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Authentication of Panax ginseng and Panax quinquefolius products is important to be able to mitigate instances of adulteration and substitution that exist within the international supply chain of ginseng. To address this issue, species-specific hydrolysis probe qPCR assays were developed and validated for both P. ginseng and P. quinquefolius herbal dietary supplements. Performance of the probe-based assays was evaluated using analytical validation criteria, which included evaluation of: (1) specificity, in selectively identifying the target species, (2) sensitivity, in detecting the lowest amount of the target material, and (3) repeatability and reproducibility of the method in detecting the target species in raw materials on a real-time PCR platform (reliability). The species-specific probes were developed and successfully passed the validation criteria with 100% specificity, 80–120% efficiency and 100% reliability. The methods developed in this study are fit for purpose, rapid, and easy to implement in quality assurance programs, authentication of ginseng herbal supplements is possible, even with extracts where DNA is fragmented and of low quality and quantity.

et al., Fast, high-throughput methods for measuring the level and duration of protective immune responses to SARS-CoV-2 are needed to anticipate the risk of breakthrough infections. Here we report the development of two quantitative PCR assays for SARS-CoV-2-specific T cell activation. The assays are rapid, internally normalized and probe-based: qTACT requires RNA extraction and dqTACT avoids sample preparation steps. Both assays rely on the quantification of CXCL10 messenger RNA, a chemokine whose expression is strongly correlated with activation of antigen-specific T cells. On restimulation of whole-blood cells with SARS-CoV-2 viral antigens, viral-specific T cells secrete IFN-γ, which stimulates monocytes to produce CXCL10. CXCL10 mRNA can thus serve as a proxy to quantify cellular immunity. Our assays may allow large-scale monitoring of the magnitude and duration of functional T cell immunity to SARS-CoV-2, thus helping to prioritize revaccination strategies in vulnerable populations., Research reported in this publication was supported in part by an ISMMS seed fund to E.G. and a Dean’s office grant to E.G. and I.M. We gratefully acknowledge use of the services and facilities of the Tisch Cancer Institute supported by the National Cancer Institute (NCI) Cancer Center Support grant (no. P30 CA196521), in particular the Hess sequencing core and the BiNGS shared facility. M.S. was supported by an NCI training grant (no. T32CA078207). J.S.Y.H. is supported by the Charles H. Revson Foundation. We acknowledge the technical contribution of D.A. Sánchez, J. Baranda, S. Baztan-Morales, M. Castillo de la Osa, A. Comins-Boo, C. del Álamo Mayo, S. Gil-Manso, B. Gonzalez, S. Hatem, J. Irure-Ventura, I. Miguens, S. Muñoz Martinez, M. Pereira, C. Rodrigues-Guerreiro, M. Rodriguez-Garcia, M.P. Rojo-Portolés and D. San Segundo. We also acknowledge Beckman Coulter for donating the equipment required for the determination of spike-specific IgG antibodies. W.M. was supported by grant no. NCI K00CA212474. This work was supported by ISMMS seed fund to J.O.; Instituto de Salud Carlos III, grant no. COV20-00668 to R.C.R.; Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call grant no. COV20/00181) cofinanced by European Development Regional Fund ‘A way to achieve Europe’ to E.P.-A.; Instituto de Salud Carlos III, Spain (grant no. COV20/00170); Government of Cantabria, Spain (grant no. 2020UIC22-PUB-0019) to M.L.H.; Instituto de Salud Carlos III (grant no. PI16CIII/00012) to P.P.; Fondo Social Europeo e Iniciativa de Empleo Juvenil YEI (grant no. PEJ2018-004557-A) to M.P.E. and grant no. REDInREN 016/009/009 ISCIII. This project has received funding from the European Union’s Horizon 2020 research and innovation program VACCELERATE under grant agreement no. 101037867 to J.O. S.G. is supported by grant nos. U24CA224319, U01DK124165 and P30 CA196521.

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

The ongoing outbreak of the novel coronavirus (SARS‐CoV‐2) infection is creating serious challenges for health laboratories that seek to identify viral infections as early as possible, optimally at the earliest appearance of symptom. Indeed, there is urgent need to develop and deploy robust diagnostic methodologies not only to use in health laboratory environments but also directly in places where humans circulate and spread the virus such as airports, trains, boats, and any public aggregation places. The success of a reliable and sensitive asymptomatic diagnosis relies on the identification and measurement of informative biomarkers from human host and virus in a rapid, sensitive, and inexpensive manner. The objective of this article is to describe an innovative multidisciplinary approach to develop an efficient, inexpensive, and easy‐to‐use portable instrument (bCUBE® by Hyris Ltd) that can be employed as a surveillance system for the emergency caused by SARS‐CoV‐2. A solution for Coronavirus testing, compliant with CDC guidelines, is scheduled to be released in the next weeks. In addition, we will describe a workflow and path of an integrated multi‐omic approach that will lead to host and pathogen biomarker discovery in order to train the instrument to provide reliable results based on a specific biomarker's fingerprint of SARS‐CoV‐2 infection.

Background: PCR methods are the most commonly used DNA-based identity tool in the commercial food, beverage, and natural health product markets. These methods are routinely used to identify foodborne pathogens and allergens in food. Proper validation methods for some sectors have been established, while there are none in other markets, such as botanicals. Results: A survey of the literature indicates that some validation criteria are not addressed when developing PCR tests for botanicals. Objective: We provide recommendations for qualitative real-time PCR methods for validating identity tests for botanical ingredients. Methods: These include common criteria that underpin the development and validation of rigorous tests, including (1) the aim of the validation test, (2) the applicability of different matrix variants, (3) specificity in identifying the target species ingredient, (4) sensitivity in detecting the smallest amount of the target material, (5) repeatability of methods, (6) reproducibility in detecting the target species in both raw and processed materials, (7) practicability of the test in a commercial laboratory, and (8) comparison with alternative methods. In addition, we recommend additional criteria, according to which the practicability of the test method is evaluated by transferring the method to a second laboratory and by comparison with alternative methods. Conclusions and Highlights: We hope that these recommendations encourage further publication on the validation of PCR methods for many botanical ingredients. These properly validated PCR methods can be developed on small, real-time biotechnology that can be placed directly into the supply chain ledger in support of highly transparent data systems that support QC from the farm to the fork of the consumer.

Xylella fastidiosa subsp. pauca (Xfp) is a gram-negative pathogenic bacteria responsible for serious diseases (Purcell, 2013) that inflicts considerable economic loss (Li et al., 2007; Luvisi et al., 2017). The pathogen has been linked to olive quick decline syndrome (OQDS). This devastating olive disease was first observed in Salento (Apulia, southeastern Italy) in 2009. Infected trees respond to Xfp infection with scattered desiccation of twigs and small branches in the upper crown, which extend to the rest of the canopy, showing the characteristic blight effect. The disease causes tree death within a few years from the onset of symptoms (Martelli, 2016). The primary agronomic procedure for counteracting the infection is by heavy pruning to stimulate new growth (Martelli et al., 2016).However, this does not prevent the withering and desiccation of upper vegetation in the infected tree. Lignin deposition increases the tolerance of some hosts to Xylella fastidiosa. Elevated concentration of quinic acid, a lignin precursor, less concentration of hydroxytyrosolglucoside and the up-regulation of cinnamoyl-CoA reductase and polyphenol oxidase were observed in the most tolerant olive cultivar, Leccino (Sabella et al., 2018). In this opinion article, we explore the use of a portable instrument to detect OQDS, based on the host responses at the transcript level. This approach was proposed previously to detect Huanglongbing, a severe disease affecting Citrus worldwide (Dandekar et al., 2010;Martinelli et al., 2014b). These innovative mthods of plant disease detection had been reviewed recently (Martinelli et al., 2014a).

The Coiled-Coil Domain Containing 80 (CCDC80) gene has been identified as strongly induced in rat thyroid PC CL3 cells immortalized by the adenoviral E1A gene. In human, CCDC80 is a potential oncosoppressor due to its down-regulation in several tumor cell lines and tissues and it is expressed in almost all tissues. CCDC80 has homologous in mouse, chicken, and zebrafish. We cloned the zebrafish ccdc80 and analyzed its expression and function during embryonic development. The in-silico translated zebrafish protein shares high similarity with its mammalian homologous, with nuclear localization signals and a signal peptide. Gene expression analysis demonstrates that zebrafish ccdc80 is maternally and zygotically expressed throughout the development. In particular, ccdc80 is strongly expressed in the notochord and it is under the regulation of the Hedgehog pathway. In this work we investigated the functional effects of ccdc80-loss-of-function during embryonic development and verified its interaction with gadd45β2 in somitogenesis. J. Cell. Physiol. 230: 821–830, 2015. © 2014 Wiley Periodicals, Inc.

Previous work demonstrated that a commercial formulation of piperonyl butoxide (PBO) did inhibit the activity of some plant proteolytic enzymes. In this paper, the effect of pure PBO and nine pure PBO homologues (PBOH) appropriately synthesized toward bromelain and papain was studied in hydrocarbon solution using the bis(2-ethylhexyl)sodium sulfosuccinate (AOT) reverse micellar system. This study establishes that the majority of these compounds show, in vitro, interesting protease inhibition activities. The benzodioxole and dihydrobenzofuran structures, in particular, 5-[2-(2-butoxyethoxy)ethoxymethyl]-benzo[1,3]dioxole (EN 1-40) and 6-[2-(2-butoxyethoxy)ethoxymethyl]-5-propyl-2,3-dihydrobenzofuran (EN 16-5), respectively, appear to be responsible for protease inhibition. Measures of octanol/water partition coefficients on PBO and PBOH have demonstrated that water solubility plays a fundamental role in the expression of protease inhibition activity.