Exploring Sorbus torminalis Leaves: Unveiling a Promising Natural Resource for Diverse Chemical and Biological Applications.

Sorbus torminalis (L.) Crantz has a rich history of versatile applications spanning the fields of medicine and nutrition. It is noteworthy that the decoction obtained from S. torminalis leaves is a traditional treatment method against both diabetes and stomach disorders. Phytochemical profiling determined by HPLC/MS-MS. The effects of the extracts on cell viability were investigated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method against MDA-MB-231 cell line (human breast adenocarcinoma).The ethanol/water extract contained more concentration of total phenolic (91.41 mg gallic acid (GAE) equivalent /gr) and flavanoid (29.10 mg rutin (RE) equivalent/gr) in the tested extract (p<0.05). Resulting of HPLC analysis, the chemical constituents varied depending on the solvents and chlorogenic acid, hyperoside, isoquercetin, delphindin-3,5-diglucoside, procyanidin B2, epicatechin, neochlorogenic acid, 3,5-dicaffeoylquinic acid were identified in all extracts. Overall, ethanol, n-hexane and ethyl acetate extracts showed the highest inhibition for the tyrosinase enzyme. The effect of leaf extracts of S. torminalis on antimicrobial, biofilm inhibitory, and anticancer activities was examined. Based on outcomes of our study recognize this plant as a critical source of medically active chemicals for feasible phytopharmaceutical and nutraceutical applications, providing the first scientific insight into the detailed biological and chemical profiles of S. torminalis.

Comprehensive investigation of cucumber heat shock proteins under abiotic stress conditions: A multi-omics survey.

Heat-shock proteins (Hsps) are a family of proteins essential in preserving the vitality and functionality of proteins under stress conditions. Cucumber (Cucumis sativus) is a widely grown plant with high nutritional value and is used as a model organism in many studies. This study employed a genomics, transcriptomics, and metabolomics approach to investigate cucumbers' Hsps against abiotic stress conditions. Bioinformatics methods were used to identify six Hsp families in the cucumber genome and to characterize family members. Transcriptomics data from the Sequence Read Archive (SRA) database was also conducted to select CsHsp genes for further study. Real-time PCR was used to evaluate gene expression levels under different stress conditions, revealing that CssHsp-08 was a vital gene for resistance to stress conditions; including drought, salinity, cold, heat stresses, and ABA application. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of plant extracts revealed that amino acids accumulate in leaves under high temperatures and roots under drought, while sucrose accumulates in both tissues under applied most stress factors. The study provides valuable insights into the structure, organization, evolution, and expression profiles of the Hsp family and contributes to a better understanding of plant stress mechanisms. These findings have important implications for developing crops that can withstand environmental stress conditions better.

HSF and Hsp Gene Families in sunflower: a comprehensive genome-wide determination survey and expression patterns under abiotic stress conditions.

Sunflowers belong to the Asteraceae family, which comprises nutrimental and economic oilseed plants. Heat shock proteins (Hsps) are protein families vital for all organisms' growth and survival. Besides the ordinary conditions, the expression of these proteins ascends during abiotic stress factors such as high temperature, salinity, and drought. Using bioinformatics approaches, the current study identified and analyzed HSF and Hsp gene family members in the sunflower (Helianthus annuus L.) plant. HSF, sHsp, Hsp40, Hsp60, Hsp70, Hsp90, and Hsp100 domains were analyzed in the sunflower genome, and 88, 72, 192, 52, 85, 49, and 148 genes were identified, respectively. The motif structures of the proteins in the same phylogenetic tree were similar, and the α-helical form was dominant in all the protein families except for sHsp. The estimated three-dimensional structure of 28 sHsp proteins was determined as ß-sheets. Considering protein-protein interactions, the Hsp60-09 protein (38 interactions) was found to be the most interacting protein. The most orthologous gene pairs (58 genes) were identified between Hsp70 genes and Arabidopsis genes. The expression analysis of selected genes was performed under high temperature, drought, and high temperature-drought combined stress conditions in two sunflower cultivars. In stress conditions, gene expressions were upregulated for almost all genes in the first half and first hours at large. The expressions of HanHSF-45 and HanHsp70-29 genes were raised in two cultivars under high temperature and high temperature-drought combined stress conditions. This study presents a blueprint for subsequent research and delivers comprehensive knowledge of this vital protein domain.

Comparative bioinformatics analysis and abiotic stress responses of expansin proteins in Cucurbitaceae members: watermelon and melon.

Watermelon and melon are members of the Cucurbitaceae family including economically significant crops in the world. The expansin protein family, which is one of the members of the cell wall, breaks down the non-covalent bonds between cell wall polysaccharides, causing pressure-dependent cell expansion. Comparative bioinformatics and molecular characterization analysis of the expansin protein family were carried out in the watermelon (Citrullus lanatus) and melon (Cucumis melo) plants in the study. Gene expression levels of expansin family members were analyzed in leaf and root tissues of watermelon and melon under ABA, drought, heat, cold, and salt stress conditions by quantitative real-time PCR analysis. After comprehensive searches, 40 expansin proteins (22 ClaEXPA, 14 ClaEXPLA, and 4 ClaEXPB) in watermelon and 43 expansin proteins (19 CmEXPA, 15 CmEXPLA, 3 CmEXPB, and 6 CmEXPLB) in melon were identified. The greatest orthologous genes were identified with soybean expansin genes for watermelon and melon. However, the latest divergence time between orthologous genes was determined with poplar expansin genes for watermelon and melon expansin genes. ClaEXPA-04, ClaEXPA-09, ClaEXPB-01, ClaEXPB-03, and ClaEXPLA-13 genes in watermelon and CmEXPA-12, CmEXPA-10, and CmEXPLA-01 genes in melon can be involved in tissue development and abiotic stress response of the plant. The current study combining bioinformatics and experimental analysis can provide a detailed characterization of the expansin superfamily which has roles in growth and reaction to the stress of the plant. The study ensures detailed data for future studies examining gene functions including the roles in plant growth and stress conditions.

Synthesis and characterization of new 1,3,4-thiadiazole derivatives: study of their antibacterial activity and CT-DNA binding.

1,3,4-Thiadiazole molecules (1-4) were synthesized by the reaction of phenylthiosemicarbazide and methoxy cinnamic acid molecules in the presence of phosphorus oxychloride, and characterized with UV, FT-IR, 13C-NMR, and 1H-NMR methods. DFT calculations (b3lyp/6-311++G(d,p)) were performed to investigate the structures' geometry and physiochemical properties. Their antibacterial activity was screened for various bacteria strains such as Enterobacter aerogenes, Escherichia coli ATCC 13048, Salmonella kentucky, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus and Gram positive such as Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 7644, Enterococcus faecium, Enterococcus durans, Staphylococcus aureus ATCC, Serratia marcescens, Staphylococcus hominis, Staphylococcus epidermidis, alfa Streptococcus haemolyticus, Enterococcus faecium and found to have an inhibitory effect on Klebsiella pneumoniae and Staphylococcus hominis, while molecules 1, 3 and 4 had an inhibitory effect on Staphylococcus epidermidis and alpha Streptococcus haemolyticus. The experimental results were supported by the docking study using the Kinase ThiM from Klebsiella pneumoniae. All the investigated compounds showed an inhibitory effect for the Staphylococcus epidermidis protein. In addition, the mechanism of the 1-4 molecule interaction with calf thymus-DNA (CT-DNA) was investigated by UV-vis spectroscopic methods.

Comparative Content, Biological and Anticancer Activities of Heracleum humile Extracts Obtained by Ultrasound-Assisted Extraction Method.

As the safety and effectiveness of synthetic drugs remain in doubt, researchers are trying to develop natural medicines from medicinal plants. Herein, ethyl acetate, methanol and water extracts from the Heracleum humile plant were obtained by an ultrasonic-assisted extraction process and the aim was to evaluate some biological effects of the extracts due to the limited data on the pharmacological properties of Heracleum humile in the literature. Weak antibacterial activity was observed on tested bacterial species. The minimum inhibitory concentration and the minimum bactericidal concentration values ranged from 250 to 500 µg/mL. In addition, cytotoxic activity was determined using the MTT test. The strongest findings were determined for ethyl acetate extract on the MDA-MB-231 cell lines at the 48th  hour (IC50 :97.94 µg/mL), followed by the MCF-7 cell lines at the 24th  hour (IC50 :103.9 µg/mL). All extracts of Heracleum humile contained mainly flavonoids, phenolic acids and their derivatives, i. e., well-known compounds that possess numerous biological activities such as antioxidant, anti-inflammatory, anticancer, antimicrobial etc. The study results could provide important information that Heracleum humile could be a potential candidate as a natural enzyme inhibitor. It can be concluded that these extracts could be useful in the elementary step of improving novel plant-derived multifunctional pharmaceuticals.

Comparative genomic analysis of expansin superfamily gene members in zucchini and cucumber and their expression profiles under different abiotic stresses.

Zucchini and cucumber belong to the Cucurbitaceae family, a group of economical and nutritious food plants that is consumed worldwide. Expansin superfamily proteins are generally localized in the cell wall of plants and are known to possess an effect on cell wall modification by causing the expansion of this region. Although the whole genome sequences of cucumber and zucchini plants have been resolved, the determination and characterization of expansin superfamily members in these plants using whole genomic data have not been implemented yet. In the current study, a genome-wide analysis of zucchini (Cucurbita pepo) and cucumber (Cucumis sativus) genomes was performed to determine the expansin superfamily genes. In total, 49 and 41 expansin genes were identified in zucchini and cucumber genomes, respectively. All expansin superfamily members were subjected to further bioinformatics analysis including gene and protein structure, ontology of the proteins, phylogenetic relations and conserved motifs, orthologous relations with other plants, targeting miRNAs of those genes and in silico gene expression profiles. In addition, various abiotic stress responses of zucchini and cucumber expansin genes were examined to determine their roles in stress tolerance. CsEXPB-04 and CsEXPA-11 from cucumber and CpEXPA-20 and CpEXPLA-14 from zucchini can be candidate genes for abiotic stress response and tolerance in addition to their roles in the normal developmental processes, which are supported by the gene expression analysis. This work can provide new perspectives for the roles of expansin superfamily genes and offers comprehensive knowledge for future studies investigating the modes of action of expansin proteins. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01108-w.

Chemical characterization, computational analysis and biological views on Daphne gnidioides Jaub. & Spach extracts: Can a new raw material be provided for biopharmaceutical applications?

The scientific world tends to turn to natural products such as medicinal and aromatic plants because of the inadequacy of commercially available synthetic drugs as antibiotics or anticancer, and their adverse effects on healthy tissues. One of these plants is Daphne gnidioides Jaub. & Spach, which belongs to the Thymelaeaceae family, and there is no data in the literature on its biological activity. This study is aimed to elucidate the chemical profiles and in vitro anticancer, antibacterial and DNA protection and enzyme inhibitory properties of methanol extracts of root, stem, and leaf of D. gnidioides Jaub. & Spach. Polyphenolic components of the extracts were characterized by HPLC-MS/MS. The highest phenolic content was detected in the leaf extract (TIPC = 43.5 ± 0.5 mg/g DE), followed by stem (TIPC = 27.3 ± 0.7 mg/g DE) and root (TIPC = 18.3 ± 0.2 mg/g DE) extracts. Vicenin-2 and 3-O-p-coumaroyl-5-O-caffeoylquinic acid were the main identified compounds in leaf and both root and stem extracts, respectively. The extracts did not show any protective effect on DNA against experimental Fenton's reagent. The minimum inhibitory concentration and the minimum bactericidal concentration values for the root and leaf extracts against tested bacterial strains ranged from 31.25 to 500 µg/mL. After 48 h interaction of the cancer cell lines with the extracts, only the stem extract had significant cytotoxicity on HeLa cells (IC50 = 86.16 µg/mL). No remarkable activity of the extracts, which was tested against MDA-MB-231, was detected (IC50 > 1000 µg/mL). These data showed that D. gnidioides Jaub. & Spach stem extract inhibited the survival of HeLa cells in a time-dependent manner. After the treatment of IC50 concentration of stem extract with HeLa cells, an increase in LC3-II autophagic gene expression was detected. Also, the extracts exhibited significant tyrosinase inhibitory effects which were confirmed by molecular docking. To sum up, the tested extracts could be used as a starting point for the development of new multifunctional drugs.

Utilisation of Rhododendron luteum Sweet bioactive compounds as valuable source of enzymes inhibitors, antioxidant, and anticancer agents.

Ethnobotanical evidences report the use of Rhododendron luteum Sweet (Ericaceae) in traditional medicinal systems. However, R. luteum has been associated to the occurrence of 'mad honey' poisoning. In the present study, the ethyl acetate, methanol, and water extracts of R. luteum were investigated for their in vitro antioxidant, enzyme inhibition, and cytotoxic properties. The cytotoxicity of R. luteum extracts on A549 lung cancer cell line was evaluated using MTT cell viability assay. Besides, HPLC-ESI-MSn approach was employed to elucidate the secondary metabolite profiles of R. luteum in order to establish any structure-activity relationship. Methanol and water extracts of R. luteum possessed highest radical scavenging and reducing properties while the ethyl acetate extract showed highest metal chelating properties. In terms of enzyme inhibition, the methanol and ethyl acetate extracts of R. luteum, possessing epigallocatechin, were active inhibitors of cholinesterase enzymes, α-glucosidase, and tyrosinase. Water extract caused growth inhibition of A549 cells with 207.2 µg/ml IC50 value. Though R. luteum has received little scientific attention due to the occurrence of grayanotoxins in the plant, however, data presented in this work shows promising biological activity of R. luteum and highlighted its role as a potential source of antioxidant and key enzyme inhibitors.

Biological synthesis of silver nanoparticles using Rheum ribes and evaluation of their anticarcinogenic and antimicrobial potential: A novel approach in phytonanotechnology.

This paper reports the anticarcinogenic and antimicrobial properties of silver nanoparticles (Ag NPs) obtained by green synthesis using the extract of Rheum ribes (R. ribes), a medicinal plant. For the synthesis of Ag NPs, the ethanolic extracts of R. ribes were used as a reducing as well as the stabilizing agent. For the characterization of Ag NPs, advanced analytical methods such as transmission electron microscopy (TEM), X-Ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and UV-vis spectrophotometry were performed. The synthesized Ag NPs obtained from R. ribes were evaluated as a cytotoxic agent against MDA-MB-231 breast carcinoma cell line. The IC50 values of the nanoparticles were ranged from 165 to 99 µg/mL against MDA-MB 231 cell line for 24 h and 48 h, respectively. The results show that the use of Ag NPs at low concentrations show the toxic effect in the cancer cells. In addition, the results of experiments on gram-positive (Staphylococcus aureus (S. aureus), Methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus subtilis (B. subtilis)) and gram-negative (Escherichia coli (E. coli)) bacteria showed that the Ag NPs had high antimicrobial activity. The results suggest that Ag NPs can be developed as potential anticancer and antibacterial agents.

Biogenic platinum nanoparticles using black cumin seed and their potential usage as antimicrobial and anticancer agent.

Herein, the biogenic platinum nanoparticles (Pt NPs) were synthesized by using black cumin seed (Nigella sativa L.) extract as a reducing agent. The biogenic platinum nanoparticles synthesized by black cumin seed extract was characterized in detail by Transmission Electron Microscopy (TEM), UV-vis spectrophotometer, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS). According to TEM analysis, Pt nanoparticles have spherical shapes and sizes between 1-6 nm. Moreover, the biogenic Pt NPs was assessed for its cytotoxicity effect on MDA-MB-231 breast and HeLa cervical cancer lines and their antibacterial effect against selected strains of gram-positive and negative bacteria. The cytotoxicity and bacterial tests showed the effectiveness of biogenic Pt nanoparticles. Dose-dependent toxicity effects were shown in the MDA-MB-231 breast and HeLa cervical cancer lines (IC50: 36.86 µg/mL and 19.83 µg/mL, respectively). In addition, Pt NPs showed high zone diameters against gram-positive and gram-negative bacteria at concentrations of 100 and 500 µg/ml. These results contribute to the development of the pharmaceutical industry as a potential antibacterial and anticancer agent.

Pharmacological and polyphenolic profiles of Phyllanthus phillyreifolius var. commersonii Müll. Arg: An unexplored endemic species from Mauritius.

Phyllanthus phillyreifolius var. commersonii Müll. Arg is an endemic plant of Mauritius. To date, no study has been performed concerning its polyphenolic profile and pharmacological properties. In this study, a decoction (water), ethyl acetate and methanol extracts of the aerial parts of P. phillyreifolius, obtained from different extraction procedures (maceration and Soxhlet), were studied for antibacterial, antioxidant, anticancer, and enzyme inhibitory properties along with their polyphenolic profile. The ethyl acetate macerated extract showed high antibacterial activity against B. cereus (MIC = 0.293 mg/mL) and E. coli (MIC = 0.417 mg/mL) while S. epidermidis was most susceptible to the ethyl acetate-Soxhlet extract (MIC = 0.521 mg/mL). The methanol-Soxhlet extract displayed the most potent cupric and ferric reducing power, and metal chelating effect, while the macerated methanolic extract was the most effective DPPH and ABTS scavenger, and BChE inhibitor. Only the ethyl acetate-Soxhlet extract exhibited α-glucosidase inhibition. All extracts exhibited a strong anti-tyrosinase activity, which was further investigated by molecular docking and molecular dynamic. After 48 h exposure to the extracts for HeLa cell lines, the ethyl acetate-Soxhlet extract showed the highest inhibition (IC50 = 533.1 µg/mL) while the decoction extract was more cytotoxic to MDA-MB-231 cells (IC50 = 337.4 µg/mL). Treatment of cancer cell lines with all P. phillyreifolius extracts resulted in a time-dependent reduction of cell viability for HeLa and dose-and time-dependent reduction for MDA-MB-231. Gene expression ratio of Bcl-2 to Bax was higher for all Soxhlet-extracts. Total phenolics (TPC) and flavonoids (TFC) content were highest in the decoction and methanol-Soxhlet extract, respectively (122.43 mg GAE/g extract and 31.28 mg RE/g extract, respectively). The extracts were abundant in ellagitannins, although phenolic acids and flavonoids were also detected. Granatin B was detected for the first time in Phyllanthus species. Overall, the aerial parts of P. phillyreifolius exemplify a potent reservoir of bioactive phytochemicals for therapeutic applications.

Identification of watermelon heat shock protein members and tissue-specific gene expression analysis under combined drought and heat stresses.

Heat shock protein (Hsp) gene family members in the watermelon genome were identified and characterized by bioinformatics analysis. In addition, expression profiles of genes under combined drought and heat stress conditions were experimentally analyzed. In the watermelon genome, 39 genes belonging to the sHsp family, 101 genes belonging to the Hsp40 family, 23 genes belonging to the Hsp60 family, 12 genes belonging to the Hsp70 family, 6 genes belonging to the Hsp90 family, and 102 genes belonging to the Hsp100 family were found. It was also observed that the proteins in the same cluster in the phylogenetic trees had similar motif patterns. When the estimated 3-dimensional structures of the Hsp proteins were examined, it was determined that the α-helical structure was dominant in almost all families. The most orthologous relationship appeared to be between watermelon, soybean, and poplar in the ClaHsp gene families. For tissue-specific gene expression analysis under combined stress conditions, expression analysis of one representative Hsp gene each from root, stem, leaf, and shoot tissues was performed by real-time PCR. A significant increase was detected usually at 30 min in almost all tissues. This study provides extensive information for watermelon Hsps, and can enhance our knowledge about the relationships between Hsp genes and combined stresses.

Analysis of DNA protection, interaction and antimicrobial activity of isatin derivatives.

Isatin, thiosemicarbazone and their derivatives have been widely used in biological applications such as antimicrobial, antiviral and anticancer therapies. Herein, eight isatin and thiosemicarbazone derivative compounds were re-synthesized and evaluated for DNA binding analysis including DNA protection studies using plasmid DNA (pUC19) and DNA interaction experiments using calf thymus DNA (CT-DNA). All compounds were also utilized in vitro assay to assess the antimicrobial activity of compounds against different pathogenic bacterial strains. All isatin and thiosemicarbazone derivative compounds exhibited DNA protection activity which ranged from 23.5 to 59.5%. Among them, I3-(N-2-MP)-TSC had the greatest DNA protective activity. For DNA binding analysis, all compounds had the same constant concentration (40 µM), which interacts with CT-DNA. It was also observed that DNA interactions gave a high intrinsic binding constant (Kb = 1.72 × 104 M-1-9.73 × 105 M-1). Besides, several derivatives of isatin thiosemicarbazone exhibited significant and selective antibacterial activity with low concentration. These compounds primarily affected Gram-positive bacteria, but were not effective against P. vulgaris and E. coli. The Gram-positive methicillin-resistant S. aureus ATCC 43300 (MRSA) was the most influenced strain by these compounds. It was found that methyphenyl group at isatin was essential for its antibacterial activity for MRSA.

Investigations into the therapeutic potential of Asphodeline liburnica roots: In vitro and in silico biochemical and toxicological perspectives.

This study aims to establish the biological and chemical profile of Asphodeline liburnica (Scop.) Rchb. root. The antioxidant, antimicrobial, enzyme inhibitory, DNA protection, apoptotic DNA ladder fragmentation analysis, and anti-proliferative of A. liburnica were established using standard assays. In silico study was also performed to understand interactions between quantified anthraquinones and key enzymes of clinical relevance. Total phenolic and flavonoid contents were found to be 9.67 mgGAE/g and 1.48 mgRE/g extract, respectively. Chrysophanol was detected as a major anthraquinone. The extract exhibited radical scavenging ability against DPPH and ABTS with values of 13.23 and 66.99 mgTE/g extract, respectively. Good inhibitory activity against tyrosinase was recorded. In silico experiments showed that the anthraquinones were able to establish coordinative bonds with the copper atoms present in the enzymatic cavity of tyrosinase. MTT cell viability test on MDA-MB-231 cells showed that at 0.1 and 1 µg of extracts induced anti-proliferative effect. Apoptotic DNA fragmentation analysis indicated nuclear condensation resulting in DNA fragmentation, which exhibited apoptotic cell death in the presence of A. liburnica. This study has provided insights on the potential usage of A. liburnica which could open new avenues for research and stimulate future interest for the development of safe novel biopharmaceuticals.

Novel in vitro and in silico insights of the multi-biological activities and chemical composition of Bidens tripartita L.

Bidens tripartita L. is a traditional phyto-remedy used in several countries, yet there is still a paucity of data on its biological potential. We aimed to provide new insights on the pharmacological potential of extracts prepared from B. tripartita via highlighting its antioxidant, key enzymes inhibitory potency, and DNA protecting effects. Phytochemical profile was established using High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) and bioactive compound(s) docked against target enzymes using in silico methods. Cytotoxicity against three cancer cell lines was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) cell viability test. The main compounds were luteolin-7-glucoside (cynaroside), chlorogenic acid, and epicatechin in the extracts. The methanol extract exhibited the highest radical scavenging activity. Ethyl acetate extract showed strongest α-amylase inhibitory activity, while the best α-glucosidase inhibitory effect recorded for the methanol extract. Molecular docking showed that cynaroside strongly interact to α-glucosidase cavity by establishing six hydrogen bonds. B. tripartita extracts were found to protect supercoiled form of pUC19 plasmid (>70%) and also showed anti-proliferative properties. Results amassed in the present study add on to a growing body of literature on the multi-pharmacological potency of B. tripartita which can be applied to bio-products development geared towards management of common diseases.

Innate immune and growth promoting responses to caper (Capparis spinosa) extract in rainbow trout (Oncorhynchus mykiss).

Cytokine responses, non-specific immune activity and growth promotion effect of dietary caper (Capparis spinosa) supplementation were examined in rainbow trout (Oncorhynchus mykiss). Rainbow trout (12.04 ± 0.71 g) were fed diets containing three doses of caper methanolic extract [0 (Control), 0.1 and 0.5 g kg(-1) of feed] for 30 days. At the end of the feeding trial, expression levels of cytokine genes that included IL-1ß, IL-8, TGF-ß, IL-12p40, TNF-α1 and IL-10 in head kidney was analyzed using qRT-PCR, and blood and serum were collected to determine superoxide anion production (SAP), phagocytic, lysozyme and myeloperoxidase activities. Expression levels of all cytokines, except TNF-α1 were elevated in the 0.1 g kg(-1) caper extract fed fish group compared to other groups. In 0.5 g kg(-1) caper extract treated fish, only IL-12p40 and IL-10 genes were up-regulated compared to control group fish. SAP was increased in both caper extract treated groups compared to the control, and the highest level was observed in the 0.1 g kg(-1) group. Phagocytic activity in both the caper extract treated groups was increased compared to control with no differences observed between those groups. Lysozyme and myeloperoxidase activities were recorded to be the highest in the 0.1 g kg(-1) fed fish group compared to other groups. Growth promotion was affected positively when caper doses were increased. Survival rate was significantly higher in 0.1 and 0.5 g kg(-1) caper extract treated fish groups compared to control (P < 0.05) after challenged with Aeromonas hydrophila. These results indicate that caper extract stimulates innate immunity through cytokine-mediated responses and promote growth in rainbow trout.

Free circulating nucleic acids in plasma and serum as a novel approach to the use of internal controls in real time PCR based detection.

Internal controls (ICs), are the main components of any real-time PCR based amplification methods, which are co-purified and co-amplified with the actual target. The existence of free circulating nucleic acids in plasma and serum (CNAPS) has been known for many years. The aim of this study was to verify whether CNAPS can be used as ICs in real-time PCR based detection and quantification of DNA or RNA targets in plasma and serum samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene, was chosen at random as CNAPS to serve as an intrinsic internal control in two different real-time PCR based quantification models in plasma and serum. Viral loads of hepatitis B virus (HBV) DNA and hepatitis delta virus (HDV) RNA were quantified as actual targets in parallel to GAPDH as IC in a total of 519 serum or plasma samples including 21 healthy controls, 202 positive chronic hepatitis delta patients, 37 chronic hepatitis C patients, 168 chronic hepatitis B patients, 52 patients with hepatocellular carcinoma, and 39 patients with non-alcoholic steatohepatitis/non-alcoholic fatty liver disease. GAPDH levels did not show significant variance in different patient groups and yielded positive signals in all 519 patients with persistent cycle threshold (CT) values 27.85±1.57 (mean±standard deviation (SD)). Reproducibility of the GAPDH amplification in HDV RNA and HBV DNA quantifications was shown with a SD value of CT ranging from 0.42 to 2.14 (mean SD; 1.18) and 0.24 to 1.75 (mean SD; 1.03), respectively. In conclusion, the freely circulating nucleic acids can clearly be used as internal controls for real-time PCR based detection and quantification of any RNA and mainly DNA targets (pathogens) in serum or plasma and this simply excludes the compulsory external addition of any IC molecules into the reaction.

A one step real time PCR method for the quantification of hepatitis delta virus RNA using an external armored RNA standard and intrinsic internal control.

BACKGROUND: Hepatitis delta virus (HDV) RNA viral load measurement is critical in diagnosis and monitoring the response to antiviral treatment. OBJECTIVES: Our aim is to design a real time PCR method for accurate quantitation of HDV RNA in clinical specimens using an armored RNA as external standard, and an intrinsic internal control. STUDY DESIGN: A plasmid bearing delta antigen region of genotype I HDV genome was used to develop an armored RNA. Serial dilutions of the armored HDV RNA standard with 10(12)copy/mL were used as standards for quantitation. A primer-probe set derived from HDAg region was used in one step EZ RT PCR kit chemistry which uses rTth enzyme allowing reverse transcription and polymerization in the same tube. The kit also uses the advantage of uracil-N-glycosylase (UNG) enzyme treatment to prevent PCR contamination. RESULTS: The established assay has a dynamic range of 10(2)-10(11)copy/mL with a PCR efficiency of 96.9%. Detection limit was 858±32copy/mL with 95% confidence interval. Intra- and inter-assay variabilities were low for high, medium and low levels of viremia. Incorporation of freely circulating GAPDH in serum into the assay as an intrinsic internal control prevented false negative results and failures in PCR amplifications due to inhibitors, inefficient extraction procedures or enzymatic reactions. CONCLUSION: In conclusion, this study defines a novel assay for sensitive and reliable quantification of HDV RNA using an armored HDV RNA as a standard and GAPDH in plasma or serum as an intrinsic internal control in a single tube.