optimizing the amplification system, the N enzyme with the best amplification performance was selected, and the optimal concentration of Mg2+ in the multiamplification system was 3 mmol/L; the final concentrations of SARS-CoV-2 NP probe and primer were 0.15 μmol/L and 0.2 μmol/L, respectively; the final concentrations of SARS-CoV-2 ORF probe and primer were both 0.15 μmol/L; the final concentrations of FluA probe and primer were 0.2 μmol/L and 0.3 μmol/L, respectively; the final concentrations of FluB probe and primer were 0.15 μmol/L and 0.25 μmol/L, respectively. A multiplex real-time quantitative fluorescence RT-PCR system for three respiratory viruses of SARS-CoV-2, FluA, and FluB was established with a high amplification efficiency and sensitivity reaching 200 copies/mL for all samples. Combined with the automated microfluidic nucleic acid detection system, the system can achieve rapid detection in 30 minutes.

Expression of lymphocyte activation gene-3 on CD4+T cells is regulated by cytokine interleukin-18 in myasthenia gravis

Myasthenia gravis (MG) is a T cell-dependent, B cell-mediated, and complement-dependent autoimmune disease. Lymphocyte activation gene-3 (LAG-3; CD223) is an immune checkpoint protein that plays an important role in maintaining autoimmune tolerance and homeostasis. To investigate the cytokine-regulated expression pattern of LAG-3, CD4+T cells were sorted from the peripheral blood of healthy volunteers by density gradient centrifugation and stimulated with various cytokines in vitro. The expression of membrane LAG-3 (mLAG-3), membrane a disintegrin and metallopeptidase domain10 (mADAM10) and membrane ADAM17 (mADAM17) on CD4+T cells was detected by flow cytometry; the concentration of soluble LAG-3 (sLAG-3) was detected by ELISA; and the relative expression of genes at the transcriptional level was detected by fluorescence quantitative RT-PCR (qRT-PCR). sLAG-3 levels were significantly increased in the peripheral plasma of AChR Ab-positive patients with MG compared to healthy volunteers, while the percentage of mLAG-3 expression on CD4+T lymphocytes in the peripheral blood of patients with MG was significantly reduced. IL-18 inhibited mLAG-3 levels on CD4+T cells in a concentration-dependent manner. Additionally, the concentration of sLAG-3 in the supernatant increased. After PHA and IL-18 stimulation, ADAM10 and ADAM17 also increased compared to those in the PHA-active group. Moreover, there were significant differences in the expression of mADAM10 and mADAM17 in CD4+T lymphocytes between patients with MG and healthy volunteers. These results suggest that IL-18 may regulate the expression pattern of mLAG-3 in CD4+T cells and sLAG-3 via ADAM10- and ADAM17-mediated pathways, thus affecting the immune effects of CD4+T cells. This study provides a preliminary exploration of the upstream regulatory molecules of the LAG-3 and IL-18/LAG-3 signalling pathways for potential targeted therapy of autoimmune diseases in the future.

Sensitivity, Specificity, Positive Rate and Consistency of Two Kits in SARS-CoV-2 Nucleic Acid Detection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was a highly transmissible and pathogenic coronavirus and it emerged in late 2019. SARS-CoV-2 had caused a pandemic of acute respiratory disease and its name was coronavirus disease 2019 (COVID-19). It threatened human health and public safety. This study would analyze and evaluate two different kinds of SARS-CoV-2 nucleic acid detection reagents which could provide value for accurate detection. 80 patients were randomly selected in the First Affiliated Hospital of Anhui Medical University in December 2022 and 80 oropharyngeal swabs were collected. Nucleic acid was extracted first, and then two real-time fluorescent quantitative RT-PCR nucleic acid amplification reagents were used to detect ORF1ab and N genes. Statistical software was used to compare and analyze the results. Among 80 patients, 57 were males and 23 were females with an age range of 5 - 90 years with an average age of 65.7 years. Most of the specimens were collected from Department of Infectious Diseases and Department of Respiratory and Critical care. Compared with regent Reference, the sensitivity of regent A and B was 88.3% and 93.3% and the specificity was 90.0% and 95.0%, respectively. The positive rates of the double target genes were 85.0% and 93.3%, the positive rates of ORF1ab gene were 86.7% and 95.0% and the positive rates of N gene were 88.3% and 96.7%, respectively. The Kappa values were all greater than 0.75, indicating high consistency. Between two nucleic acid detection reagents, reagent B had higher sensitivity and specificity. And the positive rate and consistency of reagent B were also higher than that of reagent A, with statistical significance. For weakly positive specimens with low viral load, it was recommended to use another reagent with higher sensitivity to retest and ensure the accuracy and repeatability of the results.

Nasal polyps show decreased mucociliary transport despite vigorous ciliary beating

ObjectiveMucociliary transport function in the airway mucosa is essential for maintaining a clean mucosal surface. This function is impaired in upper and lower airway diseases. Nasal polyps are a noticeable pathological feature that develop in some of the patients with chronic rhinosinusitis. Like ordinary nasal mucosae, nasal polyps have a ciliated pseudostratified epithelium with vigorous ciliary beating. We measured ex vivo Mucociliary Transport Velocity (MCTV) and Ciliary Beat Frequency (CBF) and explored the expressions of Planar Cell Polarity (PCP) proteins in nasal polyps in comparison with turbinate mucosae. MethodsInferior turbinates and nasal polyps were surgically collected from patients with chronic rhinosinusitis. Ex vivo MCTV and CBF were measured using a high-speed digital imaging system. Expressions of PCP proteins were explored by fluorescence immunohistochemistry and quantitative RT-PCR. ResultsThe MCTV of nasal polyps was significantly lower than that of the turbinates (7.43 ± 2.01 vs. 14.56 ± 2.09 μm/s; p = 0.0361), whereas CBF did not differ between the two tissues. The MCTV vector was pointed to the posteroinferior direction in all turbinates with an average inclination angle of 41.0 degrees. Immunohistochemical expressions of Dishevelled-1, Dishevelled-3, Frizzled3, Frizzled6, Prickle2 and Vangl2 were lower in the nasal polyps than in the turbinates. Confocal laser scanning microscopy showed that Frizzled3 was localized along the cell junction on the apical surface. The expression levels of mRNAs for Dishevelled-1, Dishevelled-3 and Frizzled3 in the nasal polyps were also decreased in comparison with the turbinates. ConclusionThese results indicate that muco ciliary transport in nasal polyps is impaired although vigorous ciliary beating is maintained, and that the impairment may be caused by a decrease in Dishevelled/Frizzled proteins and resultant PCP disarrangement. Level of evidenceLevel 3.

KLF14 activates the JNK-signaling pathway to induce S-phase arrest in cervical cancer cells.

To explore the role of Krüppel-like factor 14 (KLF14) and its underlying mechanism(s) of action in cell-cycle regulation in cervical cancer. Lentiviral infection was used to construct KLF14, KLF14 zinc-finger structural mutations, and empty vector controls in SiHa and HeLa cervical cancer cells. The effect of KLF14 on cervical cancer cell cycle was detected by flow cytometry. The effect of KLF14 on the expression of cyclin-dependent kinase 2 (CDK2), cyclin A2 (CCNA2), and MAPK signalling pathway-related molecules was detected by fluorescence quantitative RT-PCR (qRT-PCR) and western blot. Cervical cancer cells were treated with JNK-pathway inhibitors/agonists before we assessed changes in the cell cycle and the expression of the CDK2, CCNA2, and p-JNK/JNK. Subcutaneous xenograft studies to explore the effects of KLF14 on cervical cancer cell proliferation in vivo, and western blotting was implemented to measure the expression of CCNA2, CDK2, and the activation levels of the MAPK-signaling pathway proteins in tumours. The proportion of cells in the S phase was increased in the KLF14-overexpressing group compared with the control group (P<0.001); CDK2, CCNA2, and p-JNK/JNK expression levels were elevated in the KLF14-overexpressing group relative to the control group (all P<0.05). When JNK-pathway activation was inhibited/promoted, the proportion of cells in the S phase was reduced/increased (P<0.05) and CDK2 and CCNA2 expression levels were reduced/decreased, respectively (all P<0.05). Vivo experiments revealed that KLF14 inhibited cervical cancer cell proliferation (P<0.01) and that p-JNK/JNK, CDK2, and CCNA2 expression levels were augmented in tumours in the overexpression group (P<0.01). KLF14 induced S-phase arrest in cervical cancer cells and inhibited the proliferation of cervical cancer cells in vivo; the induction of S-phase arrest was related to its zinc-finger structure. KLF14 also activated the JNK pathway to induce S-phase arrest and promote the expression of CDK2 and CCNA2. In summary, KLF14 activates the JNK-signaling pathway to induce S-phase arrest in cervical cancer cells.

Characterization of the G-quadruplexes in the transthyretin gene and its role in silencing transthyretin mRNA transcription

Transthyretin Amyloidosis arises from the misfolding of monomers or oligomers of the normal transthyretin protein. Our investigation revealed that certain guanine-rich regions within the 5′ UTR sequence of the transthyretin gene possess the ability to form G2-quadruplex structures, as determined through analysis with QGRS mapper. We demonstrated that small molecule ligands, including TMPyP4, Braco-19, NMM, and TO, have a significant impact on the stabilization of transthyretin G-quadruplexes. The objective of this study was to confirm the effect of ligands on transthyretin gene transcription through the stabilization of G-quadruplexes. To comprehend the interaction between ligands and transthyretin G-quadruplexes, a range of analytical techniques were employed, includingUV titration, fluorescence titration assays, circular dichroism, quantitative RT-PCR and cytotoxicity tests. The results revealed the presence of four putative G2-quadruplex sequences, which formed stable anti-parallel, parallel, and hybrid G2-quadruplex structures. Notably, Ttrg 3 (5′-GGAAGGAAGGGAGGGAGGG-3′) exhibited the highest stability to form G-quadruplex. Furthermore, TmPyP4, Braco-19, NMM and TO were found to stabilize the parallel topology of Ttrg 3. After 48 h of incubation, the RT-PCR experiments revealed a significant reduction in transthyretin mRNA transcription in HepG2 cells when treated with 20 μM TmPyP4 and Braco-19, without inducing apoptosis. Our findings suggested that ligand-mediated stabilization of G-quadruplexes within the 5′-UTR can effectively silence transthyretin expression, highlighting the potential of G-quadruplex as a novel therapeutic target for Transthyretin Amyloidosis. This study might shed valuable lights for the development of innovative therapeutic approach against Transthyretin Amyloidosis.

Effect of Neurokinin-1 Receptor Knockdown on the Expression of RANTES in Allergic Rhinitis.

Neurokinin-1 receptor (NK-1R) and normal T cell expressed and secreted (RANTES) have been shown to play important roles in allergic rhinitis (AR). However, whether the regulating effect of NK-1R in AR is achieved via RANTES remains unknown. In the present study, Sprague-Dawley rats were sensitized and challenged with ovalbumin to make AR models. During the challenge period, the rats were treated intranasally with NK-1R-specific small interfering RNA (siRNA) for NKR group, negative siRNA for NCS group, rats in NSAR group and NS group were given saline. The amount of nasal secretion and the numbers of nose rubs and sneezes were measured in each rat. The levels of NK-1R and RANTES in the nasal mucosal tissues were determined through real-time fluorescence quantitative RT-PCR and immunohistochemical staining. The numbers of eosinophils in the collected nasal lavage fluid (NLF) were counted, and the concentration of RANTES in NLF was determined by enzyme-linked immunosorbent assay. Compared with that in the NS group, the expression of NK-1R and RANTES was significantly higher in the nasal mucosa of NSAR and NCS group rats. The sneezing and nose rubbing counts and the amount of nasal secretions were increased significantly in the NSAR and NCS groups. Rats in the NKR group experienced greater relief from AR symptoms than rats in the NSAR and NCS groups. Furthermore, knockdown of NK-1R expression also significantly eliminated RANTES expression and eosinophil infiltration in the nasal mucosa of NKR group rats. For the first time, we show that intranasal treatment with NK-1R-specific siRNA can significantly decrease RANTES expression, AR-related symptoms, and eosinophil inflammation, suggesting that the regulating effect of NK-1R in the development of AR occurs via alteration of RANTES expression.

Transcriptome-Wide Integrated Analysis of the PgGT25-04 Gene in Controlling Ginsenoside Biosynthesis in Panax ginseng.

Panax ginseng is a valuable medicinal herb of the Araliaceae family with various pharmacological activities. The Trihelix transcription factors family is involved in growth and secondary metabolic processes in plants, but no studies have been reported on the involvement of Trihelix genes in secondary metabolic processes in ginseng. In this study, weighted co-expression network analysis, correlation analysis between PgGTs and ginsenosides and key enzyme genes, and interaction network analysis between PgGTs and key enzyme genes were used to screen out the PgGT25-04 gene, which was negatively correlated with ginsenoside synthesis. Using ABA treatment of ginseng hair roots, PgGT genes were found to respond to ABA signals. Analysis of the sequence characteristics and expression pattern of the PgGT25-04 gene in ginseng revealed that its expression is spatiotemporally specific. The interfering vector pBI121-PgGT25-04 containing the PgGT25-04 gene was constructed, and the ginseng adventitious roots were transformed using the Agrobacterium-mediated method to obtain the pBI121-PgGT25-04 positive hairy root monocot line. The saponin contents of positive ginseng hair roots were measured by HPLC, and the changes in PgGT25-04 and key enzyme genes in positive ginseng hair roots were detected via fluorescence quantitative RT-PCR. These results preliminarily identified the role of the PgGT25-04 gene in the secondary metabolism of ginseng in Jilin to provide a theoretical basis for the study of Trihelix transcription factors in Panax ginseng.

Soybean balanced the growth and defense in response to SMV infection under different light intensities.

Light is essential for the growth and defense of soybean. It is not clear how soybeans adjust their defenses to different light environments with different cropping patterns. The mechanism of soybean response to Soybean mosaic virus (SMV) infection under different light intensities was analyzed by RNA-seq sequencing method. Enrichment analysis illustrated that most defense-related genes were down-regulated in the dark and the shade, and up-regulated under hard light and normal light. Soybean can resist SMV infection mainly by activating salicylic acid signaling pathway. Light is essential for activating salicylic acid defense signaling pathways. With the increase of light intensity, the oxidative damage of soybean leaves was aggravated, which promoted the infection of virus. When light was insufficient, the growth of soybean was weak, and the plant-pathogen interaction pathway, MAPK pathway and hormone defense pathway in infected soybean was inhibited. Under hard light, some defense genes in infected soybean were down-regulated to reduce the degree of oxidative damage. The expression of differentially expressed genes was verified by real-time fluorescence quantitative RT-PCR. In order to adapt to the change of light intensity, soybean balanced allocation of resources between growth and defense through a series regulation of gene expression. The results of this study will provide a theoretical basis for the research of SMV resistance in intercropping soybean.

Rapid two‐stage amplification in a single tube for simultaneous detection of norovirus GII and group a rotavirus

The most prevalent viruses currently causing diarrhea are norovirus and rotavirus, and rapid and sensitive detection methods are essential for the early diagnosis of disease. The purpose of this study was to establish a sensitive single-tube two-stage nucleic acid amplification method-reverse transcription recombinase-assisted PCR (RT-RAP)-for simultaneous detection of norovirus GII and group A Rotavirus, with the first stage consisting of isothermal reverse transcription recombinase-aided amplification (RT-RAA) and the second stage consisting of qPCR (quantitative PCR). RT-RAP is more sensitive than either RT-RAA or qRT-PCR (quantitative RT-PCR) alone. And the addition of a barrier that can be disassembled after heating enabled the detection of samples within 1h in a single closed tube. Sensitivity was 10copies/reaction of norovirus (Novs) GII and group A rotavirus (RVA). In parallel, two hundred fecal specimens were used to evaluate the method and compare it with a commercial fluorescent quantitative RT-PCR. The data showed kappa values of 0.957 and 0.98 (p < 0.05) for detecting Novs GII and RVA by the two methods, indicating the potential of the newly established assay to be applied to clinical and laboratory testing.

Development of a reverse-transcription droplet digital PCR method for quantitative detection of Cucumber green mottle mosaic virus

Cucumber green mottle mosaic virus (CGMMV) is a re-emerging threat to the production of greenhouse cucumber and other Cucurbitaceae crops worldwide. This seed-borne virus can easily spread from a contaminated seed to seedlings and adjacent plants by mechanical contact between the foliage of diseased and healthy plants, causing extensive yield losses. An accurate method for detecting and quantifying this virus is urgently needed to ensure the safety of the global seed trade. Here, we report the development of a reverse-transcription droplet digital polymerase chain reaction (RT-ddPCR)-based method for specific and high-sensitive detection of CGMMV. By testing three primer–probe sets and optimizing reaction conditions, we showed that the newly developed RT-ddPCR method is highly specific and sensitive, with a detection limit of 1 fg/μL (0.39 copy/μL). The sensitivity of the RT-ddPCR method was compared with that of real-time fluorescence quantitative RT-PCR (RT-qPCR) using a series of plasmid dilutions and total RNAs extracted from infected cucumber seeds, and the detection limit of RT-ddPCR was 10 times higher than RT-qPCR with plasmid dilutions and 100 times higher than RT-qPCR for detecting CGMMV from infected cucumber seeds. The RT-ddPCR method was further assessed for detecting CGMMV from a total of 323 samples of Cucurbitaceae seeds, seedlings, and fruits as compared with the RT-qPCR method. We found that the infection rate of CGMMV on symptomatic fruits was as high as 100%, whereas infection rates were lower for seeds and lowest for seedlings. Notably, the results of two methods in detecting CGMMV from different cucurbit tissues showed the high consistency with Kappa value from 0.84 to 1.0, demonstrating that the newly developed RT-ddPCR method is highly reliable and practically useful for large-scale CGMMV detection and quantification.

Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR

The natural host range for brassica yellows virus (BrYV) is generally limited to Cruciferae. However, we found that BrYV can naturally infect strawberry. The full-length genome sequences of BrYV-MB (accession No. MZ666129) and BrYV-HY (accession No. ON060762) identified in strawberry from Yantai and Beijing, China, were obtained by high-throughput sequencing (HTS) combined with the RT-PCR and RACE techniques. The complete genome sequences of BrYV-MB and BrYV-HY are 5666 nt and contain six open reading frames (ORFs). The two isolates have the highest nucleotide (nt) sequence identity of 99.0%. The infectious cDNA clone of BrYV-HY was constructed through homologous recombination and used to agroinfiltrate Nicotiana benthamiana and Arabidopsis thaliana. The inoculated leaves of N. benthamiana showed necrotic symptoms after 4 days of inoculation (dpi), and the systematic leaves of A. thaliana exhibited purple symptoms at 14 dpi. To develop a rapid and high-sensitive method for the detection of BrYV, a TaqMan real-time fluorescence quantitative RT-PCR method (TaqMan RT-qPCR) was established. Under optimum reaction conditions, the sensitivity of the detection was as low as 100 fg and approximately 100-fold more sensitive than the conventional RT-PCR, so it can be used in large-scale testing.

Evaluation of Mean Percentage of Full-Length SMN Transcripts as a Molecular Biomarker of Spinal Muscular Atrophy.

The elevation of SMN transcript and protein level remains the principal aim of SMA therapy. Still, there is no standard molecular biomarker for the assessment of its efficacy. In the current study, we tested three methods of SMN transcript level measurement using real-time RT-PCR, quantitative fluorescent RT-PCR, and a semiquantitative RT-PCR gel densitometric assay. We examined several potential mRNA-based biomarkers and examined their sensitivity and reliability by comparing the obtained values in peripheral blood mononuclear cells of SMA patients, SMA carriers, and healthy individuals. We found that the mean percentage of full-length (FL-SMN) transcripts relative to the total sum of FL-SMN and exon 7-deleted (Δ7 SMN) transcripts detected by semiquantitative and quantitative fluorescence RT-PCR differed significantly between the three analyzed groups. The relevance of this biomarker was proven in an SMN2-targeting therapeutic experiment. We showed that the values of the biomarker changed significantly in SMA fibroblast cell cultures after treatment with therapeutic antisense oligonucleotides targeting the ISS-N1 site in intron 7 of the SMN2 gene. The obtained results indicate the convenience of using the mean percentage of FL-SMN transcripts determined by semiquantitative and quantitative fluorescence RT-PCR as a putative biomarker for the assessment of SMA therapy efficacy in vitro.

RhEPO Upregulates the PPARγ Pathway in Long-term Cultured Primary Nerve Cells via PI3K/Akt to Delay Cell Senescence.

Previous studies have confirmed that both recombinant human erythropoietin (rhEPO) and peroxisome proliferator-activated receptors γ (PPARγ) activator pioglitazone can protect senescent nerve cells, and their mechanisms involve enhancing cell antioxidant capacity and reducing cell apoptosis. However, whether the PPARγ pathway is involved in the rhEPO anti-aging process in neuronal cells is still unclear. In this study, to explore the relationship between rhEPO and the PPARγ pathway at the cellular level, primary nerve cells cultured for 22days were used to simulate the natural aging process of nerve cells. Starting on the 11th day of culture, rhEPO, LY294002, and GW9662 were added for treatment. Immunochemical methods and SA-β-gal staining were used to observe the changes in cellular antioxidant capacity and the fraction of senescent cells. The results showed that PPARγ blockade retarded the effect of rhEPO on the cellular antioxidant capacity and altered the fraction of senescent cells. It was confirmed that PPARγ was involved in rhEPO's anti-aging process in neuronal cells. Real-time fluorescent quantitative RT-PCR, Western blotting, and immunofluorescence staining were used to observe the changes in PPARγ pathway-related factors in nerve cells after rhEPO treatment. The results showed that rhEPO significantly upregulated the expression of PPARγ coactivator-1α (PGC-1α), PPARγ, and nuclear PPARγ in cells but did not affect the level of phosphorylated PPARγ protein, confirming that rhEPO has the ability to upregulate the PPARγ pathway. PI3K/Akt and PPARγ pathway blockade experiments were used to explore the relationships among rhEPO, PI3K/Akt, and PPARγ. The results showed that after PPARγ blockade, rhEPO had no significant effect on the PI3K/Akt pathway-related factor p-Akt, while after PI3K/Akt blockade, rhEPO's effects on PPARγ-related factors (PGC-1α, PPARγ, and nuclear PPARγ) were significantly decreased. It is suggested that rhEPO delays the PI3K/Akt pathway in the process of neuronal senescence, which is located upstream of PPARγ regulation. In conclusion, this study confirmed that rhEPO can upregulate the expression of PGC-1α and PPARγ in cells and the level of PPARγ protein in the nucleus to enhance the antioxidant capacity of cells and delay the senescence of nerve cells through the PI3K/Akt pathway. These findings will provide ideas for finding new targets for neuroprotection research and will also provide a theoretical basis and experimental evidence for rhEPO anti-aging research in neural cells.

EV71 was Detected by Real-Time Fluorescence Quantitative RT-PCR Combined with Hand-Foot-Mouth Disease Rhesus Monkey Model

In this study, a real-time fluorescence quantitative RT-PCR method was developed for the detection of enterovirus 71 (EV71) and the detection of EV71 viral load during the establishment of hand-foot-mouth disease (HFMD) model in rhesus monkeys. The model of hand-foot-mouth macaque was established by injecting newborn macaques infected with the virus venom, and the dynamic changes of virus content in blood samples from 0 to 14 days after infection were monitored by real-time quantitative RT-PCR. The linear range of the standard curve was between 103 and 108 copies/µL, and the PCR amplification rate was 99.37%. The results showed that the established method could detect a minimum of 102 copies of viral RNA, 100 times higher than the conventional RT-PCR method. It has the characteristics of strong specificity, high sensitivity and good stability. The results of blood sample analysis showed that the three groups of rhesus monkey models were successfully infected with EV71, and the copy number of virus proliferation was correlated with time and challenge dose. The results showed that real-time quantitative PCR could be used for quantitative detection of EV71 viral load.

Decreased CFTR/PPARγ and increased transglutaminase 2 in nasal polyps

ObjectiveTransglutaminase (TGM)2 and peroxisome proliferator-activated receptor (PPAR)γ are thought to participate in the pathogenesis of nasal polyp formation in cystic fibrosis (CF). We herein investigated expressions of cystic fibrosis transmembrane conductance regulator (CFTR), TGM2, PPARγ and isopeptide bonds, a reaction product of TGM, in non-CF nasal polyps. MethodsNasal polyps and inferior turbinates were collected from chronic rhinosinusitis patients without CF during transnasal endoscopic sinonasal surgery. Expressions of CFTR, TGM2, isopeptide bonds and PPARγ were examined by fluorescence immunohistochemistry and quantitative RT-PCR. Expression of CFTR was also analyzed by Western blot. ResultsImmunohistochemical fluorescence of the nasal polyp was significantly lower for CFTR and PPARγ, and significantly higher for TGM2 and isopeptide bonds than that of the turbinate mucosa. Lower expression of CFTR in the nasal polyp than in the turbinate mucosa was also observed in Western blot. Expression of PPARG mRNA was significantly lower in the nasal polyp than in the turbinate mucosa, whereas expressions of CFTR mRNA or TGM2 mRNA did not differ between the two tissues. Immunohistochemical fluorescence for CFTR showed significant negative correlation with that for TGM2 and isopeptide bonds, and significant positive correlation with that for PPARγ. The fluorescence for TGM2 was positively correlated with that for isopeptide bonds and negatively correlated with that for PPARγ. The fluorescence for isopeptide bonds tended to be negatively correlated with that for PPARγ. ConclusionsThese results suggest a possible role of the CFTR-TGM2-PPARγ cascade in the pathogenesis of nasal polyp formation in non-CF patients as in CF patients.