Ppe.XapF: High Throughput KASP Assays to Identify Fruit Response to Xanthomonas arboricola pv. pruni (Xap) in Peach

Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Xap), is a serious peach disease with symptoms that traverse severe defoliation and black surface pitting, cracking or blemishes on peach fruit with global economic impacts. A management option for control and meeting consumer demand for chemical-free, environmentally friendly fruit production is the development of resistant or tolerant cultivars. We developed simple, accurate, and efficient DNA assays (Ppe.XapF) based on SNP genotyping with KASP technology to quickly test for bacterial spot resistance alleles in peach fruit that allows breeders to cull seedlings at the greenhouse stage. The objective of this research was to validate newly developed DNA tests that target the two major QTLs for fruit resistance in peach with diagnostic utility in predicting fruit response to bacterial spot infection. Our study confirms that only two Ppe.XapF DNA tests, Ppe.XapF1-1 and Ppe.XapF6-2, are needed to distinguish between susceptible and resistant alleles. Use of these efficient and accurate Ppe.XapF KASP tests resulted in 44% reduction in seedling planting rate in the Clemson University peach breeding program.

Cancer Therapy Guided by Mutation Tests: Current Status and Perspectives

The administration of many cancer drugs is tailored to genetic tests. Some genomic events, e.g., alterations of EGFR or BRAF oncogenes, result in the conformational change of the corresponding proteins and call for the use of mutation-specific compounds. Other genetic perturbations, e.g., HER2 amplifications, ALK translocations or MET exon 14 skipping mutations, cause overproduction of the entire protein or its kinase domain. There are multilocus assays that provide integrative characteristics of the tumor genome, such as the analysis of tumor mutation burden or deficiency of DNA repair. Treatment planning for non-small cell lung cancer requires testing for EGFR, ALK, ROS1, BRAF, MET, RET and KRAS gene alterations. Colorectal cancer patients need to undergo KRAS, NRAS, BRAF, HER2 and microsatellite instability analysis. The genomic examination of breast cancer includes testing for HER2 amplification and PIK3CA activation. Melanomas are currently subjected to BRAF and, in some instances, KIT genetic analysis. Predictive DNA assays have also been developed for thyroid cancers, cholangiocarcinomas and urinary bladder tumors. There is an increasing utilization of agnostic testing which involves the analysis of all potentially actionable genes across all tumor types. The invention of genomically tailored treatment has resulted in a spectacular improvement in disease outcomes for a significant portion of cancer patients.

Optimization of dye solutions for detecting damaged pancreatic tissues during islet isolation procedures

We previously reported that dye was effective to prevent the leakage of enzyme solutions from pancreatic glands during an islet isolation procedure. However, the dye used for islet isolation has not yet been optimized. In this study, we focused on pyoktanin blue (PB), diagnogreen (DG), and indigo carmine (IC) as potential candidates among clinically established dyes. A serial dilution assay was performed to determine minimal effective concentrations of each dye for detecting damaged pancreatic tissues. According to the outcome of serial dilution assays, double minimum effective concentrations of each dye were used for in vitro toxicity assays on islets and used in the isolation procedure to investigate whether they adversely affect islet isolation efficiency. The evaluations included islet yield, ADP/ATP, ATP/DNA, glucose stimulation test, and insulin/DNA assays. Islet viability cultured with PB contained medium was significantly lower than the other dyes. DG and IC appeared to be non-toxic to the islets. In isolation experiments, the islet yield in the DG group was considerably lower than that in the Control group, suggesting that DG might inhibit enzyme activity. The present study demonstrates that IC could be a promising candidate for an effective dye to detect damaged pancreatic tissues without affecting the enzyme activity and islet quality.

The effect of collagen hydrogels on chondrocyte behaviors through restricting the contraction of cell/hydrogel constructs

Collagen is a promising material for tissue engineering, but the poor mechanical properties of collagen hydrogels, which tend to cause contraction under the action of cellular activity, make its application challengeable. In this study, the amino group of type I collagen (Col I) was modified with methacrylic anhydride (MA) and the photo-crosslinkable methacrylate anhydride modified type I collagen (CM) with three different degrees of substitution (DS) was prepared. The physical properties of CM and Col I hydrogels were tested, including micromorphology, mechanical properties and degradation properties. The results showed that the storage modulus and degradation rate of hydrogels could be adjusted by changing the DS of CM. In vitro, chondrocytes were seeded into these four groups of hydrogels and subjected to fluorescein diacetate/propidium iodide (FDA/PI) staining, cell counting kit-8 (CCK-8) test, histological staining and cartilage-related gene expression analysis. In vivo, these hydrogels encapsulating chondrocytes were implanted subcutaneously into nude mice, then histological staining and sulfated glycosaminoglycan (sGAG)/DNA assays were performed. The results demonstrated that contraction of hydrogels affected behaviors of chondrocytes, and CM hydrogels with suitable DS could resist contraction of hydrogels and promote the secretion of cartilage-specific matrix in vitro and in vivo.

A unique molecular identifier-based and clonal hematopoiesis-aware approach for accurate mutation calling in cell-free DNA assays.

e22515 Background: Given peripheral blood cells (PBCs) matched cell-free DNA (cfDNA), accurate mutation calling in next generation sequencing (NGS)-based assays relies on discriminating artifacts and clonal hematopoiesis mutations from tissue originated somatic mutations. Although clonal hematopoiesis has been considered in previous overall error modeling, it has not been adapted to PBCs without using unique molecular identifiers (UMIs). Moreover, previous studies on background error profiling were mainly based on healthy controls without matched PBC gDNA, which may lead to potential overestimation of the error rates on those sites with clonal hematopoiesis mutations. Additionally, the fraction of tissue cells is also an important influencing factor but is usually ignored. Methods: We performed UMI-assisted capture-based DNA assays on cfDNA samples, matched PBCs, and oral epithelium cells from 150 healthy donors. A site-specific and subtype-specific background error model was first built for PBCs using the SNVs called from PBCs with matched oral epithelium cells to exclude potential clonal hematopoiesis mutations. Then a similar background model was established for cfDNA with the SNVs inferred from cfDNA to exclude clonal hematopoiesis. The SNVs identified in cfDNA and matched PBCs were separately filtered with the cfDNA and PBC background error models. In this study, we used the ultrasensitive liquid biopsy approach to evaluate paired with tissue and blood samples from 56 early-stage NSCLC patients. All samples are sequenced using NGS target-capture panels covering 29 genes. Results: The mutations were detected in 91.1% of tissue and 67.9% were discovered in plasma. The coincidence rate between tissue and plasma of the 56 early-stage NSCLC patients was 66.1%. Conclusions: We have developed a novel method tailored for UMI-assisted capture-based targeted DNA assays, which outperforms currently available methods in terms of modeling background errors and filtering clonal hematopoietic mutations.

Full pathogen characterisation: species identification including the detection of virulence factors and antibiotic resistance genes via multiplex DNA-assays

Antibiotic resistances progressively cause treatment failures, and their spreading dynamics reached an alarming level. Some strains have already been classified as highly critical, e.g. the ones summarised by the acronym ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.). To restrain this trend and enable effective medication, as much information as possible must be obtained in the least possible time. Here, we present a DNA microarray-based assay that screens for the most important sepsis-relevant 44 pathogenic species, 360 virulence factors (mediate pathogenicity in otherwise non-pathogenic strains), and 409 antibiotic resistance genes in parallel. The assay was evaluated with 14 multidrug resistant strains, including all ESKAPE pathogens, mainly obtained from clinical isolates. We used a cost-efficient ligation-based detection platform designed to emulate the highly specific multiplex detection of padlock probes. Results could be obtained within one day, requiring approximately 4 h for amplification, application to the microarray, and detection.