Catalase (CAT) Gene Family in Oil Palm (Elaeis guineensis Jacq.): Genome-Wide Identification, Analysis, and Expression Profile in Response to Abiotic Stress.

Catalases (CATs) play crucial roles in scavenging H2O2 from reactive oxygen species, controlling the growth and development of plants. So far, genome-wide identification and characterization of CAT genes in oil palm have not been reported. In the present study, five EgCAT genes were obtained through a genome-wide identification approach. Phylogenetic analysis divided them into two subfamilies, with closer genes sharing similar structures. Gene structure and conserved motif analysis demonstrated the conserved nature of intron/exon organization and motifs among the EgCAT genes. Several cis-acting elements related to hormone, stress, and defense responses were identified in the promoter regions of EgCATs. Tissue-specific expression of EgCAT genes in five different tissues of oil palm was also revealed by heatmap analysis using the available transcriptome data. Stress-responsive expression analysis showed that five EgCAT genes were significantly expressed under cold, drought, and salinity stress conditions. Collectively, this study provided valuable information on the oil palm CAT gene family and the validated EgCAT genes can be used as potential candidates for improving abiotic stress tolerance in oil palm and other related crops.

Linking antibiotic resistance gene patterns with advanced faecal pollution assessment and environmental key parameters along 2300 km of the Danube River.

The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, blaTEM with intermediate abundance and blaOXA-48like, blaCTX-M-1 group, blaCTX-M-9 group and blaKPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.

Selection and Validation of Reference Genes for Gene Expression in Bactericera gobica Loginova under Different Insecticide Stresses.

Insecticide resistance has long been a problem in crop pest control. Bactericera gobica is a major pest on the well-known medicinal plants Lycium barbarum L. Investigating insecticide resistance mechanisms of B. gobica will help to identify pesticide reduction strategies to control the pest. Gene expression normalization by RT-qPCR requires the selection and validation of appropriate reference genes (RGs). Here, 15 candidate RGs were selected from transcriptome data of B. gobica. Their expression stability was evaluated with five algorithms (Delta Ct, GeNorm, Normfinder, BestKeeper and RefFinder) for sample types differing in response to five insecticide stresses and in four other experimental conditions. Our results indicated that the RGs RPL10 + RPS15 for Imidacloprid and Abamectin; RPL10 + AK for Thiamethoxam; RPL32 + RPL10 for λ-cyhalothrin; RPL10 + RPL8 for Matrine; and EF2 + RPL32 under different insecticide stresses were the most suitable RGs for RT-qPCR normalization. EF1α + RPL8, EF1α + ß-actin, ß-actin + EF2 and ß-actin + RPS15 were the optimal combination of RGs under odor stimulation, temperature, developmental stages and both sexes, respectively. Overall, EF2 and RPL8 were the two most stable RGs in all conditions, while α-TUB and RPL32 were the least stable RGs. The corresponding suitable RGs and one unstable RG were used to normalize a target cytochrome P450 CYP6a1 gene between adult and nymph stages and under imidacloprid stress. The results of CYP6a1 expression were consistent with transcriptome data. This study is the first research on the most stable RG selection in B. gobica nymphs exposed to different insecticides, which will contribute to further research on insecticide resistance mechanisms in B. gobica.

Seasonal Dynamics of Various Scab-Causing Streptomyces Genotypes Among Potato Fields on Prince Edward Island.

Potato common scab is an important bacterial plant disease caused by numerous Streptomyces species and strains. A better understanding of the genetic diversity and population dynamics of these microorganisms in the field is crucial to develop effective control methods. Our research group previously studied the genetic diversity of scab-causing Streptomyces spp. in Prince Edward Island, one of Canada's most important potato-growing provinces. Fourteen distinct Streptomyces genotypes were identified and displayed contrasting aggressiveness toward potato tubers. To better understand the distribution and occurrence of these genotypes over time under field conditions, the population dynamics were studied in nine commercial potato fields throughout a growing season. A comparative genomic-driven approach was used to design genotype-specific primers and probes, allowing us to quantify, using quantitative polymerase chain reaction, the abundance of each of the 14 genotypes in field soil. Thirteen of the previously identified genotypes were detected in at least one soil sample, with various frequencies and population sizes across the different fields under study. Interestingly, weakly virulent genotypes dominated, independent of time or location. Among them, three genotypes accounted for more than 80% of the genotypes' combined population. Although the highly virulent genotypes were detected in lower relative abundance than the weakly virulent ones, an increase in the highly virulent genotypes' population size was observed over the growing season in most fields. The results will ultimately be useful for the development of targeted common scab control strategies.

Reference Genes Screening and Gene Expression Patterns Analysis Involved in Gelsenicine Biosynthesis under Different Hormone Treatments in Gelsemium elegans.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an accurate method for quantifying gene expression levels. Choosing appropriate reference genes to normalize the data is essential for reducing errors. Gelsemium elegans is a highly poisonous but important medicinal plant used for analgesic and anti-swelling purposes. Gelsenicine is one of the vital active ingredients, and its biosynthesis pathway remains to be determined. In this study, G. elegans leaf tissue with and without the application of one of four hormones (SA, MeJA, ETH, and ABA) known to affect gelsenicine synthesis, was analyzed using ten candidate reference genes. The gene stability was evaluated using GeNorm, NormFinder, BestKeeper, ∆CT, and RefFinder. The results showed that the optimal stable reference genes varied among the different treatments and that at least two reference genes were required for accurate quantification. The expression patterns of 15 genes related to the gelsenicine upstream biosynthesis pathway was determined by RT-qPCR using the relevant reference genes identified. Three genes 8-HGO, LAMT, and STR, were found to have a strong correlation with the amount of gelsenicine measured in the different samples. This research is the first study to examine the reference genes of G. elegans under different hormone treatments and will be useful for future molecular analyses of this medically important plant species.

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla.

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

Eugenol isolated from supercritical fluid extract of Ocimum sanctum: a potent inhibitor of DENV-2.

Dengue is one of the fairly prevalent viral infections at the world level transmitted through mosquitoes (Aedes aegypti and Aedes albopictus). Due to various environmental factors, dengue cases surged rapidly at the global level in recent decades, with 193245 cases in 2021 and an increment of 110473 cases in 2022. There is no antidote available against dengue and other flaviviruses. In the absence of a dengue vaccine or specific antiviral, medicinal plants or their products can be the only choice for its effective management. Ocimum sanctum is known as ''The Incomparable One,'' ''Mother Medicine of Nature'' and ''Queen of Herbs'' in Ayurveda, and is considered an "elixir of life" supreme in both healthcare and spiritual terms. In present study eugenol was isolated in O.sanctum. Eugenol (1-hydroxy-2-methoxy-4-allylbenzene) has been substantially responsible for its therapeutic potential. High-performance thin-layer chromatography, Fourier transform infrared spectroscopy and ultraviolet-visible spectroscopy were applied to identify the compound. The Rf value of isolated compound was same in the chromatogram (0.69 + 0.05) with compare to standard. The safe dose of plant and eugenol were found as < 31.25 µg/ml and < 15.62 µg/ml. The anti-dengue activity was assessed in C6/36 cell lines, their effect was determined through Quantitative PCR. The NMR of the isolated eugenol showed similar properties as the commercial marker compound. The eugenol and SFE extract of O. sanctum showed the inhibition of 99.28% and completely against Dengue-2, respectively. Docking study exposed that the interaction of eugenol with NS1 and NS5 dengue protein showed the binding energy as - 5.33 and - 5.75 kcal/mol, respectively. The eugenol from the O. sanctum plant has the potential to be a good source of future treatment medications for dengue illness, as well as a valuable tool in its successful management.

Experimental sickness reduces hypocretin receptor 1 expression in the lateral hypothalamus and ventral tegmental area of female mice.

Recent studies have focused on how sickness behaviours, including lethargy, are coordinated in the brain in response to peripheral infections. Decreased hypocretin (orexin) signalling is associated with lethargy and previous research suggests that hypocretin signalling is downregulated during sickness. However, there are studies that find increases or no change in hypocretin signalling during sickness. It is further unknown whether hypocretin receptor expression changes during sickness. Using lipopolysaccharide (LPS) to induce sickness in female mice, we investigated how LPS-injection affects gene expression of hypocretin receptors and prepro-hypocretin as well as hypocretin-1 peptide concentrations in brain tissue. We found that hypocretin receptor 1 gene expression was downregulated during sickness in the lateral hypothalamus and ventral tegmental area, but not in the dorsal raphe nucleus or locus coeruleus. We found no changes in hypocretin receptor 2 expression. Using a gene expression calculation that accounts for primer efficiencies and multiple endogenous controls, we were unable to detect changes in prepro-hypocretin expression. Using radioimmunoassay, we found no change in hypocretin-1 peptide in rostral brain tissue. Our results indicate that hypocretin receptor expression can fluctuate during sickness, adding an additional level of complexity to understanding hypocretin signalling during sickness.

Stress-associated ß -glucan administration stimulates the TLR - MYD88 - NFKB1 signaling pathway in Nile tilapia (Oreochromis niloticus).

There is evidence that the administration of ß-glucan can effectively activate several defense mechanisms, such as the Tlr-Myd88-Nfkb1 pathway that induces the expression of immune cytokines. Thus, the objective of this work was to evaluate whether ß-glucan acts on the mechanisms of gene transcription via the Tlr-Myd88-Nfkb1 pathway in Nile tilapia under stress after challenge with Streptococcus agalactiae. Therefore, we evaluated the expression of immune system genes such as toll-like receptors 1 (tlr1), toll-like receptors 2 (tlr2), primary myeloid differentiation response gene (myd88) and nuclear factor kappa B1 (nfkb1). A total of 408 fish were distributed in 24 polyethylene boxes and randomly divided into eight groups with 3 replications each: C15: Tilapias received a control diet (free of ß-glucan) for 15 days and were sampled after the 15th day of the experiment; C15D: Tilapias received a control diet (free of ß-glucan) for 15 days, were challenged on the 14th day and were sampled at the 15th day of the experiment; ß15: Tilapias received experimental diet (1g kg-1 of ß-glucan) for 15 days and were sampled after 15 days; ß15D: Tilapias received an experimental diet (1g kg-1 of ß-glucan) for 15 days, were challenged on the 14th day and were sampled at the 15th day of the experiment; C30: Tilapias received a control diet (free of ß-glucan) for 30 days and were sampled on the 30th day of the experiment; C30D: Tilapias received a control diet (free of ß-glucan) for 30 days, were challenged on the 29th day and were sampled at the 30th day of the experiment; ß30: Tilapias received experimental diet (1g kg-1 of ß-glucan) for 30 days and were sampled after 30 days and ß30D: Tilapias received experimental diet (1g kg-1 of ß-glucan) for 30 days, were challenged on the 29th day and were sampled at 30 of the experiment. In the fish sampled at 15 and 30 days of the experiment, after being anesthetized and killed by brain section, cranial kidney and spleen were collected for gene expression analysis. The analyzes showed that the association of ß-glucan and stressful management modulated the immune system, using the Tlr-Myd88-Nfkb1 signaling pathway, indicating that this compound can be used to promote early defense and protect fish against diseases.

Expression of MicroRNA-200a/b/c in the Mediobasal Hypothalamic Nuclei with Aging.

BACKGROUND: MicroRNAs (miRNAs) belong to small non-coding RNAs that coordinate the expression of cellular genes at the post-transcriptional level. The hypothalamus is a key regulator of homeostasis, biological rhythms and adaptation to different environmental factors. It also participates in the aging regulation. Variations in miRNA expression in the hypothalamus can affect the aging process. OBJECTIVE: Our objective of this study is to examine the expression of miR-200a-3p, miR-200b-3p, miR-200c-3p in the dorsomedial (DMN), ventromedial (VMN) and arcuate (ARN) nuclei of the hypothalamus in male and female rats during aging. METHODS: The expression of miR-200a-3p, miR-200b-3p, and miR-200c-3p in DMN, VMN and ARN was studied by qPCR-RT. The results were presented using the 2-ΔΔCq algorithm. RESULTS: The expression of miR-200a-3p, miR-200b-3p, miR-200c-3p microRNAs decreases with aging in the DMN of males and in the VMN of females. The level of miR-200b-3p expression decreased in aged males in the VMN and females in the DMN. The expression of miR-200c-3p declined in aged males in the ARN and in females in the DMN. The expression of miR-200a-3p, miR-200b-3p, and miR-200c-3p did not change in females in the ARN in aging. CONCLUSION: We found a decrease in the expression of members of the miR-200a-3p, miR-200b-3p, and miR-200c-3p in the tuberal hypothalamic nuclei and their sex differences in aging rats.

The Effect of Delivery Matrix on Bifidobacterium animalis subsp. lactis HN019 Survival through In Vitro Human Digestion.

Bifidobacterium animalis subsp. lactis HN019 is a probiotic with several documented human health benefits. Interest in probiotics has led to the development of new formats that probiotics, including HN019, can be supplemented into. In this study, we looked at common HN019 formats such as frozen culture and freeze-dried powder as well as supplementing it into the following food matrices: yogurts (dairy, soy, and oat based), xanthan gum-based tablets, pulpless orange juice, whey sports drink, and dark chocolate (70% cocoa). In this work, our aim was to investigate whether the food matrix that carried HN019 via simulated human digestion (a dual model system mimicking both upper and lower gastrointestinal digestion) influenced probiotic delivery. To that end, we validated and used a real-time qPCR assay to detect HN019 after simulated digestion. In addition, we also measured the effect on a panel of metabolites. After simulated digestion, we were able to detect HN019 from all the matrices tested, and the observed changes to the metabolite profile were consistent with those expected from the food matrix used. In conclusion, this work suggests that the food matrix supplemented with HN019 did not interfere with delivery to the colon via simulated human digestion.

Quantification of arbuscular mycorrhizal fungi root colonization in wheat, tomato, and leek using absolute qPCR.

Arbuscular mycorrhizal fungi (AMF) form symbioses with most terrestrial plants and are known to have a positive effect on plant growth and health. Different methodologies have been developed to assess the AMF-plant symbiosis. The most applied method, which involves staining of roots and microscopic observation of the AMF structures, is tedious and time-consuming and the results are highly dependent on the observer. Using quantitative polymerase chain reaction (qPCR) to quantify AMF root colonization represents a reliable, high-throughput technique that allows the assessment of numerous samples. Quantification with qPCR can be performed through two methods: relative quantification and absolute quantification. In relative quantification, the target gene is normalized with a reference gene. On the other hand, absolute quantification involves the use of a standard curve, for which template DNA is serially diluted. In a previous paper, we validated the primer pair AMG1F and AM1 for a relative quantification approach to assess AMF root colonization in Petunia. Here, we tested the same primers with an absolute quantification approach and compared the results with the traditional microscopy method. We evaluated the qPCR method with three different crops, namely, wheat (cv. Colmetta and Wiwa), tomato, and leek. We observed a strong correlation between microscopy and qPCR for Colmetta (r = 0.90, p < 0.001), Wiwa (r = 0.94, p < 0.001), and tomato (r = 0.93, p < 0.001), but no correlation for leek (r = 0.27, p = 0.268). This highlights the importance of testing the primer pair for each specific crop.

Identification of sex-specific markers based on gene expression in adult Colorado potato beetles.

The Colorado potato beetle, Leptinotarsa decemlineata, (Coleoptera: Chrysomelidae) is an economically important pest insect of potatoes. Understanding how the mechanisms driving its invasiveness vary between sexes will be critical for developing modern control methods. However, the currently available methods for sexing adult Colorado potato beetles are either inefficient or unsuitable for projects that require RNA as an input, like those measuring gene expression. Therefore, the development of simple molecular tools that are tailored to these studies is important. In this study, we used publicly available RNA-seq data to select 5 candidate genes for sex-specific markers in adult Colorado potato beetles. We confirmed that our 5 marker candidates exhibit a sex-specific expression pattern and can be used as PCR markers for sex determination. This method of sex detection will allow researchers to distinguish the sex of the individual with a simple PCR reaction using cDNA as the template and assign sex to RNA-seq samples post hoc.

The effects of intracellular iron availability on the outcome of Toxoplasma gondii infection in mice.

Toxoplasma gondii (T. gondii) is a parasite that obtains the iron it needs for its own metabolism from the host-cell iron pool. In this work, we aimed to investigate if iron supplementation or deficiency affected the course of T. gondii infection. Eighty mice were divided into four groups, each with 20 animals: Group (I): Uninfected control group. Group (II): Infected control group: injected with Phosphate buffered saline. Group (III): Infected group: received iron sucrose treatment. Group (IV): Infected group: treated with deferoxamine. Quantitative PCR studies were performed on days 3 and 8 post-infection to detect the expression of iron metabolism genes (hamp and ferroprotin) and immune-histochemical analysis to study the percentage of TNF-α and TGF-ß tissue expression. Iron supplementation induced progressions of infection evident by increased tissue expression of pro-inflammatory cytokine TNF-α and downregulation of TGF-ß which is mostly linked to suppression of the inflammatory process caused by T. gondii. Increased expression of TGF-ß and decreased expression of TNF-α was noticed when iron deprivation occurred. On day 3, we noticed increased expression in the hamp gene with iron supplementation while it decreases when the iron supply is low. On the contrary, iron deficiency increased ferroprotin gene expression whereas supplementing decreased it. On day 8, the level of expression of these genes returned to normal levels. These observations document the potential role of iron in controlling toxoplasmosis infection and indicate that the transcription of hamp and ferroprotin in T. gondii-infected cells appears to be regulated by a sophisticated indirect mechanism.

Bacterial Inhibition Mechanism of Rhamnolipid-Modified ß-Carotene/Rutinoside Complex Liposomes.

In this study, a new cholesterol-free delivery system named RL-ßC-Rts was developed using rhamnolipid (RL) as the surfactant and encapsulating both ß-carotene (ßC) and rutinoside (Rts). The purpose was to examine its antibacterial properties against four food-borne pathogenic microorganisms including Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. m), and Salmonella typhimurium (S. typhimurium) and to investigate the mechanism behind the inhibition. Results from bacterial viability tests and minimum inhibitory concentration (MIC) showed RL-ßC-Rts possessed antibacterial activity. Upon further examination of the cell membrane potential, it was observed that E. coli, S. aureus, L. m, and S. typhimurium exhibited a reduction in mean fluorescence intensity by 50.17%, 34.07%, 34.12%, and 47.05%, respectively. These decreases suggested damage to the structure of the cell membrane, which subsequently resulted in the discharge of proteins from the bacteria and the consequential impairment of crucial functions. This was supported by alterations in protein concentration. The results of the RT-qPCR showcased that the expression of genes associated with energy metabolism, tricarboxylic acid cycle, DNA metabolism, virulence factor formation and cell membrane formation could be suppressed by RL-ßC-Rts. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01077-6.

Genome-wide identification and expression analysis of terpene synthases in Gossypium species in response to gossypol biosynthesis.

Cottonseed is an invaluable resource, providing protein, oil, and abundant minerals that significantly contribute to the well-being and nutritional needs of both humans and livestock. However, cottonseed also contains a toxic substance called gossypol, a secondary metabolite in Gossypium species that plays an important role in cotton plant development and self-protection. Herein, genome-wide analysis and characterization of the terpene synthase (TPS) gene family identified 304 TPS genes in Gossypium. Bioinformatics analysis revealed that the gene family was grouped into six subgroups TPS-a, TPS-b, TPS-c, TPS-e, TPS-f, and TPS-g. Whole-genome, segmental, and tandem duplication contributed to the evolution of TPS genes. According to the analysis of selection pressure, it was predicted that TPS genes experience predominantly negative selection, with positive selection occurring subsequently. RT-qPCR analysis in TM-1 and CRI-12 lines revealed GhTPS48 gene as the candidate gene for silencing experiments. To summarize, comprehensive genome-wide studies, RT-qPCR, and gene silencing experiments have collectively demonstrated the involvement of the TPS gene family in the biosynthesis of gossypol in cotton.

Selection and Validation of Optimal RT-qPCR Reference Genes for the Normalization of Gene Expression under Different Experimental Conditions in Lindera megaphylla.

Lindera megaphylla, a broad-leaved evergreen that is used as a landscape ornamental plant and medicinal plant, is an ecologically important and dominant tree species. However, little is known about the molecular mechanisms of its growth, development, and metabolism. The selection of suitable reference genes is critical for molecular biological analyses. To date, no research on reference genes as a foundation for gene expression analysis has been undertaken in L. megaphylla. In this study, 14 candidate genes were selected from the transcriptome database of L. megaphylla for RT-qPCR assay under different conditions. Results showed that helicase-15 and UBC28 were most stable in different tissues of seedlings and adult trees. For different leaf developmental stages, the best combination of reference genes was ACT7 and UBC36. UBC36 and TCTP were the best under cold treatment, while PAB2 and CYP20-2 were the best under heat treatment. Finally, a RT-qPCR assay of LmNAC83 and LmERF60 genes were used to further verify the reliability of selected reference genes above. This work is the first to select and evaluate the stability of reference genes for the normalization of gene expression analysis in L. megaphylla and will provide an important foundation for future genetic studies of this species.

Detection of Hazelnut and Almond Adulteration in Olive Oil: An Approach by qPCR.

Virgin olive oil (VOO), characterized by its unique aroma, flavor, and health benefits, is subject to adulteration with the addition of oils obtained from other edible species. The consumption of adulterated olive oil with nut species, such as hazelnut or almond, leads to health and safety issues for consumers, due to their high allergenic potential. To detect almond and hazelnut in olive oil, several amplification systems have been analyzed by qPCR assay with a SYBR Green post-PCR melting curve analysis. The systems selected were Cora1F2/R2 and Madl, targeting the genes coding the allergenic protein Cor a 1 (hazelnut) and Pru av 1 (almond), respectively. These primers revealed adequate specificity for each of the targeted species. In addition, the result obtained demonstrated that this methodology can be used to detect olive oil adulteration with up to 5% of hazelnut or almond oil by a single qPCR assay, and with a level as low as 2.5% by a nested-qPCR assay. Thus, the present research has shown that the SYBR-based qPCR assay can be a rapid, precise, and accurate method to detect adulteration in olive oil.

Molecular quantification for differentiation of fresh and dried Jinqian Baihua She.

Freshly-used crude drugs have unique functions and advantages in TCM practice of treating diseases. Jinlong Capsule is a patent traditional Chinese medicine product effective for treatment of hepatocarcinoma, and fresh Jinqian Baihua She (JBS, the body of juvenile Bungarus multicinctus) is one of its important ingredients. The emergence of counterfeit fresh JBS, often identified as dried JBS with almost identical appearance, poses a difficult problem in the quality control of the product. Herein we report a molecular quantification-based method for differentiation of fresh and dried JBS by determining the copy number of a specific DNA marker in the samples. Using species-specific primers and TaqMan probes, we established a real-time quantitative PCR system for amplification of a fragment in the 658-bp cytochrome oxidase subunit I (COI) region from JBS specimens. The amplicon copy number in the muscle tissues ranged from 1.14 × 107 to 4.83 × 107 copies/mg in fresh JBS samples, as compared with 1.13 × 105-8.91 × 106 copies/mg in dried JBS samples. Based upon Fisher discriminant analysis, we used 1.27 × 107 copies/mg as the cut-off value for differentiating fresh and dried JBS, which was validated in the single-blinded validation test of fresh and dried JBS samples. This qPCR system may provide an efficient means for accurate identification of fresh JBS to improve the quality control of the medicinal product.

Exploring the Effect of Functional Diets Containing Phytobiotic Compounds in Whiteleg Shrimp Health: Resistance to Acute Hepatopancreatic Necrotic Disease Caused by Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis (AHPND) is an emerging severe disease caused by strains of Vibrio parahaemolyticus (VpAHPND) in whiteleg shrimp (Litopenaeus vannamei). Mitigating its negative impact, and at the same time minimizing antibiotics treatments, is the major challenge in shrimp aquaculture. A sustainable strategy could be to include immunostimulants in diet. Phytobiotics, harmless plant extracts with immunostimulatory and biocidal activities, are promising candidates. In this study, we evaluated the effectiveness of two diets (E and F) supplemented with phytobiotics (functional diets) in terms of protecting shrimp against AHPND. For this purpose, groups of animals were fed functional or control diets for 4 and 5 weeks and, subsequently, they were challenged with VpAHPND by immersion. We compared the mortality in infected groups and estimated the percentage of carriers by using a specific qPCR in hepatopancreas tissue. The results showed that mortality was significantly lower in the group fed functional diet E and, after a 5-week feeding schedule. This group also showed the lowest percentage of carriers. The pathological effects were also reduced with diet F. Thus, feeding shrimp with phytobiotic-enriched diets in critical periods will be highly beneficial because it increases the host's resistance to AHPND pathology.