CodY: An Essential Transcriptional Regulator Involved in Environmental Stress Tolerance in Foodborne Staphylococcus aureus RMSA24.

Staphylococcus aureus (S. aureus), as the main pathogen in milk and dairy products, usually causes intoxication with vomiting and various kinds of inflammation after entering the human body. CodY, an important transcriptional regulator in S. aureus, plays an important role in regulating metabolism, growth, and virulence. However, little is known about the role of CodY on environmental stress tolerance. In this research, we revealed the role of CodY in environmental stress tolerance in foodborne S. aureus RMSA24. codY mutation significantly reduced the tolerance of S. aureus to desiccation and oxidative, salt, and high-temperature stresses. However, S. aureus was more tolerant to low temperature stress due to mutation of codY. We found that the expressions of two important heat shock proteins-GroEL and DanJ-were significantly down-regulated in the mutant codY. This suggests that CodY may indirectly regulate the high- and low-temperature tolerance of S. aureus by regulating the expressions of groEL and danJ. This study reveals a new mechanism of environmental stress tolerance in S. aureus and provides new insights into controlling the contamination and harm caused by S. aureus in the food industry.

Application of a modified tetra-primer ARMS-PCR assay for rapid Panax species identity authentication in ginseng products.

Panax ginseng products can be adulterated with materials from other Panax species. The purpose of this study is to provide a rapid P. ginseng authentication method for simultaneous identification of P. ginseng and detection of adulteration in ginseng products at different processing stages. First, a tetra-primer ARMS-PCR assay was designed based on a single-nucleotide polymorphism (SNP) within the trnL-trnF region and was tested at 28 PCR cycles with DNA extracted from Botanical Reference Materials (BRMs). Next, 5' end random nucleotide and 3' terminus phosphorothioates linkage modifications were incorporated into the inner primers to improve sensitivity and specificity at 40 PCR cycles. Finally, the modified assay was validated using characterized market ginseng materials and the detection limit was determined. The modified tetra-primer ARMS-PCR assay can achieve the desired sensitivity and specificity using one set of reaction conditions in ginseng materials at different stages. In validation, it was able to correctly identify target species P. ginseng and differentiate it from closely related species. This study suggests that the modified tetra-primer ARMS-PCR assay can be used for the rapid, species identity authentication of P. ginseng material in ginseng products. This assay can be used to complement chemical analytical methods in quality control, so both species identity and processing attributes of ginseng products can be efficiently addressed.

Geographic dispersion and corporate resilience during the COVID-19 pandemic.

This study demonstrates the positive role of geographic dispersion in corporate resilience to the COVID-19 pandemic in the context of China. This association is more pronounced when firms are highly dependent on the domestic market, less likely to obtain enough financing, highly apply digital technology, and have low customer concentration. This association is attributable to the following three channels: a diversified portfolio, the maintenance of business relationships, and access to non-local resources. Overall, our findings provide a more nuanced picture of the potential impacts of corporate diversification on corporate resilience.

Validation of a Real-Time PCR Assay for Identification of Fresh and Processed Carica papaya Botanical Material: Using Synthetic DNA to Supplement Specificity Evaluation.

Several commercially important botanicals have a lack of diagnostic testing options that can quickly and unambiguously identify materials of different matrices. Real-time PCR can be a useful, orthogonal approach to identification for its exceptional specificity and sensitivity. Carica papaya L. is a species with a lack of available identification methods, and one which features two distinct commercially relevant matrices: fresh fruit and powdered fruit extract. In this study, we demonstrate the successful design and validation of a real-time PCR assay for detection of papaya DNA extracted from the two matrices. We also propose a technique that can be used during exclusivity panel construction, when genuine botanical samples are not available for certain species: substitution with synthetic DNA. We demonstrate the use of this material to complete a comprehensive specificity evaluation and confidently determine suitable Ct cutoff values. Further, we demonstrate how ddPCR can be used to determine the copy number of the target sequence in a set amount of genomic DNA, to which synthetic DNA samples can be corrected, and how it can verify specificity of the primers and probe. Through the presentation of successful assay validation for papaya detection, this work serves as a guideline for how to approach specificity evaluation when non-target botanical samples are difficult to obtain and otherwise may not have been included in the exclusivity panel.

Development and Validation of a Species-specific PCR Method for the Identification of Ginseng Species Using Orthogonal Approaches.

When testing botanical ingredients of herbal medicines and dietary supplements, the complexity of botanical matrixes often requires the use of orthogonal methods to establish identification procedures suitable for quality control purposes. Genomic-based botanical identification methods are evolving and emerging as useful quality control tools to complement traditional morphological and chemical identification methods. Species-specific polymerase chain reaction methods are being evaluated for botanical quality control and as a cost-effective approach to identify and discriminate between closely related botanical species. This paper describes orthogonal identification of Panax ginseng, P. quinquefolius, and P. notoginseng materials in commerce as an example of the development and validation of a set of species-specific polymerase chain reaction methods to establish botanical identity in ginseng roots. This work also explored the possibility of extending the application of species-specific polymerase chain reaction methods to provide species identity information for processed materials, such as steamed roots and hydroalcoholic extracts, and showed success with this approach. Finally, the paper provides recommendations for an out-of-specification investigation of samples that may pass some of the orthogonal tests and fail others.

Development of Hydrolysis Probe-Based qPCR Assays for Panax ginseng and Panax quinquefolius for Detection of Adulteration in Ginseng Herbal Products.

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.

Validation of a Triplex Quantitative Polymerase Chain Reaction Assay for Detection and Quantification of Traditional Protein Sources, Pisum sativum L. and Glycine max (L.) Merr., in Protein Powder Mixtures.

Several botanicals have been traditionally used as protein sources, including the leguminous Pisum sativum L. and Glycine max (L.) Merr. While a rich history exists of cultivating these plants for their whole, protein-rich grain, modern use as powdered supplements present a new challenge in material authentication. The absence of clear morphological identifiers of an intact plant and the existence of long, complex supply chains behoove industry to create quick, reliable analytical tools to identify the botanical source of a protein product (many of which contain multiple sources). The utility of molecular tools for plant-based protein powder authentication is gaining traction, but few validated tools exist. Multiplex quantitative polymerase chain reaction (qPCR) can provide an economical means by which sources can be identified and relative proportions quantified. We followed established guidelines for the design, optimization, and validation of qPCR assay, and developed a triplex qPCR assay that can amplify and quantify pea and soy DNA targets, normalized by a calibrator. The assay was evaluated for analytical specificity, analytical sensitivity, efficiency, precision, dynamic range, repeatability, and reproducibility. We tested the quantitative ability of the assay using pea and soy DNA mixtures, finding exceptional quantitative linearity for both targets - 0.9983 (p < 0.0001) for soy and 0.9915 (p < 0.0001) for pea. Ratios based on mass of protein powder were also tested, resulting in non-linear patterns in data that suggested the requirement of further sample preparation optimization or algorithmic correction. Variation in fragment size within different lots of commercial protein powder samples was also analyzed, revealing low SD among lots. Ultimately, this study demonstrated the utility of qPCR in the context of protein powder mixtures and highlighted key considerations to take into account for commercial implementation.

Field Identification of Matricaria chamomilla using a Portable qPCR System.

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.

Development and validation of a probe-based qPCR method to prevent parsley leaf material misidentification.

Parsley (Petroselinum crispum) leaf is an herb widely consumed for its health benefits. Due to similar morphological and chemical profiles, celery leaf may be a source of substitution in commercial parsley materials. In order to detect this substitution, the present work characterized parsley and celery leaf at the cultivar level by physical, chemical and DNA approaches. In contrast to the variations observed in physical appearances and chemical profiles that make verification of authenticity difficult, consistent differences observed between their DNA sequences are suitable for verifying parsley material authenticity. To identify parsley and detect celery simultaneously, a multiplex qPCR assay was developed and validated with respect to efficiency and specificity. Further testing indicated the assay can be used to detect 1% (w/w) celery in parsley materials with a probability of detection greater than 0.9. The developed method is well-suited for routine quality control to prevent parsley material misidentification in commercial trade.

Development of a Differential Multiplex PCR Assay for the Supplemental Identification of Different Sources of Proteins.

BACKGROUND: Differentiation of proteins from multiple sources provides challenges in the accuracy using multiple and often disputed protein identification methods. The U.S. Pharmacopeia Food Chemical Codex does not include monographs for many protein sources, including milk proteins and soy protein isolate. Monographs that are included for proteins do not list a single comprehensive identification method but instead rely on a combined assessment of ash (total), fat, lactose, loss on drying, and protein content. A fast, inexpensive, and accurate protein source assay is tantamount to prevention of economic adulteration in protein powders. OBJECTIVE: This study describes the development of a novel method to identify and differentiate animal proteins (cow protein powders as milk protein and whey protein) and plant proteins (soy protein powders). These proteins powders are of high importance to the food and dietary supplement industries, as they encompass the highest grossing and fastest growing protein sources in the global protein powder market. METHODS: The developed method uses PCR amplification and gel electrophoresis of short chain DNA fragments found in processed protein powders to identify and differentiate the source of each powder. The original development was performed using reference materials of known identity and tested against an inclusivity panel of protein powders from commercial sources. Bands were identified using the Agilent Tapestation 4200 and Tapestation Analysis Software A.02.02 (SR1) using proprietary band analysis. RESULTS: The developed method was found to be specific for the identification of each protein source, passing a computational (National Center for Biotechnology Information Basic Local Alignment Search Tool) exclusivity panel and an experimental inclusivity panel. The method was also able to detect multiple adulterants in concentrations as low as 1% (w/w). CONCLUSIONS: The developed method is fast, inexpensive, and accurate (100%) for the supplemental identification of cow and soy proteins and able to detect adulteration as low as 1% (w/w). HIGHLIGHTS: A new method can identify cow and soy proteins, and detect low levels of adulteration using DM-PCR.

Validation of a Targeted PCR Method for Raw and Processed Botanical Material Identification: An Example Using Matricaria chamomilla (Chamomile).

Background: A requirement of current good manufacturing practices for dietary supplements is that manufacturers must identify their dietary ingredients. DNA-based methods can provide species-level authentication that may sometimes be difficult to achieve using conventional morphological and chemical analysis methods. However, because of varying levels of DNA degradation in botanical materials, many commercial tests fail to generate consistent test results across all types of botanical materials. AOAC published guidelines for validation of botanical identification methods and proposed probability of identification (POI) as a method performance parameter. However, few DNA-based botanical authentication methods in the literature follow these guidelines and evaluate POI. Objective: To provide a targeted PCR method validation example that follows AOAC guidelines for validation of botanical identification methods. Methods: Using Matricaria chamomilla (chamomile) as an example, we performed a single-laboratory validation for a targeted PCR method that aimed to identify both raw and processed chamomile materials. The performance parameters of the test were evaluated by carrying out an inclusivity/exclusivity study and a Specified Superior Test Material/Specified Inferior Test Material study to demonstrate that the method's POI meets industry requirements. Results: The chamomile samples were identified by the method and achieved a POI greater than 0.9 with respect to all types of chamomile botanical materials. Conclusions: The method was validated for DNA-based identification of raw and processed chamomile materials, such as sterilized powders and extracts. Highlights: This work will provide insight for laboratories and manufacturers that aim to develop and validate DNA-based botanical identification methods.

DNA Quality and Quantity Analysis of Camellia sinensis Through Processing from Fresh Leaves to a Green Tea Extract.

Background: Although there has been some success using DNA barcoding to authenticate raw natural health product (NHP) botanical ingredients, there are many gaps in our understanding of DNA degradation, which may explain low PCR and sequencing success in processed NHPs. Objective: In this study, we measured multiple DNA variables after each step in the processing of a green tea extract in order to document DNA quality and quantity. Methods: We sampled plant material after each step of green tea extract processing: five steps at a Chinese tea farm (n = 10) and five at an NHP processing facility (n = 3). We hypothesized that processing treatments degrade and remove DNA from NHPs, reflected by decreasing quantities of extractable genomic DNA (gDNA), an increasing proportion of small DNA fragments in genomic extracts, and decreasing quantitative PCR (QPCR) efficiency [higher cycle threshold (Ct) values]. DNA from end-production green tea extract was sequenced in order to try to validate material as the botanical of interest. Results: We saw a 41.1% decrease in mean extractable gDNA through farm processing (P < 0.01) and a 99.7% decrease through facility processing (P < 0.05). There was a 26.3% decrease in mean DNA fragment size through farm processing (P < 0.001) and an 82.0% decrease through facility processing (P < 0.05). QPCR efficiency was reduced through processing, marked by significant increases in Ct values with 100 base pair (bp) and 200 bp PCR targets (P < 0.05), and an inability to amplify 300 bp targets when using DNA template from end-production green tea extract. Conclusions: Although there was significant degradation and removal of DNA through processing, sufficiently intact DNA was able to be recovered from highly processed green tea extract for further sequencing and identification. Highlights: This work addresses a key gap in the understanding of DNA degradation through processing and provides useful information to consider when designing molecular diagnostic techniques for NHP identification.

Recommendations for Validation of Real-Time PCR Methods for Molecular Diagnostic Identification of Botanicals.

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.

Single-Laboratory Validation of a Two-Tiered DNA Barcoding Method for Raw Botanical Identification.

Background: The applications of deoxyribonucleic acid (DNA) barcoding methods have been extended from authenticating taxonomic provenance of animal products to identifying botanicals used as herbal medicine and in botanical dietary supplements. DNA barcoding methods for botanical identification must be adequately validated to meet regulatory compliance. Objective: The goal of this study is to provide a validation protocol for a two-tiered DNA barcoding method that aims to identify raw botanicals. Methods: A barcode database was computationally validated to define the barcode combinations that can unambiguously identify botanicals in the database. A maximum variation sampling technique was used to capture a wide range of perspectives relating to DNA barcode-based botanical identification, including plant parts and species distance, for the experimental validation. Twenty-two authenticated botanicals were purposively sampled from different plant parts-covering both closely related and distantly related species-to validate the two-tiered DNA barcoding method. The performance of the method was assessed on accuracy, precision, ruggedness, and uncertainty. Results: High accuracy (100%) and precision (1.0) were obtained from the validation samples. The method was also found to be rugged and have acceptable uncertainty. Conclusions: The method was validated and suitable for DNA-based identification of botanical raw materials listed in the current database. Highlights: This work will provide support guidance for manufacturers and regulatory policy makers to implement equivalent validated and compliant DNA-based testing in quality control processes to improve botanical raw material identification and authentication.

Visualization of DNA in highly processed botanical materials.

DNA-based methods have been gaining recognition as a tool for botanical authentication in herbal medicine; however, their application in processed botanical materials is challenging due to the low quality and quantity of DNA left after extensive manufacturing processes. The low amount of DNA recovered from processed materials, especially extracts, is "invisible" by current technology, which has casted doubt on the presence of amplifiable botanical DNA. A method using adapter-ligation and PCR amplification was successfully applied to visualize the "invisible" DNA in botanical extracts. The size of the "invisible" DNA fragments in botanical extracts was around 20-220 bp compared to fragments of around 600 bp for the more easily visualized DNA in botanical powders. This technique is the first to allow characterization and visualization of small fragments of DNA in processed botanical materials and will provide key information to guide the development of appropriate DNA-based botanical authentication methods in the future.

Enhanced Cancer Immunotherapy by Chimeric Antigen Receptor-Modified T Cells Engineered to Secrete Checkpoint Inhibitors.

Purpose: Despite favorable responses of chimeric antigen receptor (CAR)-engineered T-cell therapy in patients with hematologic malignancies, the outcome has been far from satisfactory in the treatment of solid tumors, partially owing to the development of an immunosuppressive tumor microenvironment. To overcome this limitation, we engineered CAR T cells secreting checkpoint inhibitors (CPI) targeting PD-1 (CAR.αPD1-T) and evaluated their efficacy in a human lung carcinoma xenograft mouse model.Experimental Design: To evaluate the effector function and expansion capacity of CAR.αPD1-T cells in vitro, we measured the production of IFNγ and T-cell proliferation following antigen-specific stimulation. Furthermore, the antitumor efficacy of CAR.αPD1-T cells, CAR T cells, and CAR T cells combined with anti-PD-1 antibody was determined using a xenograft mouse model. Finally, the underlying mechanism was investigated by analyzing the expansion and functional capacity of TILs.Results: Human anti-PD-1 CPIs secreted by CAR.αPD1-T cells efficiently bound to PD-1 and reversed the inhibitory effect of PD-1/PD-L1 interaction on T-cell function. PD-1 blockade by continuously secreted anti-PD-1 attenuated the inhibitory T-cell signaling and enhanced T-cell expansion and effector function both in vitro and in vivo In the xenograft mouse model, we demonstrated that the secretion of anti-PD-1 enhanced the antitumor activity of CAR T cells and prolonged overall survival.Conclusions: With constitutive anti-PD-1 secretion, CAR.αPD1-T cells are more functional and expandable, and more efficient at tumor eradication than parental CAR T cells. Collectively, our study presents an important and novel strategy that enables CAR T cells to achieve better antitumor immunity, especially in the treatment of solid tumors. Clin Cancer Res; 23(22); 6982-92. ©2017 AACR.

SCR7 is neither a selective nor a potent inhibitor of human DNA ligase IV.

DNA ligases are attractive therapeutics because of their involvement in completing the repair of almost all types of DNA damage. A series of DNA ligase inhibitors with differing selectivity for the three human DNA ligases were identified using a structure-based approach with one of these inhibitors being used to inhibit abnormal DNA ligase IIIα-dependent repair of DNA double-strand breaks (DSB)s in breast cancer, neuroblastoma and leukemia cell lines. Raghavan and colleagues reported the characterization of a derivative of one of the previously identified DNA ligase inhibitors, which they called SCR7 (designated SCR7-R in our experiments using SCR7). SCR7 appeared to show increased selectivity for DNA ligase IV, inhibit the repair of DSBs by the DNA ligase IV-dependent non-homologous end-joining (NHEJ) pathway, reduce tumor growth, and increase the efficacy of DSB-inducing therapeutic modalities in mouse xenografts. In attempting to synthesize SCR7, we encountered problems with the synthesis procedures and discovered discrepancies in its reported structure. We determined the structure of a sample of SCR7 and a related compound, SCR7-G, that is the major product generated by the published synthesis procedure for SCR7. We also found that SCR7-G has the same structure as the compound (SCR7-X) available from a commercial vendor (XcessBio). The various SCR7 preparations had similar activity in DNA ligation assay assays, exhibiting greater activity against DNA ligases I and III than DNA ligase IV. Furthermore, SCR7-R failed to inhibit DNA ligase IV-dependent V(D)J recombination in a cell-based assay. Based on our results, we conclude that SCR7 and the SCR7 derivatives are neither selective nor potent inhibitors of DNA ligase IV.

The repetitive portion of the Xenopus IgH Mu switch region mediates orientation-dependent class switch recombination.

Vertebrates developed immunoglobulin heavy chain (IgH) class switch recombination (CSR) to express different IgH constant regions. Most double-strand breaks for Ig CSR occur within the repetitive portion of the switch regions located upstream of each set of constant domain exons for the Igγ, Igα or Igϵ heavy chain. Unlike mammalian switch regions, Xenopus switch regions do not have a high G-density on the non-template DNA strand. In previous studies, when Xenopus Sµ DNA was moved to the genome of mice, it is able to support substantial CSR when it is used to replace the murine Sγ1 region. Here, we tested both the 2kb repetitive portion and the 4.6 kb full-length portions of the Xenopus Sµ in both their natural (forward) orientation relative to the constant domain exons, as well as the opposite (reverse) orientation. Consistent with previous work, we find that the 4.6 kb full-length Sµ mediates similar levels of CSR in both the forward and reverse orientations. Whereas, the forward orientation of the 2kb portion can restore the majority of the CSR level of the 4.6 kb full-length Sµ, the reverse orientation poorly supports R-looping and no CSR. The forward orientation of the 2kb repetitive portion has more GG dinucleotides on the non-template strand than the reverse orientation. The correlation of R-loop formation with CSR efficiency, as demonstrated in the 2kb repetitive fragment of the Xenopus switch region, confirms a role played by R-looping in CSR that appears to be conserved through evolution.

Human lymphoid translocation fragile zones are hypomethylated and have accessible chromatin.

Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones were found to be more accessible than the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more-refined models for why these zones are 100- to 1,000-fold more prone to undergo chromosomal translocation than the adjacent regions.