R-Biopharm (Germany)

D-Glucose and D-fructose are present in honey, wine and beer, and in a range of other foodstuffs such as bread, pastries, or chocolate. Both sugars can occur as a monosaccharide or in di-, oligo-, and polysaccharides. To validate the performance of the Enzytec™ Liquid D-Glucose/D-Fructose test kit for the determination of D-glucose and D-fructose in food and beverages such as fruit and vegetable juices, soft drinks, wines, and beer. The method is based on enzymes that are part of a prepackaged kit that contains three ready-to-use components. Both sugars are phosphorylated by a hexokinase. Glucose-6-phosphate (G-6-P) and nicotinamide adenine dinucleotide (NAD) are converted to gluconate-6-phosphate and reduced nicotinamide adenine dinucleotide (NADH). NADH is measured at 340 nm. Phosphoglucose isomerase converts fructose-6-phosphate to G-6-P, which in turn is converted to gluconate-6-phosphate and NADH. The test is specific to D-glucose and D-fructose and shows no side-activities or interferences with the exception of mannose and sulphite that interfere at 5.1 and 1.25 g/L or more, respectively. The measurement range is from 6.1 to 2000 mg/L for D-glucose and 5.6 to 1000 mg/L for D-fructose (100 µL test volume). Trueness was evaluated using NIST SRM 3282 (cranberry juice) and one reference wine. The recoveries ranged from 101 to 102%. Spiking of wine, beer, soft drinks, and juices resulted in recoveries between 93 and 105%. Intermediate precision is below 6% for concentrations at 25 mg/L and below 4% for higher concentrations. For automation, three applications with different test volumes and different measurement ranges were validated. Linearity is given from 5.0 to 10 000 mg/L for the sum of both sugars. The method is robust and accurate for manual and automated applications and was approved as an AOAC Official Method of Analysis℠. The ready-to-use components of the test kit have a shelf life of at least 29 months.

BackgroundThe SureFast® SARS-CoV-2 PLUS Test is a reverse transcription qPCR (RT-qPCR) assay for the direct, qualitative detection of novel coronavirus (SARS-CoV-2) RNA from stainless-steel environmental sample swabs.ObjectiveTo validate the SureFast SARS-CoV-2 PLUS Kit as part of the AOAC Research Institute’s Emergency Response Validation Performance Tested Method(s)SM program.MethodThe SureFast SARS-CoV-2 PLUS Kit was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms (both near neighbors and background organisms) using the ThermoBLAST program. The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the US Centers for Disease Control and Prevention 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020).ResultsResults of the in silico analysis demonstrated 99.99% selectivity of the method in being able to detect target sequences of the known CoV-2 genomes and discriminate them from near neighbors. In the matrix study, the candidate method demonstrated statistically significant better recovery of the target analyte than the PCR detection reference method.ConclusionsThe SureFast SARS-CoV-2 PLUS Kit is a rapid and accurate method that can be utilized by food producers to detect the causative agent of COVID-19 on stainless-steel contact surfaces.HighlightsSureFast SARS-CoV-2 PLUS test method is highly specific for primer/probe binding to the E target genome region for the SARS-CoV-2 virus, 99.99% binding specificity using in silico analysis.

The growth and differentiation factor 5 (GDF‐5) is known to play a key role in cartilage morphogenesis and homeostasis, and a single‐nucleotide polymorphism in its promoter sequence was found to be associated with osteoarthritis (OA). In addition, GDF‐5 was shown to promote extracellular matrix (ECM) production in healthy chondrocytes, to stimulate chondrogenesis of mesenchymal stem cells (MSCs) and to protect against OA progression in vivo. Therefore, GDF‐5 appears to be a promising treatment for osteoarthritis. However, GDF‐5 also promotes osteogenesis and hypertrophy, limiting its therapeutic utility. To circumvent this, a GDF‐5 mutant with lower hypertrophic and osteogenic properties was engineered: M1673. The present study aimed to evaluate and compare the effects of GDF‐5 and M1673 on primary porcine and human OA chondrocytes. We found that both GDF‐5 and M1673 can robustly stimulate ECM accumulation, type II collagen and aggrecan expression in porcine and human OA chondrocytes in 3D culture. In addition, both molecules also down‐regulated MMP13 and ADAMTS5 expression. These results suggest that M1673 retained the anabolic and anti‐catabolic effects of GDF‐5 on chondrocytes and is an alternative to GDF‐5 for osteoarthritis.

Bone regeneration of sheep lumbar osteopenia is promoted by targeted delivery of bone morphogenetic proteins (BMPs) via a biodegradable, brushite-forming calcium-phosphate-cement (CPC) with stabilizing poly(l-lactide-co-glycolide) acid (PLGA) fibers. The present study sought to quantify the release and bioactivity of BMPs from a specific own CPC formulation successfully used in previous in vivo studies. CPC solid bodies with PLGA fibers (0%, 5%, 10%) containing increasing dosages of GDF5, BB-1, and BMP-2 (2 to 1000 µg/mL) were ground and extracted in phosphate-buffered saline (PBS) or pure sheep serum/cell culture medium containing 10% fetal calf serum (FCS; up to 30/31 days). Released BMPs were quantified by ELISA, bioactivity was determined via alkaline phosphatase (ALP) activity after 3-day exposure of different osteogenic cell lines (C2C12; C2C12BRlb with overexpressed BMP-receptor-1b; MCHT-1/26; ATDC-5) and via the influence of the extracts on the expression of osteogenic/chondrogenic genes and proteins in human adipose tissue-derived mesenchymal stem cells (hASCs). There was hardly any BMP release in PBS, whereas in medium + FCS or sheep serum the cumulative release over 30/31 days was 11–34% for GDF5 and 6–17% for BB-1; the release of BMP-2 over 14 days was 25.7%. Addition of 10% PLGA fibers significantly augmented the 14-day release of GDF5 and BMP-2 (to 22.6% and 43.7%, respectively), but not of BB-1 (13.2%). All BMPs proved to be bioactive, as demonstrated by increased ALP activity in several cell lines, with partial enhancement by 10% PLGA fibers, and by a specific, early regulation of osteogenic/chondrogenic genes and proteins in hASCs. Between 10% and 45% of bioactive BMPs were released in vitro from CPC + PLGA fibers over a time period of 14 days, providing a basis for estimating and tailoring therapeutically effective doses for experimental and human in vivo studies.

Abstract Western society is facing an increase in the number of food-allergic individuals, with rising incidence in the past years. Therefore, allergen-free food and accurate and reliable analysis of allergen contamination are essential for the protection of consumers. Yet, there is limited understanding on the effect of food processing on allergenicity and on the ability of available methods to detect trace contamination in processed food. Available studies addressing this have relied on sample processing on a laboratory scale. In this study, industry-like processing under precisely defined conditions (ranging from 110 to 150°C roasting temperatures) was employed to better understand the limitations of state-of-the-art methods for detecting traces of hazelnut and almond in processed food. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis indicated an overall reduction in extracted proteins from roasted nut samples, and with matrix-assisted laser desorption ionization time-of-flight Cor a 9 and Prunin, were identified as majorly expressed proteins for hazelnut and almond, respectively. A commercial ELISA kit detected nut traces only up to a 130°C roasting temperature. Untargeted MS (Orbitrap) analysis was able to detect traces of nuts roasted up to 150°C while also confirming Cor a 9 and Prunin as the major expressed proteins for hazelnut and almond, respectively. Preparing cookie dough spiked with roasted nut samples, a complex food matrix was simulated. Analysis by ELISA showed the same limitations encountered for pure nuts samples, hardly detecting traces of nuts roasted above 130°C. Targeted MS (linear ion trap) using multiple reaction monitoring methods for one proteotypic peptide for Cor a 9 and Prunin, respectively, enabled a detection of nut traces up to 150°C. The results indicated that a reduced extractability because of temperature-related effects (e.g., protein denaturation, cross-linking, poor solubility) caused the significant differences between the ELISA and MS analysis. Overall, the results of this study may form the basis to improve allergen detection after roasting through improved extraction methods and refined ELISA formats.

of the 14th Congress of ECCO -European Crohn's and Colitis Organisation S369 of Crohn's patients. While the use of biologic therapy has led to improvements in patient outcomes, loss of response is common, with subsequent worsening of fistula tracts and new abscess formation. 2 Previous studies suggest that patients who achieve deeper healing, and eradication of the tract radiologically, have longer duration of response. The aim of this study was to compare the clinical course of patients achieving MRI healing with those achieving clinical remission, with the hypothesis that radiological healing will lead to a longer duration of response. Methods: A retrospective analysis of perianal fistulising Crohn's patients treated at St Vincent's Hospital, Melbourne was performed. Records were reviewed for patient demographics, disease history, clinical assessments (including PDAI scores), investigation results (including MRI pelvises), and disease flares. Clinical remission was defined as closure of all baseline fistula openings, on examination. Radiological healing was defined as the absence of any T2-hyperintense sinuses, tracts or collections. Primary endpoint was flare-free time, defined as time between achieving healing (clinical or radiological) and a patient's first signs or symptoms requiring escalation in medical and/ or surgical therapy. Statistical analysis consisted of Mann-Whitney U tests, Wilcoxon Signed Rank, and Log-rank tests. Significant parameters were entered into a multi-variate Cox regression model. Results: 93 patients were included, with a median follow-up of 4.75 years (IQR, 2.4-6 years). 85/93 (91%) received treatment with a biologic agent. PDAI and van Assche scores were significantly lower following biologic treatment. Twenty-two/44 (50)% of patients achieved clinical remission, while 15/93 (16%) achieved radiological healing. Ten/22 (45%) of patients with clinical remission had a subsequent disease flare, at a median of 7 months, compared with the 3/15 (20%) patients with MRI healing, who flared at a median of 3.6 years. Radiological healing was associated with a significantly longer flare-free period (p = 0.01). Conclusions: Radiological healing is a less common, but deeper form of healing, associated with improved clinical outcomes. Further prospective trials are required to assess the benefit of earlier, and more regular imaging, with escalation of therapy based on radiological findings.

Until recently, analytical tests for food were performed primarily in laboratories, but technical developments now enable consumers to use devices to test their food at home or when dining out. Current consumer devices for food can determine nutritional values, freshness, and, most recently, the presence of food allergens and substances that cause food intolerances. The demand for such products is driven by an increase in the incidence of food allergies, as well as consumer desire for more information about what is in their food. The number and complexity of food matrixes creates an important need for properly validated testing devices with comprehensive user instructions (definitions of technical terms can be found in ISO 5725-1:1994 and the International Vocabulary of Metrology). This is especially important with food allergen determinations that can have life-threatening consequences. Stakeholders-including food regulators, food producers, and food testing kit and equipment manufacturers, as well as representatives from consumer advocacy groups-have worked to outline voluntary guidelines for consumer food allergen- and gluten-testing devices. These guidelines cover areas such as kit validation, user sampling instructions, kit performance, and interpretation of results. The recommendations are based on (1) current known technologies, (2) analytical expertise, and (3) standardized AOAC INTERNATIONAL allergen community guidance and best practices on the analysis of food allergens and gluten. The present guidance document is the first in a series of papers intended to provide general guidelines applicable to consumer devices for all food analytes. Future publications will give specific guidance and validation protocols for devices designed to detect individual allergens and gluten, as statistical analysis and review of any validation data, preferably from an independent third party, are necessary to establish a device's fitness-for-purpose. Following the recommendations of these guidance documents will help ensure that consumers are equipped with sufficient information to make an informed decision based on an analytical result from a consumer device. However, the present guidance document emphasizes that consumer devices should not be used in isolation to make a determination as to whether a food is safe to eat. As advances are made in science and technology, these recommendations will be reevaluated and revised as appropriate.