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Introduction: High‐quality nucleic acids are the basis for molecular biology experiments. Traditional RNA extraction methods are not suitable for Eleutherococcus senticosus Maxim. Objective: To find a suitable method to improve the quality of RNA extracted, we modified the RNA extraction methods of Trizol. Methodology: Based on the conventional Trizol method, the modified Trizol method 1 and modified Trizol method 2 were used as the control for extraction of RNA from E. senticosus Maxim leaves. The modified Trizol method 1 added β‐mercaptoethanol on the conventional Trizol method. After RNA was dissolved, a mixed solution of phenol, chloroform, and isoamyl alcohol was added to denature protein and inhibit the degradation of RNA. The modified Trizol method 2 adds PVPP to grind on the basis of modified Trizol method 1, so as to better remove phenols from leaves, and eliminates the step of incubation at −20°C to reduce extraction time and RNA degradation. Chloroform, CTAB, and CH3COONa were used instead of a phenol, chloroform, and isoamyl alcohol mixed solution to ensure complete separation of nucleic acid from plant tissues and to obtain high‐purity RNA. Results: The research results showed that the quality of RNA extracted by conventional Trizol method, modified Trizol method 1, was incomplete, accompanied with different degrees of contamination of polysaccharides, polyphenols, and DNA. The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. The ratio of A260/A280 was in the range of 1.8–2.0, and the yield of RNA was the highest, which was 1.68 and 1.15 times compared with that by conventional Trizol method and modified Trizol method 1 extraction, respectively. The reverse transcription cDNA was further tested through PCR with the specific primers. The amplified fragments are displayed in clear and bright bands in accordance with the expected size. Conclusion: The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. High‐quality RNA has more advantages in molecular biology study of E. senticosus Maxim. The conventional Trizol method was modified by adding PVPP for grinding, as well as using Chloroform, CTAB, and CH3COONa instead of a phenol, chloroform, and isoamyl alcohol mixed solution. The present investigation assesses and enhances the methods employed for extracting RNA from E. senticosus. The modified Trizol method 2, which has facilitated the successful isolation of premium RNA from E. senticosus leaves, stands as a convenient and cost‐effective extraction protocol and can promote research in plant molecular biology. [ABSTRACT FROM AUTHOR]

This article presents a study that compares different techniques for estimating gene copy numbers in genetically modified crops. The researchers evaluated Southern blotting, qPCR, dPCR, and PE-WGS and found that all four methods were suitable to varying degrees for determining gene copy numbers. The strengths and limitations of each technique are discussed, and the authors suggest prioritizing dPCR and PE-WGS for precise analysis. The document also provides references and supporting information for three related articles, which can be helpful for library patrons researching agricultural biotechnology, PCR techniques, and structural variation studies in plants. [Extracted from the article]

Low-temperature (LT) is one of the major abiotic stresses that restrict the growth and development of maize seedlings. Brassinolides (BRs) have been shown to enhance LT tolerance in several plant species; the physiological and molecular mechanisms by which BRs enhance maize tolerance are still unclear. Here, we characterized changes in the physiology and transcriptome of N192 and Ji853 seedlings at the three-leaf stage with or without 2 μM 2,4-epibrassinolide (EBR) application at 25 and 15 °C environments via high-performance liquid chromatography and RNA-Sequencing. Physiological analyses revealed that EBR increased the antioxidant enzyme activities, enhanced the cell membrane stability, decreased the malondialdehyde formation, and inhibited the reactive oxygen species (ROS) accumulation in maize seedlings under 15 °C stress; meanwhile, EBR also maintained hormone balance by increasing indole-3-acetic acid and gibberellin 3 contents and decreasing the abscisic acid level under stress. Transcriptome analysis revealed 332 differentially expressed genes (DEGs) enriched in ROS homeostasis, plant hormone signal transduction, and the mitogen-activated protein kinase (MAPK) cascade. These DEGs exhibited synergistic and antagonistic interactions, forming a complex LT tolerance network in maize. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that 109 hub genes involved in LT stress regulation pathways were discovered from the four modules with the highest correlation with target traits. In conclusion, our findings provide new insights into the molecular mechanisms of exogenous BRs in enhancing LT tolerance of maize at the seedling stage, thus opening up possibilities for a breeding program of maize tolerance to LT stress. [ABSTRACT FROM AUTHOR]

Simple Summary: This study focused on understanding how radiation therapy, a common treatment for cancer, affects the skin of dogs. While radiation is effective at destroying cancer cells, it can also cause damage to healthy skin, leading to various side effects like redness, peeling, changes in skin color, and sores. Over nine weeks, we monitored these skin changes in dogs, observing that radiation led to increased inflammation and stress in the skin cells, as well as significant disruptions in how skin cells grow and heal. We also noticed changes in specific proteins and genes related to skin inflammation, healing, and cell death. These findings help us better understand how radiation therapy impacts the skin and provide valuable information for managing these side effects in dogs. Radiation therapy is a crucial cancer treatment, but it can damage healthy tissues, leading to side effects like skin injuries and molecular alterations. This study aimed to elucidate histological and molecular changes in canine skin post-radiation therapy (post-RT) over nine weeks, focusing on inflammation, stem cell activity, angiogenesis, keratinocyte regeneration, and apoptosis. Four male beagles received a cumulative radiation dose of 48 Gy, followed by clinical observations, histological examinations, and an RT-qPCR analysis of skin biopsies. Histological changes correlated with clinical recovery from inflammation. A post-RT analysis revealed a notable decrease in the mRNA levels of Oct4, Sox2, and Nanog from weeks 1 to 9. VEGF 188 levels initially saw a slight increase at week 1, but they had significantly declined by week 9. Both mRNA and protein levels of COX–2 and Keratin 10 significantly decreased over the 9 weeks following RT, although COX–2 expression surged in the first 2 weeks, and Keratin 10 levels increased at weeks 4 to 5 compared to normal skin. Apoptosis peaked at 2 weeks and diminished, nearing normal by 9 weeks. These findings offer insights into the mechanisms of radiation-induced skin injury and provide guidance for managing side effects in canine radiation therapy. [ABSTRACT FROM AUTHOR]

The RNA-based study provides an excellent indication of an organism’s gene expression profile. Obtaining high-yield and high-purity RNA from Gram-positive and acid-fast bacteria is difficult without high-end kits and facilities. We optimised effective and simple protocol for RNA isolation that is a combination of enzymatic, physical and chemical treatment to disrupt cells. We successfully isolated high quality intact total RNA with yields ranging from 23.13 ± 0.40 to 61.51 ± 0.27 µg and the 260/280 purity ratio of 1.95 ± 0.01 to 2.05 ± 0.01 from Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Mycobacterium smegmatis. These results represents a significantly enhanced yield and purity compared to other combination of techniques which we performed. Compared to previous studies the yield obtained by this method is high for the studied organisms. Furthermore the yielded RNA was successfully used for downstream applications such as quantitative real time PCR. The described method can be easily optimised and used for various bacteria. [ABSTRACT FROM AUTHOR]

Background The emergence and rapid spread of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), poses a significant threat to human health and public safety. While next-generation sequencing (NGS) is capable of detecting and tracking new COVID-19 variants for disease diagnosis and prevention, its high cost and time-consuming nature limit its widespread use. In this study, our aim was to develop a highly adaptable and accurate RT-PCR method for identifying the Delta or BA.1 variants in inactivated COVID-19 vaccine. We devised three two-plex RT-PCR methods targeting specific mutation sites: S: Δ156–157, S: N211-, L212I, and S: Δ142–144, Y145D. The RT-PCR method targeting the S: Δ156–157 mutation site was able to distinguish the Delta variant from other COVID-19 virus strains, while the RT-PCR methods targeting the S: N211-, L212I or S: Δ142–144, Y145D mutation sites were able to distinguish the BA.1 variant from other COVID-19 virus strains. We separately validated these three two-plex RT-PCR methods, and the results demonstrated good linearity, repeatability, reproducibility, and specificity for each method. Moreover, all three methods can be applied in the production of SARS-CoV-2 variant inactivated vaccines, enabling the identification of Delta or BA.1 variants in virus cultures as well as in inactivated vaccine stocks. This study presents a systematic approach to identify COVID-19 variants using multiple RT-PCR methods. We successfully developed three two-plex RT-PCR methods that can identify Delta and BA.1 variants based on specific mutation sites, and we completed the validation of these three methods. [ABSTRACT FROM AUTHOR]

Background: Classifying diverse skin types is crucial for promoting skin health. However, efficiently identifying and analyzing relevant biomarkers from a vast array of available genetic data is challenging. Therefore, this study aimed to develop a precise and efficient platform for analyzing specific skin biomarkers using quantitative real‐time PCR (qRT‐PCR) with the minimal invasive skin sampling method (MISSM). Materials and methods: MISSM was used for RNA extraction from skin samples, followed by qRT‐PCR analysis to quantify the expression of 20 biomarkers associated with skin characteristics (four biomarkers each for five skin characteristics). Noninvasive measurements from 299 Korean participants were utilized to correlate biomarker expression with skin parameters. Statistical analyses were conducted between biomarker expression levels and noninvasive skin measurements to select the relatively best‐performing biomarker for each skin characteristic. Results: Collagen type 1 alpha 1 (COL1A1) and moesin (MSN) were identified as skin aging biomarkers. Krüppel‐like factor 4 (KLF4) and serine peptidase inhibitor Kazal type 5 (SPINK5) were identified as skin dryness biomarkers, whereas melan‐A (MLANA) was selected as a biomarker for understanding pigmentation dynamics. Myelin protein zero like 3 (MPZL3) and high mobility group box 2 (HMGB2) were identified as markers of oily skin and skin sensitivity, respectively. Statistically significant correlations were found between the biomarker expression levels and noninvasive skin characteristic measurements. Conclusion: This study successfully developed a platform for the precise evaluation of individual skin characteristics using MISSM and qRT‐PCR biomarker analysis. By selecting biomarkers that correlate with noninvasive measurements of skin characteristics, we demonstrated the platform's efficacy in assessing diverse skin conditions. [ABSTRACT FROM AUTHOR]

Background: The lack of reference genes makes it difficult to conduct molecular biology research on the plant genus, Clematis L. Clematis lanuginosa belongs to Sect. Viticella DC of Clematis L. It is also an important ornamental cultivated variety parent of the early and late large-flowered groups. Studying the reference genes of C. lanuginosa in different tissues will provide a theoretical basis for the reference selection of early and late large-flowered groups of Clematis, which could promote research progress on molecular biology of ornamental Clematis. Results: The roots, stems, leaves, sepals, stamens, and carpels of C. lanuginosa were used as research materials, and seven candidate reference genes were used for quantitative real-time PCR analysis. Comprehensive stability analysis using geNorm, NormFinder, BestKeeper, and RefFinder software showed that suitable reference genes in C. lanuginosa root, stem, and leaf were PP2A-2 and UBC34; and in floral tissue were UBC34, PP2A-2, and ARP7. These reference genes can be used as internal reference either alone or in combination. The pairwise variation value evaluated with geNorm software showed that two internal reference genes were needed for gene expression correction in the tissues. In the floral organs, three reference genes were required for gene expression correction. Conclusions: Our results provide a foundation for future gene expression analysis of C. lanuginosa and guidance for the screening of reference genes in Clematis.How to cite: Li Q, Wang S, Lv F, et al. Selection and validation of reference genes for quantitative real-time PCR in different tissues of Clematis lanuginosa. Electron J Biotechnol 2024;70. https://doi.org/10.1016/j.ejbt.2024.04.005.

Abstract Background Colorectal cancer (CRC) is considered as one of the most common malignancy and the fourth leading cause of cancer-related deaths, worldwide. Here, we aimed to investigate the expression of miR–663b and miR–650 in CRC tissue and plasma specimens. Methods In this case–control study, tumor specimens, non-tumoral adjacent tissues, and matched-plasma samples were obtained from forty patients with CRC living in the northwestern of Iran. Plasma of healthy patients was also collected as control. Total RNA was extracted from all specimens and studied by real-time PCR. Furthermore, the correlation between the expression of microRNAs and clinico-pathological features were also studied. Results Our data illustrated that miR-650 and miR-663b are down-regulated and up-regulated in tumor samples compared to non-tumoral margins, respectively (p

Objectives: Infertility is inability to conceive after 12 months of regular unprotected sex. MiRNA expression changes can serve as potential biomarkers for infertility in males due to impaired spermatogenesis. This research was conducted to measure the expression level of miR‐211 in plasma samples as a factor identifying infertility in comparison with the control group. Methods: In this study, blood plasma were taken from the infertile men (n = 103) nonobstructive azoospermia (NOA) or severe oligozoospermia (SO) and the control group (n = 121). The expression of circulating miR‐211 in plasma was assessed by qRT‐PCR. A relative quantification strategy was adopted using the 2−ΔΔCT method to calculate the target miR‐211 expression level in both study groups. Results: Plasma miR‐211 levels were significantly lower in infertile men compared to the control group (0.544 ± 0.028 and 1.203 ± 0.035, respectively, p < 0.001). Pearson's correlation analysis showed that miR‐211 expression level has a positive and significant correlation with sperm parameters, including sperm concentration, sperm total motility, progressive motility, and normal morphology (p < 0.001). Conclusions: Decreased expression of miR‐211 in blood plasma seems to be associated with male infertility. This experiment showed that miR‐211 can be considered as a biomarker for evaluation, diagnosis, and confirmation of the results of semen analysis in male infertility. [ABSTRACT FROM AUTHOR]

In this study, a genetic linkage map was developed based on simple sequence repeat (SSR) markers to aid in the identification and expression analysis of gene loci underlying the quantitative trait loci (QTL) of starch content (SC) in cassava (Manihot esculenta). A total of 912 polymorphic loci from 4,515 tested SSRs were mapped into 18 linkage groups. QTL analysis revealed four loci with highly significant associations with starch. Gene annotation was performed, and potential genes related to starch synthesis were identified. Of these, expression of four genes related to starch synthesis (Raffinose synthase [RS], Phosphoenolpyruvate kinase [PK], Glucose-6-phosphate [G6P], and Glycogen synthase kinase 3 [GSK-3]) was investigated by real-time PCR assays. The results found a statistically significant difference (p < 0.05) at 3 months after planting (MAP) in all the tested genes, whereas a higher expression was detected in the low-SC group with specific genotypes of linked markers. A higher level of expression was observed in the RS, PK, and G6P genes at six and nine MAP, indicating that starch accumulation mainly occurs after 3 MAP, during which the root system begins to form. The potential genes identified in this study could provide a better understanding of the starch biosynthesis mechanism, and could be useful for applications in marker-assisted selection to facilitate cassava breeding programs in the future. [ABSTRACT FROM AUTHOR]

Dōng líng căo, the dried aboveground parts of Isodon rubescens (Hemls.) Hara. is commonly consumed as a medicinal decoction or tea beverage. Natural beverages can be an important source of human dietary selenium (Se). However, how I. rubescens plants respond to exogenous Se remains unknown. In this study, a pot cultivation experiment was employed to investigate the phenotypic and physiological responses of I. rubescens plants exposed to Se. Fifteen days after applying different concentrations of sodium selenate to the soil, the Se enrichment capacity, growth indices, antioxidant capacities, and the content of flavonoids and diterpenoids were measured in the plants. Further, the oridonin content was quantified using the high-performance liquid chromatography method, and the expression levels of key diterpenoid synthesis genes were analyzed by quantitative real-time PCR (qRT-PCR). I. rubescens plants efficiently accumulated Se, with the Se content increasing proportionally to the applied dose, reaching levels of nearly 200 mg·kg dry leaves as Se concentration increased. None of the three Se treatments significantly altered the phenotypic indices, except a longer root length occurred in the 3 μM·kg Se group. Among three Se doses, 6 μM·kg Se gave the highest accumulation of flavonoids, diterpenoids, and oridonin, with the increase of 2.0-, 1.8-, and 1.9-fold in aboveground parts, respectively. Selenium application boosted the activities of antioxidant enzymes and antioxidant capacities according to 2,2-Diphenyl-1-picrylhydrazyl (DPPH), ferric reducing/antioxidant power, and tea brewing color experiments. Four key synthase genes were upregulated significantly by 6 μM·kg Se treatment, notably 1-deoxy-D-xylulose 5-phosphate reductoisomerase (IrDXR), with a 5-fold increase, and kaurene synthase-like 4 (IrKSL4), with a 6-fold increase. Thus, Se application in I. rubescens cultivation may be a potential biofortification method to supplement Se while increasing flavonoid and diterpenoid contents. Supplementary Material Supplementary Material File [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is considered as one of the most common malignancy and the fourth leading cause of cancer-related deaths, worldwide. Here, we aimed to investigate the expression of miR–663b and miR–650 in CRC tissue and plasma specimens. Methods: In this case–control study, tumor specimens, non-tumoral adjacent tissues, and matched-plasma samples were obtained from forty patients with CRC living in the northwestern of Iran. Plasma of healthy patients was also collected as control. Total RNA was extracted from all specimens and studied by real-time PCR. Furthermore, the correlation between the expression of microRNAs and clinico-pathological features were also studied. Results: Our data illustrated that miR-650 and miR-663b are down-regulated and up-regulated in tumor samples compared to non-tumoral margins, respectively (p < 0.001). However, the results did not show any significant difference in patient's plasmas compared to controls. Further analysis disclosed that the expression of miR-663b is significantly associated with tumor size, lymph node metastasis, and tumor stage, while miR-650 is remarkably related to TNM stage, lymph node metastasis, distant metastasis, tumor size, and age. (p < 0.05) Furthermore, receiver operating characteristic (ROC) analyses revealed that miR-650 and miR-663b are potential biomarkers in differentiating CRC patients from healthy controls. Conclusion: In conclusion, our data illustrated the potential of miR-650 and miR-663b as biomarkers in colorectal cancer. However, further studies are needed to confirm the employment of these microRNAs in the diagnosis and/or prognosis of colorectal malignancies. [ABSTRACT FROM AUTHOR]

In transgenic plants, the transgene copy numbers can highly affect the level of expression and genetic stability of the transgene. Hence, the first step in their characterization is the estimation of transgene copy numbers integrated in the plant genome. Quantitative real-time PCR (qRT-PCR) was used to determine the copy numbers of human interferon-γ (hIFN-γ) and hygromycin phosphortransferase II (hptII) transgenes in the genome of the T0 generation of 18 transgenic tobacco lines using the axi1 gene as an endogenous control. With optimized PCR conditions, we attained highly exact estimates of one, two, three, and/or four transgene copies in the T0 transformants. Moreover, estimation of copy numbers of the hIFN-γ transgene and the hptII selective marker gene indicated that rearrangements of the T-DNA has regularly happened in transgenic tobacco. Transgene copy number was also estimated using Southern blot analysis of gDNA derived from transformants. The transcript level and expression amount of recombinant hIFN-γ protein were evaluated in various events using RT-PCR and enzyme-linked immunosorbent assay (ELISA) techniques. A disagreement between the transcript level and the amount of recombinant protein with an inverse correlation between transgene copy number and expression level observed in some events, probably showing translational gene silencing and co-suppression or silencing, respectively. These results were also compared with segregation ratios of hygromycin-resistant phenotype in T1 plants of each line and found to be, in general, consistent. [ABSTRACT FROM AUTHOR]

Simple Summary: Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. In this study, we developed a droplet digital polymerase chain reaction (ddPCR) system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. The ddPCR test showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR was higher than that of qPCR. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. (1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections. [ABSTRACT FROM AUTHOR]

Dianchi Lake is a eutrophic lake in Yunnan, China with many endangered or extinct native fish species. We sought to explore the fish community using traditional netting and trapping methods and, for the critically endangered golden-line barbel Sinocyclocheilus grahami, environmental DNA (eDNA) to detect its presence and distribution in the lake. Using traditional netting and trapping methods, we found that the fish community has been almost completely converted to non-native species (~ 97% of abundance). Loss of native fishes was serious, with ~ 77% of historical species not detected. We designed sensitive species-specific primers for the mitochondrial COI gene specific to S. grahami and sampled eDNA throughout the lake. While only two individuals of S. grahami were caught using traditional methods, eDNA analysis revealed its presence across the central and southern regions of the lake. While it is unclear if the fish detected represent a recovering population or recently stocked individuals, occurrence records for the fish were inversely related to regions of the lake that suffered serious cyanobacteria blooms. This study highlights the sensitivity and utility of eDNA for non-destructively detecting presence of an endangered fish species. [ABSTRACT FROM AUTHOR]

[Objectives]Polyploidy is widespread in plants kingdom. However, the lack of reference genes has severely limited the research progress of polyploidy-related gene expression. The present study aimed to improve the accuracy and reproducibility of gene expression analyses in polyploids by establishing a set of stable reference genes. [Methods]The abundance of 10 candidate reference genes(UBI-ep, ACT, ACT3, TUA, EF-1α, CACS, TIP41, F-box, CYP and UBQ)were compared by real-time fluorescence quantitative analysis(qPCR)at different ploidy levels of Cucumis including artificial allotetraploid, their diploid parents and triploid offsprings. The stability of these candidate reference genes expression was analyzed by geNorm and NormFinder software. Furthermore, the correlation of relative transcript abundance estimated by qPCR analysis and transcriptome sequencing(RNA-seq)data was calculated to evaluate the stability of candidate reference genes. [Results]The qPCR results showed that the reference gene CYP in cucumber had the highest expression with a CT value of 15.8, and the reference gene F-box in tetraploid material had the lowest expression with a CT value of 30.5. Four stable reference genes were selected by combining the results of geNorm and NormFinder, which were F-box, TIP41, ACT3 and ACT. Among them, F-box and TIP41 had a lower stability value M than ACT3 and ACT genes at the polyploid level, but their expression abundance were low. When ACT3 was used as the internal reference, the relative transcript abundance correlation coefficient between qPCR and RNA-seq was extremely significant(R2=0.844, P<0.01). But the correlation was minimum when F-box was an internal parameter. [Conclusions]Attention should be paid to the selection of reference genes when comparing transcript abundance across different ploidy levels. TIP41 could be selected as the internal reference gene for the low abundance expression genes of multiple ploidy materials in melon. For highly expressed genes, ACT3 could be used as an internal reference gene. [ABSTRACT FROM AUTHOR]

In order to investigate the tetrodotoxin（TTX） content in the tissues of Takifugu obscurus and the expression characteristics of voltage gated sodium channel genes in toxic tissues, this study used enzymelinked immunosorbent assay（ ELISA） to determine the TTX content in 8 tissues, including the liver and heart of Takifugu obscurus. Real time fluorescence quantification （RT-qPCR） was used to detect the relative expression of voltage gated sodium channels α subunit gene family, including scn1aa, scn1ab, scn4aa, scn4ab, scn5aa, scn5ab, scn8aa, and scn8ab in various tissues. The results showed that the TTX content in various tissues ranged from high to low, including brain （9. 521 ± 2. 816） μg/g, heart （4. 271 ± 1. 129） μg/g, gills（ 1. 586 ± 0. 527） μg/g, muscle（ 1. 494 ± 0. 938） μg/g, liver（ 0. 913 ± 0. 206） μg/g, skin（ 0. 902 ± 0. 235） μg/g, intestine （0. 894 ± 0. 215） μg/g, and eye （0. 864 ± 0. 287） μg/g. Brain and heart tissues were weakly toxic, while liver, skin, muscle, eye, intestine, and gill tissues were slightly toxic. The RTqPCR results showed that the relative expression levels of scn1aa, scn1ab, scn4ab, scn5aa, scn5ab, scn8aa, and scn8ab genes were the highest in liver tissue. The scn4aa gene was only expressed in muscle tissue, and only the scn4ab gene was expressed in skin tissue. Moreover, the scn4ab gene was expressed in all detected tissue types, indicating widespread expression. It is discovered that there are differences in the expression characteristics of sodium channel genes between zebrafish and Takifugu obscurus tissues. [ABSTRACT FROM AUTHOR]

Leaf rust (LR) caused by Puccinia recondita f. sp. secalis (Prs) is a highly destructive disease in rye. However, the genetic mechanisms underlying the rye immune response to this disease remain relatively uncharacterised. In this study, we analysed the expression of four genes in 12 rye inbred lines inoculated with Prs at 20 and 36 h post-treatment (hpt): DXS (1-deoxy-D-xylulose 5-phosphate synthase), Glu (β-1,3-glucanase), GT (UDP-glycosyltransferase) and PR-1 (pathogenesis-related protein 1). The RT-qPCR analysis revealed the upregulated expression of the four genes in response to Prs in all inbred lines and at both time-points. The gene expression data were supported by microscopic and macroscopic examinations, which revealed that eight lines were susceptible to LR and four lines were highly resistant to LR. A relationship between the infection profiles and the expression of the analysed genes was observed: in the resistant lines, the expression level fold changes were usually higher at 20 hpt than at 36 hpt, while the opposite trend was observed in the susceptible lines. The study results indicate that DXS, Glu, GT and PR-1 may encode proteins crucial for the rye defence response to the LR pathogen. [ABSTRACT FROM AUTHOR]

Chicken infectious anaemia virus (CIAV) causes severe anemia and immunosuppression through horizontal or vertical transmission in young chickens. Especially, vertical transmission of virus through the egg can lead to significantly economic losses due to the increased mortality in the broiler industry. Here, 28 CIAV complete sequences circulating in Korea were first characterized using the newly designed primers. Phylogenetic analysis based on the complete sequences revealed that CIAV isolates were divided into four groups, IIa (2/28, 7.1%), IIb (9/28, 32.1%), IIIa (8/28, 28.6%) and IIIb (9/28, 32.1%), and exhibited a close relationship to each other. The major groups were IIb, IIIa and IIIb, and no strains were clustered with a vaccine strain available in Korea. Also, for viral titration, we newly developed a quantitative PCR assay that is highly sensitive, reliable and simple. To investigate the pathogenicity of three major genotypes, 18R001(IIb), 08AQ017A(IIIa), and 17AD008(IIIb) isolates were challenged into one-day-old specific-pathogen-free (SPF) chicks. Each CIAV strain caused anaemia, severe growth retardation and immunosuppression in chickens regardless of CIAV genotypes. Notably, a 17AD008 strain showed stable cellular adaptability and higher virus titer in vitro as well as higher pathogenicity in vivo. Taken together, our study provides valuable information to understand molecular characterization, genetic diversity and pathogenicity of CIAV to improve management and control of CIA in poultry farm. [ABSTRACT FROM AUTHOR]

Brassica rapa, a major oilseed crop in high-altitude areas, is well known for its indeterminate inflorescences. However, this experiment revealed an intriguing anomaly within the plot: a variant displaying a determinate growth habit (520). Determinate inflorescences have been recognized for their role in the genetic enhancement of crops. In this study, a genetic analysis in a determinate genotype (520) and an indeterminate genotype (515) revealed that two independently inherited recessive genes (Brdt1 and Brdt2) are responsible for the determinate trait. BSA-seq and SSR markers were employed to successfully locate the Brdt1 gene, which is localized within an approximate region 72.7 kb between 15,712.9 kb and 15,785.6 kb on A10. A BLAST analysis of these candidate intervals revealed that Bra009508 (BraA10.TFL1) shares homology with the A. thaliana TFL1 gene. Then, BraA10.TFL1 (gene from the indeterminate phenotype) and BraA10.tfl1 (gene from the determinate phenotype) were cloned and sequenced, and the results indicated that the open reading frame of the alleles comprises 537 bp. Using qRT-PCR, it was determined that BraA10.TFL1 expression levels in shoot apexes were significantly higher in NIL-520 compared to 520. To verify the function of BraA10.TFL1, the gene was introduced into the determinate A. thaliana tfl1 mutant, resulting in the restoration of indeterminate traits. These findings demonstrate that BraA10.tfl1 is a gene that controls the determinate inflorescence trait. Overall, the results of this study provide a theoretical foundation for the further investigation of determinate inflorescence. [ABSTRACT FROM AUTHOR]

The ever-increasing applications of metabarcoding analyses for environmental samples demand a well-designed assessment of the stability of DNA and RNA contained in cells that are deposited or buried in marine sediments. We thus conducted a qPCR quantification of the DNA and RNA in the vegetative cells of three microalgae entrapped in facsimile marine sediments and found that >90% of DNA and up to 99% of RNA for all microalgal species were degraded within 60 days at 4 °C. A further examination of the potential interference of the relic DNA of the vegetative cells with resting cyst detection in sediments was performed via a metabarcoding analysis in artificial marine sediments spiked with the vegetative cells of two Kareniaceae dinoflagellates and the resting cysts of another three dinoflagellates. The results demonstrated a dramatic decrease in the relative abundances of the two Kareniaceae dinoflagellates in 120 days, while those of the three resting cysts increased dramatically. Together, our results suggest that a positive detection of microalgae via metabarcoding analysis in DNA or RNA extracted from marine sediments strongly indicates the presence of intact or viable cysts or spores due to the rapid decay of relic DNA/RNA. This study provides a solid basis for the data interpretation of metabarcoding surveys, particularly in resting cyst detection. [ABSTRACT FROM AUTHOR]

Chicken coccidiosis, caused by Eimeria protozoa, affects poultry farming. Toll‐like receptors (TLRs) and host defence peptides (HDPs) help host innate immune responses to eliminate invading pathogens, but their roles in Eimeria tenella infection remain poorly understood. Herein, 14‐day‐old chickens were treated orally with 50,000 E. tenella oocysts and the cecum was dissected at different timepoints. mRNA expression of 10 chicken TLRs (chTLRs) and five HDPs was measured by quantitative real‐time PCR. chTLR7 and chTLR15 were upregulated significantly at 3 h post‐infection while other chTLRs were downregulated (p <.05). chTLR1a, chTLR1b, chTLR2b and chTLR4 peaked at 36 h post‐infection, chTLR3, chTLR5 and chTLR15 peaked at 72 h post‐infection and chTLR21 expression was highest among chTLRs, peaking at 48 h post‐infection (p < 0.05). For HDPs, cathelicidin (CATH) 1 to 3 and B1 peaked at 48 h post‐infection, liver‐expressed antimicrobial peptide 2 peaked at 96 h post‐infection, and CATH 2 expression was highest among HDPs. CATH2 and CATH3 were markedly upregulated at 3 h post‐infection (p <.05). The results provide insight into innate immune molecules during E. tenella infection in chicken, and indicate that innate immune responses may mediate resistance to chicken coccidiosis. [ABSTRACT FROM AUTHOR]

objective: Pseudomonas fluorescens is the dominant spoilage organism in frozen foods, and its spoilage-caus¬ing genes are regulated by quorum sensing system. In order to accurately quantify the expression of spoilage genes and thus investigate the mechanism of quorum sensing regulation in food spoilage, it is required to screen reference genes of Pseudomonas fluorescens. Methods: Using Pseudomonas fluorescens PF-08 as the research object, eight common reference genes (dsbA, carA, rpsL, gyrB, atpD, rpoD, gltA, 16S rRNA) were selected and their gene expression was measured by quantitative real-time PCR (qPCR) after incubation with different types of quorum sensing signal molecules. geNorm, Normfinder, BestKeeper and RefFinder were used to evaluate the expression stability of the candidate reference genes and to screen out the most appropriate reference genes. Results: Under different types of exogenous signal molecules incuba¬tion, the least change in Ct value of Pseudomonas fluorescens was gltA and the Ct value of 16S rRNA was too low. The most stable reference genes by geNorm analysis were atpD and rpoD and the combination of the two could more accu¬rately quantify the expression levels of target genes. gltA was the most stable reference gene analyzed by Normfinder and BestKeeper. Further comprehensive evaluation with RefFinder showed rpoD and gltA were the most stable reference genes. Conclusion: Both rpoD and gltA were stably expressed in Pseudomonas fluorescens after incubation with different QS sig¬nal molecules. They could be used for the subsequent study of Pseudomonas fluorescens spoilage gene expression, and could also provide reference genes for studying the expression of QS-related genes in other spoilage bacteria. [ABSTRACT FROM AUTHOR]

Background: Seoul virus (SEOV) is a significant causative pathogen of hemorrhagic fever with renal syndrome (HFRS). Accurate discrimination of SEOV infection from other viral or bacterial infections holds vital clinical importance. Methods: Our study utilized quantitative real-time PCR (qRT-PCR), metagenomic next-generation sequencing (mNGS), and immunological assays to identify the pathogen causing HFRS. Results: For the case, mNGS identified SEOV and suspected host or environmental microorganisms at 5 days from symptom onset. qRT-PCR detected SEOV between 5 to 8 days from symptom onset. Anti-hantavirus IgM antibodies reached positive criteria at 7 days and IgG antibodies at 9 days from symptom onset. Conclusions: qRT-PCR, mNGS, and immunological assays each have merits and drawbacks. Optimal selection depends on laboratory conditions and clinical requirements. [ABSTRACT FROM AUTHOR]

Quantitative Real-time PCR (qRT-PCR) is one of the most widely used methods for gene quantification because it is extremely sensitive, can be highly sequence specific, and has high precision while being easily reproducible in samples. The stability of the reference genes used for data normalization is crucial for exact experimental results and conclusions. In this study, qRT-PCR was used to analyze the expression of eight candidate reference genes including β-actin (ACTB), α-tubulin (TUBA), Elongation factor-1-α (EF1A), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH1, GAPDH2), Hypoxanthine guanine phosphoribosyl transferase1 (HPRT), Ribosomal protein L7 (RPL7), and RNA polymerase II (RNA PoL Ⅱ) in largemouth bass. The expression and stability of above genes were evaluated among eight tissues with or without largemouth bass virus (LMBRaV) infection and assessed using BestKeeper, GeNorm, NormFinder statistical softwares and comparative Ct method, and then analyzed the comprehensive ranking by ReFinder. Our results showed that ACTB was identified as the most suitable genes of different tissues, while TUBA was ranked as the best reference gene in tissues infection with LMBRaV. To verify the screened reference genes, the expression pattern of IFN in spleen during LMBRaV infection was examined. This study provides useful information for gene expression characterization using qRT-PCR, which will benefit for future studies of gene regulation in largemouth bass.

Escherichia albertii is an emerging zoonotic foodborne pathogen. The clinical significance of this bacterium has increasingly been recognized worldwide. However, diagnostic method has not yet been established and its clinical manifestations are not fully understood. Here, we show that an Eacdt gene-based quantitative real-time PCR (qRT-PCR) developed in this study is 100% specific and sensitive when tested with 39 E. albertii and 36 non-E. albertii strains, respectively. Detection limit of the real-time PCR was 10 colony forming unit (CFU) and 1 pg of genomic DNA per PCR tube. When E. albertii was spiked with 4 × 100–106 CFU per mL to stool of healthy person, detection limit was 4.0 × 103 and 4.0 CFU per mL before and after enrichment culture, respectively. Moreover, the qRT-PCR was able to detect E. albertii in five children out of 246 (2%) but none from 142 adults suffering from gastroenteritis. All five E. albertii strains isolated carried eae and paa genes, however, only one strain harbored stx2f genes. Long-term shedding of stx2f gene-positive E. albertii in a child stool could be detected because of the qRT-PCR developed in this study which might have been missed if only conventional PCR and culture methods were employed. Furthermore, E. albertii isolated from siblings with diarrhea showed clonality by PFGE analysis. Taken together, these data suggest that the Eacdt gene-based qRT-PCR developed for the detection of E. albertii is useful and will assist in determining the real burden and clinical manifestation of E. albertii infections.

Gelsemium elegans is a traditional Chinese medicinal plant, with indole alkaloids as its main active ingredient. The plant can be used as medicine, mainly for pain relief, anti-inflammation and anti-tumor. Exogenous stress, such as low temperature, methyl jasmonate (MeJA), and salicylic acid (SA), can affect the growth of G. elegans and the synthesis of secondary metabolites. Under these conditions, there are only a few reports on the selection and validation of internal reference genes in G. elegans. In this study, seven candidate internal reference genes that showed stable expression abundance in the transcriptome database of G. elegans were selected. The stability and reliability of these genes were analyzed in different G. elegans tissues and under low temperature, MeJA, and SA stresses. The results showed that CUL was the optimal reference gene for expression analysis in different G. elegans tissues and under cold stress, SA stress, followed by eEF-1α. Under MeJA stress, the best one was eEF-1α, and the next was CUL. Furthermore, the expression patterns of three different G. elegans genes, including GPPS, ERF, and 60S, were carried out to confirm that single reference gene (CUL or eEF-1α) or double reference genes (CUL + eEF-1α) can be selected as the best or perfect combination of reference genes for gene expression analysis in G. elegans. This study lays a foundation for the accurate normalization and quantification of gene expression in G. elegans.

Radiation therapy is a crucial cancer treatment, but it can damage healthy tissues, leading to side effects like skin injuries and molecular alterations. This study aimed to elucidate histological and molecular changes in canine skin post-radiation therapy (post-RT) over nine weeks, focusing on inflammation, stem cell activity, angiogenesis, keratinocyte regeneration, and apoptosis. Four male beagles received a cumulative radiation dose of 48 Gy, followed by clinical observations, histological examinations, and an RT-qPCR analysis of skin biopsies. Histological changes correlated with clinical recovery from inflammation. A post-RT analysis revealed a notable decrease in the mRNA levels of Oct4, Sox2, and Nanog from weeks 1 to 9. VEGF 188 levels initially saw a slight increase at week 1, but they had significantly declined by week 9. Both mRNA and protein levels of COX–2 and Keratin 10 significantly decreased over the 9 weeks following RT, although COX–2 expression surged in the first 2 weeks, and Keratin 10 levels increased at weeks 4 to 5 compared to normal skin. Apoptosis peaked at 2 weeks and diminished, nearing normal by 9 weeks. These findings offer insights into the mechanisms of radiation-induced skin injury and provide guidance for managing side effects in canine radiation therapy.

Introduction: The moth species Athetis lepigone (Moschler) (Lepidoptera: Noctuidae), which has recently been identified as a pest of summer maize (Zea mays L.) in China, has demonstrated a rapid proliferation with in the Huang-Huai-Hai Plain region since its initial discovery in Hebei Province in 2005. It has become a prevalent pest of corn crops, and its ability to adapt quickly to its surroundings is currently being investigated. One of the key characteristics of its siphoning mouthparts is not only the feeding apparatus itself but also the chemosensory organs that enable the detection of chemical signals from the surrounding environment. However, there is a lack of comprehensive research on the genes responsible for chemosensory and metabolic mechanisms in the proboscises of male and female A. lepigone adults. Methods: In this study, we utilized transcriptome analysis to identify a total of fifty chemosensory genes from six distinct families, including 19 odorant-binding proteins (OBPs), 22 chemosensory proteins (CSPs), one co-receptor (Orco), six odorant receptors (ORs), four ionotropic receptors (IRs), and two sensory neuron membrane proteins (SNMPs) in the proboscis. Notably, seven OBPs, two CSPs, and one OR were discovered for the first time. Additionally, fourteen genes related to metabolism, including cytochrome P450 (CYPs) and carboxylesterases (CXEs), were also identified. Furthermore, a qualitative analysis was conducted on the relative transcript levels of eight related genes. The expression of 21 annotated chemosensory and metabolic genes was compared between A. lepigone adults and larvae using qRT-PCR, revealing tissue specificity. The majority of genes exhibited predominant expression in the antennae and proboscis during the adult stage, while showing slight expression in the combination of sixth-instar larval head oral appendages (maxilla, labium, and antenna) and pheromone gland-ovipositors of female adults. Results/discussion: Our study points to a new pest control strategies that these newly discovered genes have the potential to serve as targets for enhancing future pest control, including mating disruption and the use of food attractants. And it would be advantageous to ascertain the distribution of chemosensory gene expression and gain insights into the functionalities of these genes, thereby establishing a novel theoretical framework for the advancement of eco-friendly pesticides and efficient pest management strategies in the future. [ABSTRACT FROM AUTHOR]

A diet with additional nutrient regimens accelerates the physiological and metabolic processes in organisms, which results in better survival. There is a lot of research being done on model species to determine the locomotion and olfactory memory of organisms. Although, how dietary content affects behaviour paradigms is not much studied. In the present study, we used Drosophila ananassae subgroup of fruit flies to study behavioural paradigms of manipulated diet on male and female flies using climbing assay, Y maze assay, and T maze assay. We fed D. ananassae and D. bipectinata adult flies with media composition enriched with low and high doses of Sucrose and tryptophan in three replicates. We also aimed to find the variation in the gene expression of Sh and ORCO genes, responsible for behavioural traits. Our results discovered that dietary composition significantly affects these physiological parameters. Tryptophan (100 mg dose) was the most effective in the climbing assay. Drosophila ananassae male (70%) and D. bipectinata female (80%) were most active at this dose. Y-maze assay results showed that tryptophan (100 mg) is the most effective diet for D. ananassae males (65%) and females (60%) whereas, sucrose (30 mg) is the optimum diet for D. bipectinata males and females olfactory response (75% and 65%, respectively). T maze studies found diet enriched with sucrose is the most effective dose for males and females of both species. Gene expression analysis using RTqPCR found these behavioural changes are not based on a mutation in genes. The underlying reason can be modulation in the levels of biological compounds due to diet-induced physiological changes. So, these species grown on sucrose and tryptophanrich diet can be used as a valuable tool for identifying the molecular mechanisms underlying the difference in depressive and memory disorders prevalence between men and women. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Tea Science is the property of Journal of Tea Science Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In general, QuantAS provides an accurate quantitative method for isoform quantification, reduces systematic errors in experiments, and demonstrates high compatibility and specificity, while other methods, such as HiFENS, based on Fluorescence I in situ i hybridization (FISH) technique for endogenous splicing isoforms detection, which still relies on isoform-specific regions (Shilo I et al i ., [21]). Keywords: absolute quantification; alternative splicing; digital PCR; QuantAS; quantitative real-time PCR EN absolute quantification alternative splicing digital PCR QuantAS quantitative real-time PCR 928 939 12 10/09/23 20231101 NES 231101 Data availability All data supporting the findings are contained in this manuscript and Supporting Information. The method utilizes isoform-specific primers to overcome the isoform identification difficulty caused by different AS events and is designed by using the functional coding region as the isoform structure classification unit to ensure isoform independence (Fig. [Extracted from the article]

Tropaeolum majus (nasturtium) is an important ornamental and medicinal plant due to its colorful flowers, shield-shaped leaves, and richness in mineral elements and bioactive compounds. However, the key genes related to these important biological traits, as well as their expression patterns and functions, remain obscure. In this study, to choose appropriate reference genes for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, we screened 14 candidate genes from the transcriptome of T. majus and evaluated their expression stability. Through evaluation with four commonly used algorithms (geNorm, NormFinder, BestKeeper, and RefFinder), EXP1, EXP2, and TUB6 were found to be the most stably expressed genes among different organs, while EXP1 combined with CYP2 was identified as the optimal reference gene combination for seeds at different development stages. For all the tested samples, EXP1, EXP2, CYP2, and ACT2 were the most suitable reference genes. Moreover, the target gene KCS11 involved in very-long-chain fatty acid biosynthesis was employed to confirm the most and least stable reference genes in different organs, seeds at different development stages, and all the tested samples. The expression profiles of KCS11 were similar, with minor differences based on the analysis of different stable reference genes (either alone or in combination), while the expression profiles were diverse and the relative expression level was overestimated when using the least stable ones. These results suggest that the appropriate selection of reference genes is critical for the normalization of gene expression. Furthermore, the reference genes screened in this study will greatly improve the accuracy of the qRT-PCR quantification of candidate genes involved in the many biological characteristics of nasturtium. [ABSTRACT FROM AUTHOR]

The selection of proper reference genes is vital for ensuring precise quantitative real-time PCR (qPCR) assays. This study evaluates the stability of the expression of nine candidate reference genes in different tissues and during testicular development in H. labeo. The results show that eef1a is recommended as a reference gene for qPCR analysis in tissues and during testicular development. Furthermore, we evaluated the optimal number of reference genes needed when calculating gene expression levels using the geomean method, revealing that two reference genes are sufficient. Specifically, eef1a and rps27 are recommended for analysis of gene expression in tissues, whereas eef1a and actb are advised for evaluating gene expression during testicular development. In addition, we examined the expression pattern of kifc1, a kinesin involved in the reshaping of spermatids. We detected peak expression levels of kifc1 in testes, with its expression initially increasing before decreasing throughout testicular development. The highest expression of kifc1 was observed in stage IV testes, the active period of spermiogenesis, suggesting a possible role for kifc1 in the regulation of the reshaping of spermatids and hence testicular development. This study represents the first investigation of reference genes for H. labeo, providing a foundation for studying gene expression patterns and investigating gene expression regulation during testicular development.

(1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections.

Abstract Hemibarbus maculatus is a globally important species for its value in fisheries and aquaculture. There is relatively little information available about this species' sexual development genes. Therefore, in this study, we used the technology of high‐throughput RNA sequencing (RNA‐seq), to investigate if the ovary and testis' differently expressed genes (DEGs) during sex differentiation in H. maculatus. Our results revealed a total of 43,468,248 and 39,241,362 qualified reads were generated in the female and male libraries, and 32,127 unigenes were assembled via the annotation analysis. Moreover, 17,808 unigenes were found to be DEGs between males and females, with 12,726 up‐ and 5082 downregulated genes in the ovaries and testes, respectively. The transcriptome analysis revealed there are 23 DEGs between ovary and testes of H. maculatus, which may be involved in sex determination. The expression patterns of these genes were validated by quantitative real‐time PCR (qPCR). The results of qPCR were generally consistent with the RNA‐seq results. This work was the first to annotate genes linked with gonadal development and reproduction in H. maculatus using gonadal transcriptome analysis, which provides useful molecular data for a better understanding of sexual development, as well as functional genomics and population genetics studies of H. maculatus.

Ribose 2′O-methylation (Nm, ribomethylation) is the most abundant RNA modification present in rRNA. It has been shown that alterations in ribosomal 2′O-methylation at individual Nm sites likely reflect regulated cellular processes. Although several analytical approaches for Nm detection and profiling have been developed, a simple and affordable method for the screening and measurement of individual Nm sites in large numbers of tissue samples is required to examine their potential for clinical translation. Here, we describe a new quantitative reverse transcription PCR-based method that can sensitively assess ribomethylation levels at specific rRNA sites at single-nucleotide resolution in low input amounts of total RNA.

Introduction: The moth species Athetis lepigone (Möschler) (Lepidoptera: Noctuidae), which has recently been identified as a pest of summer maize (Zea mays L.) in China, has demonstrated a rapid proliferation with in the Huang-Huai-Hai Plain region since its initial discovery in Hebei Province in 2005. It has become a prevalent pest of corn crops, and its ability to adapt quickly to its surroundings is currently being investigated. One of the key characteristics of its siphoning mouthparts is not only the feeding apparatus itself but also the chemosensory organs that enable the detection of chemical signals from the surrounding environment. However, there is a lack of comprehensive research on the genes responsible for chemosensory and metabolic mechanisms in the proboscises of male and female A. lepigone adults.Methods: In this study, we utilized transcriptome analysis to identify a total of fifty chemosensory genes from six distinct families, including 19 odorant-binding proteins (OBPs), 22 chemosensory proteins (CSPs), one co-receptor (Orco), six odorant receptors (ORs), four ionotropic receptors (IRs), and two sensory neuron membrane proteins (SNMPs) in the proboscis. Notably, seven OBPs, two CSPs, and one OR were discovered for the first time. Additionally, fourteen genes related to metabolism, including cytochrome P450 (CYPs) and carboxylesterases (CXEs), were also identified. Furthermore, a qualitative analysis was conducted on the relative transcript levels of eight related genes. The expression of 21 annotated chemosensory and metabolic genes was compared between A. lepigone adults and larvae using qRT-PCR, revealing tissue specificity. The majority of genes exhibited predominant expression in the antennae and proboscis during the adult stage, while showing slight expression in the combination of sixth-instar larval head oral appendages (maxilla, labium, and antenna) and pheromone gland-ovipositors of female adults.Results/discussion: Our study points to a new pest control strategies that these newly discovered genes have the potential to serve as targets for enhancing future pest control, including mating disruption and the use of food attractants. And it would be advantageous to ascertain the distribution of chemosensory gene expression and gain insights into the functionalities of these genes, thereby establishing a novel theoretical framework for the advancement of eco-friendly pesticides and efficient pest management strategies in the future.

Background & Aims Hepatitis delta virus (HDV) infection accelerates the progression of chronic hepatitis B virus (HBV) infection, posing a large economic and health burden to patients. At present, there remains a lack of accurate and portable detection methods for HDV RNA. Here, we aim to establish a convenient, rapid, highly sensitive and specific method to detect HDV RNA using CRISPR–Cas13a technology.Methods We established fluorescence (F) and lateral flow strip (L) assays based on CRISPR–Cas13a combined with RT–PCR and RT-RAA for HDV RNA detection, respectively. we conducted a cohort study of 144 patients with HDV-IgG positive to evaluate the CRISPR–Cas13a diagnostic performance for identifying HDV in clinical samples, compared to RT–qPCR and RT-ddPCR.Results For synthetic HDV RNA plasmids, the sensitivity of RT–PCR-CRISPR-based fluorescence assays was 1 copy/μL, higher than that of RT–qPCR (10 copies/μL) and RT-ddPCR (10 copies/μL); for HDV RNA-positive samples, the sensitivity of RT-RAA-CRISPR-based fluorescence and lateral flow strip assays was 10 copies/μL, as low as that of RT–qPCR and RT-ddPCR, and the assay took only approximately 85 min. Additionally, the positivity rates of anti-HDV IgG-positive samples detected by the RT–qPCR, RT-ddPCR, RT–PCR-CRISPR fluorescence and RT-RAA-CRISPR lateral flow strip methods were 66.7% (96/144), 76.4% (110/144), 81.9% (118/144), and 72.2% (104/144), respectively.Conclusions We developed a highly sensitive and specific, as well as a portable and easy CRISPR-based assay for the detection of HDV RNA, which could be a prospective measure for monitoring the development of HDV infection and evaluating the therapeutic effect.

Mytilus coruscus is one of large bivalve mollusks that is wildly distributed along the coasts of Korea, Japan and China and as a potential economic shellfish candidate. Efforts to propagate and culture M. coruscus have expanded worldwide recent years. The larval stage of bivalves is a vital life-history phase, of which the eyed veligers larva with a pigmented eyespot is the pre-settlement phase of M. coruscus. However, information on light conditions for the breeding of its larval during the eye-spotting period is still limited. In our study, based on the transcriptome analysis of the different light cycles from M. coruscus were performed with the DNBSEQ to screen for light-response genes. Comparative transcriptome analysis identified 853 differentially expressed genes (DEGs) in the 12 h vs 0 h group and 696 genes were differentially expressed between 12 h vs 24 h group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that DEGs were assigned to 1078 and 969 pathways, respectively. The DEGs were enriched in organismal systems pathways such as immune system pathway, endocrine system pathway, development pathway, and environmental adaptation pathway. Further analysis revealed that DEGs, including CRY and CLOCK, were significantly overrepresented in the circadian rhythm pathway. We also identified a visual OPIN gene, which affects the detection of relevant visual signals. Quantitative real-time PCR (qRTPCR) showed that CRY and CLOCK were down-regulated, whereas OPIN was up-regulated. The data from this study provides important insights into the light response of M. coruscus, which will aid future investigations into the management of M. coruscus breeding in Bivalvia.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different TDNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Quantitative real‐time polymerase chain reaction (qPCR) is a method widely used to determine changes and differences in gene expression. As target gene expression is most often quantified relative to the expression of reference genes, the validation of suitable reference genes is of critical importance. In practice, however, such validation might not be thoroughly conducted if the same species and the same tissue or body parts are used for qPCR experiments. Here we show, that qPCR reference genes published for workers of European honey bee (Apis mellifera) subspecies fail to be stably expressed in workers of the African subspecies Apis mellifera scutellata. This is the case even when the sampled workers are in the same life stage, the same organ was dissected and the same reagents were used. Thus, reference genes need to be thoroughly re‐tested before they can be used as suitable references even when the only thing that changes is the subspecies used. [ABSTRACT FROM AUTHOR]

Quantitative real-time polymerase chain reaction (qPCR) is one of the most widely used molecular pathological diagnostic techniques in China due to its advantages of the simple operation, short turnaround time, high sensitivity, and standardizable result analysis. However, in clinical practice, there is not yet an expert consensus to guide molecular pathological diagnostics of tumor using qPCR techniques in terms of validation and verification of method performance, quality management and interpretation of complex results. Therefore, this expert consensus aims to provide standardized opinions on the practical application of qPCR techniques, and suggestions on how to deal with common problems and abnormal results, and reach a Chinese expert consensus on the clinical practice of qPCR techniques in molecular pathological diagnostics of tumor, in order to standardize the testing process, improve the accuracy of results, and promote the clinical applications of qPCR techniques. [ABSTRACT FROM AUTHOR]

Mink circovirus disease caused by Mink Circovirus (MiCV) is a serious infectious disease of mink that has become prevalent in recent years in China, severely affecting the reproductive performance of mink and causing significant economic losses to farms. To date, there have been few studies on MiCV, its pathogenic mechanism is not clear, and there is no effective vaccine or drug to prevent and control the disease. Therefore, it is necessary to establish a rapid and reliable molecular diagnostic method, which would aid future studies of this novel virus. In our study, we developed a sensitive and specific TaqMan-based quantitative real-time PCR assay targeting the MiCV Cap gene. The assay showed no crossreaction with other tested animal viruses. The assay is highly sensitive, with a detection limit of as low as 10 plasmid DNA copies and 2.38 × 10-2 pg of viral DNA. The intra and inter--assay coefficients of variation were both low. The positive detection rate of MiCV in clinical samples from minks, foxes, and raccoon dogs were 58.8% (133/226), 50.7% (72/142), and 42.2% (54/128), respectively, giving a total positive detection rate of 52.2% (259/496). Higher contamination levels were observed in samples from the environment in direct or indirect contact with animals, with a total positive detection rate of 75.1% (220/293). These epidemiological results showed that minks, foxes, and raccoon dogs had high infection rates of MiCV. This was also the first study to detect MiCV on the ground and equipment of fur-bearing animal farms. Our assay is highly sensitive and specific for the diagnosis and quantification of MiCV, and should provide a reliable real-time tool for epidemiological and pathogenetic study of MiCV infection. [ABSTRACT FROM AUTHOR]

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is an important regulator of the stress response in mammals, influencing both the hypothalamic–pituitary–adrenal (HPA) axis and the sympathetic nervous system (SNS). PACAP has been reported to influence energy homeostasis, including adaptive thermogenesis, an energy burning process in adipose tissue regulated by the SNS in response to cold stress and overfeeding. While research suggests PACAP acts centrally at the level of the hypothalamus, knowledge of PACAP's role within the sympathetic nerves innervating adipose tissues in response to metabolic stressors is limited. This work shows, for the first time, gene expression of PACAP receptors in stellate ganglia and highlights some differential expression with housing temperature. Additionally, we present our dissection protocol, analysis of tyrosine hydroxylase gene expression as a molecular biomarker for catecholamine producing tissue and recommend three stable reference genes for the normalization of quantitative real time‐polymerase chain reaction (qRT‐PCR) data when working with this tissue. This study adds to information about neuropeptide receptor expression in peripheral ganglia of the sympathetic nervous system innervating adipose tissue and provides insight into PACAP's role in the regulation of energy metabolism. [ABSTRACT FROM AUTHOR]