Efficient Targeted Mutagenesis in Brassica Crops Using CRISPR/Cas Systems.

CRISPR/Cas has been established for targeted mutagenesis in many plant species since 2013, including Brassica napus and Brassica oleracea. Since that time, improvements have been made in terms of efficiency and choice of CRISPR systems. This protocol encompasses improved Cas9 efficiency and an alternative Cas12a system, allowing more challenging and diverse editing outcomes to be achieved.

Identification of Fusarium Head Blight Resistance in Triticum timopheevii Accessions and Characterization of Wheat-T. timopheevii Introgression Lines for Enhanced Resistance.

A diverse panel of wheat wild relative species was screened for resistance to Fusarium head blight (FHB) by spray inoculation. The great majority of species and accessions were susceptible or highly susceptible to FHB. Accessions of Triticum timopheevii (P95-99.1-1), Agropyron desertorum (9439957), and Elymus vaillantianus (531552) were highly resistant to FHB while additional accessions of T. timopheevii were found to be susceptible to FHB. A combination of spray and point inoculation assessments over two consecutive seasons indicated that the resistance in accession P95-99.1-1 was due to enhanced resistance to initial infection of the fungus (type 1 resistance), and not to reduction in spread (type 2 resistance). A panel of wheat-T. timopheevii (accession P95-99.1-1) introgression lines was screened for FHB resistance over two consecutive seasons using spray inoculation. Most introgression lines were similar in susceptibility to FHB as the wheat recipient (Paragon) but substitution of the terminal portion of chromosome 3BS of wheat with a similar-sized portion of 3G of T. timopheevii significantly enhanced FHB resistance in the wheat background.

In-planta Gene Targeting in Barley Using Cas9 With and Without Geminiviral Replicons.

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications; however, this remains inefficient especially in key crop species. Here, we describe successful, heritable gene targeting in barley at the target site of Cas9 using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase the copy number of the repair template. Without the replicon, we were able to delete 150 bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared with the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false-positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1, and subsequently, the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

CRISPR-Cas9 Based Genome Editing in Wheat.

The development and application of high precision genome editing tools such as programmable nucleases are set to revolutionize crop breeding and are already having a major impact on fundamental science. Clustered regularly interspaced short palindromic repeats (CRISPR), and its CRISPR-associated protein (Cas), is a programmable RNA-guided nuclease enabling targeted site-specific double stranded breaks in DNA which, when incorrectly repaired, result in gene knockout. The two most widely cultivated wheat types are the tetraploid durum wheat (Triticum turgidum ssp. durum L.) and the hexaploid bread wheat (Triticum aestivum L.). Both species have large genomes, as a consequence of ancient hybridization events between ancestral progenitors. The highly conserved gene sequence and structure of homoeologs among subgenomes in wheat often permits their simultaneous targeting using CRISPR-Cas9 with single or paired single guide RNA (sgRNA). Since its first successful deployment in wheat, CRISPR-Cas9 technology has been applied to a wide array of gene targets of agronomical and scientific importance. The following protocols describe an experimentally derived strategy for implementing CRISRP-Cas9 genome editing, including sgRNA design, Golden Gate construct assembly, and screening analysis for genome edits. © 2021 The Authors. Basic Protocol 1: Selection of sgRNA target sequence for CRISPR-Cas9 Basic Protocol 2: Construct assembly using Golden Gate (MoClo) assembly Basic Protocol 3: Screening for CRISPR-Cas9 genome edits Alternate Protocol: BigDye Terminator reactions for screening of CRISPR-Cas9 genome edits.

An Efficient Agrobacterium-Mediated Transformation Protocol for Hexaploid and Tetraploid Wheat.

Wheat, though a key crop plant with considerable influence on world food security, has nonetheless trailed behind other major cereals in the advancement of gene transformation technology for its improvement. New breeding technologies such as genome editing allow precise DNA manipulation, but their potential is limited by low regeneration efficiencies in tissue culture and the lack of transformable genotypes. We developed, in the hexaploid spring wheat cultivar "Fielder," a robust, reproducible Agrobacterium tumefaciens-mediated transformation system with transformation efficiencies of up to 33%. The system requires immature embryos as starting material and includes a centrifugation pretreatment before the inoculation with Agrobacterium. This high-throughput, highly efficient, and repeatable transformation system has been used effectively to introduce genes of interest for overexpression, RNA interference, and CRISPR-Cas-based genome editing. With slight modifications reported here, the standard protocol can be applied to the hexaploid wheat "Cadenza" and the tetraploid durum wheat "Kronos" with efficiencies of up to 4% and 10%, respectively. The system has also been employed to assess the developmental gene fusion GRF-GIF with outstanding results. In our hands, this technology combined with our transformation system improved transformation efficiency to 77.5% in Fielder. This combination should help alleviate the genotype dependence of wheat transformation, allowing new genome-editing tools to be used directly in more elite wheat varieties. © 2021 The Authors. Basic Protocol 1: Growing of donor plants Basic Protocol 2: Transformation of Agrobacterium with vector by electroporation Basic Protocol 3: Starting material collection, sterilization, and embryo inoculation Basic Protocol 4: Selection, regeneration, rooting, and acclimatization of transformants.

Dominant inhibition of awn development by a putative zinc-finger transcriptional repressor expressed at the B1 locus in wheat.

Awns, bristle-like structures extending from grass lemmas, provide protection against predators, contribute to photosynthesis and aid in grain dispersal. In wheat, selection of awns with minimal extension, termed awnletted, has occurred during domestication by way of loci that dominantly inhibit awn development, such as Tipped1 (B1), Tipped2 (B2), and Hooded (Hd). Here we identify and characterize the B1 gene. B1 was identified using bulked segregant RNA-sequencing of an F2 durum wheat population and through deletion mapping of awned bread wheat mutants. Functional characterization was accomplished by gene overexpression while haplotype analyses assessed B1 polymorphisms and genetic variation. Located on chromosome 5A, B1 is a C2H2 zinc finger encoding gene with ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs. Constitutive overexpression of B1 in awned wheat produced an awnletted phenotype with pleiotropic effects on plant height and fertility. Transcriptome analysis of B1 overexpression plants suggests a role as transcriptional repressor, putatively targeting pathways involved in cell proliferation. Haplotype analysis revealed a conserved B1 coding region with proximal polymorphisms and supported the contention that B1 is mainly responsible for awnletted wheats globally. B1, predominantly responsible for awn inhibition in wheat, encodes a C2H2 zinc finger protein with EAR motifs which putatively functions as a transcriptional repressor.

A versatile method for assessing pathogenicity of Hymenoscyphus fraxineus to ash foliage.

We describe a method for inoculating rachises of Fraxinus excelsior (European or common ash) with Hymenoscyphus fraxineus, which is faster than previous methods and allows associated foliar symptoms to be assessed on replicate leaves. A total of ten ash seedlings were inoculated with five isolates of H. fraxineus and lesion development assessed over four weeks. A five-point disease progress scale of symptom development was developed from no lesion (0), lesion on rachis (1), "pre-top dead," with curling of distal leaflets and bending of the rachis (2), top dead, with wilting and death of distal leaflets (3) to leaf abscission (4). The method revealed variation in aggressiveness of H. fraxinus isolates and may be suitable for assessing the resistance of F. excelsior and other Fraxinus species to dieback. The in vitro growth rate of H. fraxineus isolates was highly correlated with both disease progress and the length of rachis lesions on susceptible plants, indicating that it can be used as a preliminary step in selecting isolates with high aggressiveness for use in resistance screening.

Adamantinoma-like Ewing sarcoma mimicking basal cell adenocarcinoma of the parotid gland: a case report and review of the literature.

Adamantinoma-like Ewing sarcoma (AES) is a rare variant of the Ewing family of tumors that resembles classic adamantinoma of bone. AES shows epithelial differentiation and a more complex immunohistochemical expression profile with keratin and basal marker immunoreactivity and can resemble a variety of carcinomas. We report an unusual case of an AES of the parotid gland that mimicked a basal cell adenocarcinoma. Like basal cell adenocarcinoma, this AES showed a nested basaloid proliferation with peripheral palisading in tumor nests with 'basaloid' epithelial differentiation as highlighted by cytokeratin AE1/3 and p40 positivity. However, unlike most basal cell adenocarcinomas, this tumor demonstrated high grade morphology, showed no true ductal or myoepithelial component, and also showed a tendency towards neuroectodermal phenotype with focal rosette formation, CD99 and weak synaptophysin immunoreactivity. EWSR1 and FLI1 fluorescence in situ hybridization confirmed the presence of a translocation supporting the diagnosis of AES. This is the first case of AES presenting as a primary parotid mass highlighting the potential to be mistaken for primary salivary gland carcinomas, which in addition to basal cell adenocarcinoma include other basaloid tumors such as adenoid cystic carcinoma.

Malnourishment in a population of young children with severe early childhood caries.

PURPOSE: The purpose of this study was to describe the nutritional status of children with severe early childhood caries (S-ECC) using several clinical measurements. METHODS: Children aged 2 to 6 years with S-ECC were measured for height, weight, triceps skinfolds (TSF), and measurement of upper mid-arm circumference (MAC). Blood samples assessed: (1) hemoglobin; (2) mean corpuscular volume (MCV); (3) serum ferritin; and (4) serum albumin. Weight-for-height was converted into ideal body weight (IBW) percentiles. Body mass index (BMI) was calculated as kg/m2. TSF and MAC were converted into measurement of arm muscle circumference (MAMC). All measurements were compared with population reference values. RESULTS: Using weight for height centiles, 17% were diagnosed as being malnourished and 66% as within normal limits. Using BMI centiles, only 4% were identified as being malnourished and 75% as being normal. Conversely, the body fat of 24% was assessed as low (<10th percentile). Serum albumin was low for 16%. The majority had evidence of inadequate iron intake with low serum ferritin (80%), iron depletion (24%), iron deficiency (6%), or iron deficiency anemia (11%). CONCLUSIONS: All tests detected levels of malnutrition, with blood tests finding the most severe cases. The results suggest that severe Early Childhood Caries may be a risk marker for iron deficiency anemia. Since iron deficiency has permanent effects on growth and development, pediatric dentists should recommend assessment of iron levels in S-ECC patients regardless of their anthropometric appearance.

A comparison of the costs and patient acceptability of professionally applied topical fluoride foam and varnish.

OBJECTIVES: In Canada and the United States, professionally applied topical fluorides (PATF) are usually applied as a gel or foam. However, fluoride varnish has also been found to be effective for caries prevention and may be a preferred method because less time is required and fluoride exposure can be better controlled. The goal of this study was to compare the costs and patient acceptability of two methods of PATF (foam and varnish). METHODS: The study population was a convenience sample of high-risk children from the York Region and the city of Hamilton, Ontario, Canada, who had been identified as requiring fluoride therapy (n = 256). Children received from dental hygienists either fluoride foam applied in trays or fluoride varnish painted on tooth surfaces. An observer recorded the time to perform each procedure, adverse outcomes, and the satisfaction of children with treatment. RESULTS: The varnish technique took significantly less time compared to foam (5.81 vs 7.86 minutes; P < .0001). Significant differences between procedure times were found in all age groups, but the largest difference was for children aged 3-6 years (5.22 vs 8.61 minutes; P < .0001). Signs of gagging were observed in a lower proportion of participants who received varnish (3.8% vs 15.1%; P < .01), and this difference was largest for children aged 3-6 years (2.6% vs 29.7%; P < .01). The cost per varnish application, for children aged 3-6 years, was substantially less after labor costs were considered (dollar 3.43 vs dollar 4.43, CDN). CONCLUSIONS: Varnish applications were found to take less time and resulted in fewer signs of discomfort. These results support the use of fluoride varnish in caries prevention programs, especially for younger children.

Assessing the responsiveness of measures of oral health-related quality of life.

OBJECTIVES: This paper illustrates ways of assessing the responsiveness of measures of oral health-related quality of life (OHRQoL) by examining the sensitivity of the oral health impact profile (OHIP)-14 to change when used to evaluate a dental care program for the elderly. METHODS: One hundred and sixteen elderly patients attending four municipally funded dental clinics completed a copy of the OHIP-14 prior to treatment and 1 month after the completion of treatment. The post-treatment questionnaire also included a global transition judgement that assessed subjects' perceptions of change in their oral health following treatment at the clinics. Change scores were calculated by subtracting post-treatment OHIP-14 scores from pre-treatment scores. The longitudinal construct validity of these change scores were assessed by means of their association with the global transition judgements. Measures of responsiveness included effect sizes for the change scores, the minimal important difference, and Guyatt's responsiveness index. An receiver operating characteristic (ROC) curve was constructed to determine the accuracy of the change scores in predicting whether patients had improved or not as a result of the treatment. RESULTS: Based on the global transition judgements, 60.2% of subjects reported improved oral health, 33.6% reported no change, and only 6.2% reported that it was a little worse. These changes are reflected in mean pre- and post-treatment OHIP-14 scores that declined from 15.8 to 11.5 (P < 0.001). Mean change scores showed a consistent gradient in the expected direction across categories of the global transition judgement, but differences between the groups were not significant. However, paired t-tests showed no significant differences in the pre- and post-treatment scores of stable subjects, but showed significant declines for subjects who reported improvement. Analysis of data from stable subjects indicated that OHIP-14 had excellent test-retest reliability with an intraclass correlation coefficient (ICC) of 0.84. Effect size based on change scores for all subjects and subgroups of subjects were small to moderate. The ROC analysis indicated that OHIP-14 change scores were not good "diagnostic tests" of improvement. The minimal important difference for the OHIP-14 was of 5-scale points, but detecting this difference would require relatively large sample sizes. CONCLUSIONS: OHIP-14 appeared to be responsive to change. However, the magnitude of change that it detected in the context described here was modest, probably because it was designed primarily as a discriminative measure. The psychometric properties of the global transition judgements that often provide the "gold standard" for responsiveness studies need to be established.

Hiperexpressão e mutação do gene p53 em biópsias endoscópicas de pacientes com megaesôfago chagásico / Overexpression and mutation of gene p53 in endoscopic biopsies of patients with Chagas megaesophagus

O câncer do esôfago é mais prevalente em pacientes com megaesôfago chagáscio do que na população normal. Marcadores desse maior risco não são conhecidos. Objetivos: Correlacionar as alterações do p53 (gene supressor de tumor) e do PCNA (antígeno nuclear de células em proliferação) com as alterações histólogiacas da mucosa em pacientes com megaesôfago avançado. Métodos:Quatro carcinomas epidermóides do esôfago e áreas adjacentes correspondentes, provenietes de pacientes com megaesôfago, foram analisados através de imuno-histoquimica para p53 e PCNA. Concomitantemente, 128 biópsias, de diferentes níveis do esôfago, de 16 pacientes com megaesôfago avançado (sem tumor) foram prospectivamente coletados e avaliadas quanto ao grau de inflamação, hiperplasia, displasia e imuno-histoquímica para p53 e PCNA. Todos os tecidos positivos para o p53 foram submetidos à análise gênica topográfica através de microdissecção, amplicifacção por PCR e direito sequenciamento dos exons 5 a 8. Resultados: Reatividade forte e difusa para o p53 foi observada em 2/4 tumores (exon 7,238 CH e exon 5, 146WC). Em um paciente, a área adjacente ao tumor apresentou forte positividade para o p53. Na mucosa adjacente, as mesmas áreas mostrando hiperexpressão do p53 também apresentaram maior positividade ao PCNA. No grupo prospectivo, 7/16 (43,7por cento) pacientes ou 53/128(41,4por cento) biópsias expressaram o p53. Hiperplasia foi diagnosticada em 4/16pacientes ou em 10 biópsias e associada a hiperexpressão do p53 em todos os casos . Displasia não esteve presente nesse grupo. Foi observada positividade ao p53 em 15,4por cento das biópsias sem inflamação em 31,4por cento com inflamação leve, 78,3por cento com moderada e 88,9poe cento com intensa (p<0,00001). A expressão do PCNA foi detectada na camada basal da mucosa e aumento da positividade foi associada à hiperexpressão da p53 (p=0,00018). A genotipagem revelou mutação em exon 5 códon 213 RG, em um paciente (1/16, 6,2por cento). Conclusão: 1) A expressão e mutação do p53 em biópsias de pacientes de alto risco indicam que este gene representa um evento inicial no desenvolvimento do câncer esôfago: 2)A inflamação frequentemente observada nesses pacientes parece estar associada às alterações da proteína p53; 3) A expressão do gene supressor de tumor está aumentada em áreas mostrando proliferação celular

Cathepsin B and Cathepsin D Expression in Follicular Adenomas and Carcinomas of the Thyroid Gland.

Biologic features that distinguish follicular adenomas (FAs), minimally invasive follicular carcinomas (MICs), and extensively invasive follicular carcinomas (EICs) of the thyroid gland are not well understood. Endogenous proteases, including cathepsin B (CB) and cathepsin D (CD), have been linked to tumor progression in other malignancies and may be important in the different biologic behavior of these follicular thyroidal lesions. Archival paraffin-embedded tissue sections from 16 FAs, 12 MICs, and 4 EICs were studied for immunohistochemical expression of CR and CD. Percent of tumor staining, intensity of staining, and intracytoplasmic staining pattern were assessed. Increased intensity of staining with CD was observed in follicular carcinomas compared to adenomas and was greatest in the EIC (p < 0.04). An increase in diffuse cytoplasmic staining pattern with CD (p < 0.008) and CB (p < 0.02) was observed in follicular carcinomas compared to FAs. The increased intensity of staining with CD in the follicular thyroid carcinomas and the increased diffuse cytoplasmic staining pattern with CD and CR in these carcinomas suggest that these proteinases may play a role in their propensity to invade and metastasize and, therefore, their more aggressive behavior. Furthermore, this diffuse cytoplasmic staining suggests an altered intracellular processing of these proteinases in the carcinomas.

Proliferative Activity in Pancreatic Endocrine Tumors: Association with Function, Metastases, and Survival.

Endocrine tumors of the pancreas are slow-growing lesions, yet one-third to one-half will metastasize. It is generally accepted that histopathologic features do not reliably predict metastatic potential or outcome. We investigated whether proliferative activity, as determined by MIB-l labeling, correlated with tumor type, metastasis, or patient survival. Formalin-fixed sections of pancreatic endocrine tumors were immunohistochemically stained for the MIB-l antibody against Ki-67 using the avidin-biotin complex technique. Labeling index (LI) was determined by counting 1000 consecutive tumor cells in an area of greatest staining intensity at x400 and expressed as a percentage. The study group included 37 patients, including 10 gastrinomas, 9 insulinomas, 4 glucagonomas, 2 VlPomas, and 12 nonfunctioning tumors. Twenty-one patients had metastases, primarily to regional lymph nodes and the liver. Five patients had MEN I. MIB-1 LI was significantly greater in the nonfunctioning tumors (mean 20S%) than in the functioning tumors (mean 51%) (p = 0.01). LI for functional tumors (insulinomas 64%, glucagonoma 44%, gastrinomas 32%, VlPomas 32%) were similar to each other, MIB-1 was significantly higher in those tumors that metastasized (mean 15.6%) compared to those that did not (mean 31%), (p = 0.04). All tumors with MIB-1 LI >10% developed metastases. Logistic regression showed that MIB-1 was a significant predictor of metastases (p = 0.003) after adjusting for functional status. MIB-1 LI also correlated with outcome in that those patients with MIB-1 LI >/=10% had a mean survival of 19 mo compared to 72 mo for those with levels <10% (p = 0.0001). Results of the proportional hazards model showed that MIR-1 remained a significant (p = 0.03) and independent predictor of survival times after adjustment for tumor size and functional status. Higher MIB-1 LI values were significantly associated with shorter survival times. In conclusion, MIB-1 LI appears to be a useful indicator of metastatic potential and is predictive of outcome in PET.