LGC

Genomic selection (GS) has potential to accelerate the genetic gain in crop plants. This study was undertaken to assess the accuracy and potential of GS in faba bean [Vicia faba (L.)] and to enhance its application in breeding programs. A set of 118 diverse faba bean accessions were phenotyped for key agronomic traits under herbicide and heat stress across 16 environments in Morocco, Lebanon, Sudan, and the United States. These accessions were genotyped, revealing 170 single nucleotide polymorphisms (SNPs) strongly associated with target traits. kompetitive allele‐specific PCR (KASP) markers were subsequently designed and validated on 4512 diverse breeding lines. Prediction accuracy (PA) was assessed using the reproducing kernel Hilbert space model, with and without genotype‐by‐environment interactions and taking into consideration two cross‐validation (CV) strategies: CV1 (predicting new lines) and CV2 (predicting complete records from unbalanced data). Additionally, 75 KASP markers with known association with heat tolerance traits were prioritized to estimate the PA of the models. The results showed a comparable PA between the two models, with CV1 outperforming CV2. This highlighted the difficulty in predicting the performance of untested lines in tested environments compared to lines evaluated in some environments but not others. Moreover, SNP subset size and composition significantly impacted PA, especially under heat stress. The highest accuracies were observed for days to flowering and plant height under heat stress and for plant height and grain yield under herbicide environments, indicating that these traits are ideal for training population selection. Optimizing the size and composition of the training population will enhance the effectiveness of GS in faba bean breeding.

Boldenone is an anabolic-androgenic steroid (AAS) that is prohibited in equine sports. However, in certain situations, it is endogenous, potentially formed by the microbes in urine. An approach to the differentiation based on the detection of the biomarkers Δ1-progesterone, 20(S)-hydroxy-Δ1-progesterone and 20(S)-hydroxyprogesterone was assessed, and their concentrations were monitored in the urine of untreated female horses (n = 291) alongside boldenone, boldienone, testosterone and androstenedione. Using an ultra-sensitive analytical method, boldenone (256 ± 236 pg/mL, n = 290) and the biomarkers (Δ1-progesterone up to 57.6pg/mL, n = 8; 20(S)-hydroxy-Δ1-progesterone 85.3 ± 181 pg/mL, n = 130; 20(S)-hydroxyprogesterone 43.5 ± 92.1pg/mL, n = 158) were detected at low concentrations. The ex vivo production of Δ1-steroids was artificially induced following the storage of urine samples at room temperature for 7days in order to assess the concentrations and ratios of the monitored steroids. The administration of inappropriately stored feed source also resulted in an increase in 20(S)-hydroxy-Δ1-progesterone concentrations and the biomarker ratios. Using the results from different datasets, an approach to differentiation was developed. In situations where the presence of boldenone exceeds a proposed action limit of 5ng/mL, the presence of the biomarkers would be investigated. If Δ1-progesterone is above 50 pg/mL or if 20(S)-hydroxy-Δ1-progesterone is above 100 pg/mL with the ratio of 20(S)-hydroxy-Δ1-progesterone:20(S)-hydroxyprogesterone greater than 5:1, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration. There remains a (small) possibility of a false negative result, but the model increases confidence that adverse analytical findings reported in female horses are caused by AAS administrations.

The reproducibility of research using laboratory animals requires reliable management of their quality, in particular of their genetics, health and environment, all of which contribute to their phenotypes. The point at which these biological materials are transferred between researchers is particularly sensitive, as it may result in a loss of integrity of the animals and/or their documentation. Here, we describe the various aspects of laboratory animal quality that should be confirmed when sharing rodent research models. We also discuss how repositories of biological materials support the scientific community to ensure the continuity of the quality of laboratory animals. Both the concept of quality and the role of repositories themselves extend to all exchanges of biological materials and all networks that support the sharing of these reagents.

Throughout the history of horse racing, doping techniques to suppress or enhance performance have expanded to match the technology available. The next frontier in doping, both in the equine and human sports areas, is predicted to be genetic manipulation; either by prohibited use of genome editing, or gene therapy via transgenes. By using massively-parallel sequencing via a two-step PCR method we can screen for multiple doping targets at once in pooled primer sets. This method has the advantages of high scalability through combinational indexing, and the use of reference standards with altered sequences as controls. Custom software produces transgene-specific amplicons from any Ensembl-annotated genome to facilitate rapid assay design. Additional scripts batch-process FASTQ data from experiments, automatically quality-filtering sequences and assigning hits based on discriminatory motifs. We report here our experiences in establishing the workflow with an initial 31 transgene and vector feature targets. To evaluate the sensitivity of parallel sequencing in a real-world setting, we performed an intramuscular (IM) administration of a control rAAV vector into two horses and compared the detection sensitivity between parallel sequencing and real-time qPCR. Vector was detected by all assays on both methods up to 79 h post-administration, becoming sporadic after 96 h.

Immunogenicity assays are required to evaluate anti-drug antibody (ADA)responses that can be generated against biotherapeutic modalities. Regulatory guidelines focus on clinical requirements, yet it has become apparent that industry has applied these clinical recommendations for immunogenicity assessment to nonclinical studies in varying degrees. ADAsare an anticipated outcome of dosing a humanized or fully human biotherapeutic into an animal. However, a nonclinical ADA response is rarely predictive of the immunogenic potential in humans. The addendum to ICH S6 recommends that immunogenicity should be explicitly examined where there is: evidence of altered pharmacodynamic activity;unexpected changes in exposure in the absence of a pharmacodynamic markerorevidence of immuno-mediated reactions. The European Bioanalytical Forum has extensively discussed and reached a consensus on a minimal strategic approach of when and what to include for nonclinical immunogenicity assessments. Additionally, this paper recommends a strategy for ADA assay validation and sample analysis for those cases when it is considered necessary to include an immunogenicity assessment in nonclinical toxicology studies.

<div> <div> <p>Cross-linking mass spectrometry (XL-MS) is a structural biology technique that can provide insights into the structure and interactions of proteins and their complexes, especially those that cannot be easily assessed by other methods. Quantitative XL-MS has the potential to probe the structural and temporal dynamics of protein complexes; however, it requires further development. Until recently, quantitative XL-MS has largely relied upon isotopic labeling and data dependent acquisition (DDA) methods, limiting the number of biological samples that can be studied in a single experiment. Here, the acquisition modes available on an ion mobility (IM) enabled QToF mass spectrometer are evaluated for the quantitation of cross-linked peptides, eliminating the need for isotopic labels and thus expanding the number of comparable studies that can be conducted in parallel. Workflows were optimized using metabolite and peptide standards analyzed in biological matrices, facilitating modelling of the data and addressing linearity issues, which allow for significant increases in dynamic range. Evaluation of the DDA acquisition method commonly used in XL-MS studies indicated consistency issues between technical replicates and reduced performance in quantitative metrics. On the contrary, data independent acquisition (DIA) and parallel reaction monitoring (PRM) modes proved more robust for analyte quantitation. Mobility enabled modes exhibited an improvement in sensitivity due to the added dimension of separation, and a simultaneous reduction in dynamic range, which was largely recovered by correction methods. Hi[3] and probabilistic quantitation methods were successfully applied to the DIA data, determining the molar amounts of cross-linked peptides relative to their linear counterparts.</p></div></div>

Authentication of Basmati rice has relied on microsatellite markers since 2004, but microsatellites cannot distinguish between all of the forty-one Basmati varieties approved in 2017. This study investigated whether single nucleotide polymorphisms (SNP) and insertion/deletion (InDel) variations developed into KASP™ (Kompetitive Allele Specific PCR; LGC Biosearch Technologies) could be used to distinguish between commercial Basmati varieties. Suitable loci were identified by comparing whole genome sequences of 120 diverse rice accessions. Sequences flanking these loci were standardized across a wide range of rice genomes to produce optimal KASP designs. We selected 364 KASP designs to use for genotyping; they were either near to informative microsatellite markers, within the Badh2 and Waxy genes, or distributed throughout the rice genome. Genotypes for 327 KASP were obtained with 255 loci revealing polymorphism in up to 41 samples of approved Basmati varieties and 20 non-Basmati varieties. The varieties genotyped had not been used in the KASP design process. KASP were able to distinguish between commercial Basmati varieties that could not be distinguished with currently available microsatellites. Thirty-seven Basmati varieties could be distinguished from all others with between 3 and 8 KASP markers out of a pool of 98 informative markers. A reduced set of 24 KASP markers could determine whether a sample belongs to one of eight family groups. All of the KASP markers used in this study can be purchased from LGC Biosearch Technologies. These markers have potential to be used by industry for routine testing and regulation.

Novel synthetic opioids (NSOs) are a class of novel psychoactive substances (NPS) that are growing in popularity and presenting a significant public health risk. Included in this class are derivatives of the highly potent analgesic, fentanyl. Cyclopropylfentanyl (CycP-F) was first reported to the EU Early Warning System in August 2017, and was subsequently linked to more than 100 deaths in the US alone. Limited pharmacological, pharmacokinetic or toxicological data is available for many emerging NSOs; however we can expect novel fentanyl analogues to present limited detection windows, short onset, narrow therapeutic indices and the potential for very high potency. Knowledge of the metabolism of these drugs is essential for the identification of analytical targets for their detection. Therefore in vitro metabolites of CycP-F were produced using human liver microsomal incubations. Metabolites formed were elucidated using liquid chromatography-high resolution accurate mass analysis (LC-HRAM). Identified metabolites were added to our accurate mass screening database for NPS which was utilised for subsequent screening analysis. CycP-F and metabolites were identified in two human blood case samples. Eleven metabolites were identified in vitro, with the major metabolites produced via N-dealkylation, monohydroxylation and N-oxidation. Analysis of the positive case samples identified four in vivo metabolites, all of which were observed in vitro. The major metabolite identified in vitro and in vivo was the N-dealkylated nor-metabolite; two further mono-hydroxylated and one dihydroxylated metabolite were detected in vivo.