Single-incision percutaneous drilling technique to achieve hemiepiphysiodesis of the distal metacarpus in foals with metacarpophalangeal varus deformities.

OBJECTIVE: To describe a drilling technique for hemiepiphysiodesis of the distal lateral metacarpal physis and report the outcome of treated foals. STUDY DESIGN: Retrospective case series. SAMPLE POPULATION: Eleven thoroughbred foals. METHODS: While horses were under general anesthesia, the lateral aspect of the distal metacarpal physis was approached through a single small incision by using a power drill. The drill bit was placed at the level of the physis under radiographic guidance. A 4.5-mm drill bit was passed several times through the lateral growth plate to remove the cartilage in a fan-like pattern. Postoperative outcomes consisted of clinical assessment and farm manager/owner satisfaction. Cosmetics and radiographic appearance of the surgical site were assessed when the horses were yearlings. RESULTS: The procedure was performed in 16 limbs of 11 thoroughbred foals with a median age of 113.5 days (range, 90-129). Median age at postoperative follow-up was 422 days (range, 366 to 452). The procedure stopped the progression of a metacarpophalangeal varus deformity in all the limbs treated, determined by visual clinical evaluation and farm manager's satisfaction with subjectively excellent radiographic images and cosmetic outcomes at yearling age. CONCLUSION: Physis ablation was consistently achieved in these 11 foals with developing growth deformities of the distal metacarpus. CLINICAL SIGNIFICANCE: This drilling technique may offer a minimally invasive, safe, and simple technique to manage distal limb conformation in foals without placement of implants. Additional quantitative data are required to assess its effectiveness relative to other options.

Prospective diagnostic performance of semiconductor SPECT myocardial perfusion imaging: wall thickening analysis reduces the need for an additional prone acquisition.

PURPOSE: To determine whether the assessment of regional wall thickening (WT) in addition to myocardial perfusion from stress supine acquisitions could compensate for the lack of prone acquisition and the corresponding decrease in the diagnostic performance of SPECT myocardial perfusion imaging (MPI) in patients with known or suspected coronary artery disease (CAD). METHODS: The study group comprised 41 patients (123 vessels) with known or suspected CAD prospectively recruited for systematic prone and supine 201Tl stress SPECT MPI. The diagnostic performance of SPECT MPI was determined for various image sets including nongated supine images (supine NG), nongated combined prone and supine images (prone and supine NG) and gated supine images, allowing WT evaluation from NG images in addition to perfusion (supine NG + WT) using invasive coronary angiography and fractional flow reserve as the gold standards. RESULTS: The rate of false positives was significantly higher among the supine NG images (20.8%) than among either the prone and supine NG or the supine NG + WT images (3.3% and 2.7%, respectively, P < 0.05 vs. supine NG). Consequently, specificity was higher for the prone and supine NG images than for the supine NG images (96.1% vs. 76.1%, P < 0.01) and was highest for the supine NG + WT images (96.8%, P not significant vs. prone and supine NG), without significant differences in sensitivity (80.0%, 86.6% and 73.3%, respectively, P not significant for all comparisons). CONCLUSION: The diagnostic performance of supine stress SPECT MPI is improved when WT assessment of ischaemic segments is used as an additional diagnostic criterion to values not significantly different from those with combined prone and supine acquisitions.

Genetic and environmental factors affecting the expression of α-gliadin canonical epitopes involved in celiac disease in a wide collection of spelt (Triticum aestivum ssp. spelta) cultivars and landraces.

BACKGROUND: Celiac disease (CD) is an autoimmune disorder affecting genetically predisposed individuals whose dietary gluten proteins trigger an inflammatory reaction in the small intestine. Gluten is found in the seeds of cereals like bread wheat (Triticum aestivum ssp. aestivum) and spelt (Triticum aestivum ssp. spelta). The development of new varieties lacking immunogenic peptides is one of the strategies currently investigated to address the CD problem. Among gluten proteins, α-gliadins display the strongest immunogenicity with four main T-cell stimulatory epitopes. The objective of this work was to study the expression of α-gliadin epitopes related to CD in a wide collection of 121 spelt accessions (landraces and varieties, spring and winter accessions) from different provenances, and to analyze the correlation between the presence of epitope sequences in gDNA and their expression (cDNA). The effect of environmental factors (harvest year and N fertilization) on the epitope expression was also investigated. RESULTS: TaqMan probes targeting the canonical form of the epitopes were used to evaluate the epitope expression levels. Significant variations in the amount of epitope transcripts were identified between accessions and according to the provenances. Spring accessions showed a significantly higher immunogenicity than winter ones and no influence of spelt breeding on the epitope expression levels could be assessed when comparing landraces and varieties from Northwestern Europe. No correlation was observed between quantitative PCR results obtained from cDNA and gDNA for 45 accessions tested, stressing the need to use markers focusing on epitope transcripts rather than on genomic sequences. A relative stability of the amount of epitopes expressed by a same accession across four harvest years was detected. The fertilization strategy, evaluated through seven N fertilization modalities applied to two commercial spelt varieties, did not influence the epitope expression of the first variety, whereas it had a slight effect for the second one. CONCLUSIONS: The results obtained in this work showed that the CD-related epitope expression greatly fluctuated among the spelt accessions studied. This expression was not correlated to the epitope genomic occurrence and environmental factors had almost no influence on the amount of epitope transcripts.

How reliable is metabarcoding for pollen identification? An evaluation of different taxonomic assignment strategies by cross-validation.

Metabarcoding is a powerful tool, increasingly used in many disciplines of environmental sciences. However, to assign a taxon to a DNA sequence, bioinformaticians need to choose between different strategies or parameter values and these choices sometimes seem rather arbitrary. In this work, we present a case study on ITS2 and rbcL databases used to identify pollen collected by bees in Belgium. We blasted a random sample of sequences from the reference database against the remainder of the database using different strategies and compared the known taxonomy with the predicted one. This in silico cross-validation (CV) approach proved to be an easy yet powerful way to (1) assess the relative accuracy of taxonomic predictions, (2) define rules to discard dubious taxonomic assignments and (3) provide a more objective basis to choose the best strategy. We obtained the best results with the best blast hit (best bit score) rather than by selecting the majority taxon from the top 10 hits. The predictions were further improved by favouring the most frequent taxon among those with tied best bit scores. We obtained better results with databases containing the full sequences available on NCBI rather than restricting the sequences to the region amplified by the primers chosen in our study. Leaked CV showed that when the true sequence is present in the database, blast might still struggle to match the right taxon at the species level, particularly with rbcL. Classical 10-fold CV-where the true sequence is removed from the database-offers a different yet more realistic view of the true error rates. Taxonomic predictions with this approach worked well up to the genus level, particularly for ITS2 (5-7% of errors). Using a database containing only the local flora of Belgium did not improve the predictions up to the genus level for local species and made them worse for foreign species. At the species level, using a database containing exclusively local species improved the predictions for local species by ∼12% but the error rate remained rather high: 25% for ITS2 and 42% for rbcL. Foreign species performed worse even when using a world database (59-79% of errors). We used classification trees and GLMs to model the % of errors vs. identity and consensus scores and determine appropriate thresholds below which the taxonomic assignment should be discarded. This resulted in a significant reduction in prediction errors, but at the cost of a much higher proportion of unassigned sequences. Despite this stringent filtering, at least 1/5 sequences deemed suitable for species-level identification ultimately proved to be misidentified. An examination of the variability in prediction accuracy between plant families showed that rbcL outperformed ITS2 for only two of the 27 families examined, and that the % correct species-level assignments were much better for some families (e.g. 95% for Sapindaceae) than for others (e.g. 35% for Salicaceae).

A detailed workflow to develop QIIME2-formatted reference databases for taxonomic analysis of DNA metabarcoding data.

BACKGROUND: The DNA metabarcoding approach has become one of the most used techniques to study the taxa composition of various sample types. To deal with the high amount of data generated by the high-throughput sequencing process, a bioinformatics workflow is required and the QIIME2 platform has emerged as one of the most reliable and commonly used. However, only some pre-formatted reference databases dedicated to a few barcode sequences are available to assign taxonomy. If users want to develop a new custom reference database, several bottlenecks still need to be addressed and a detailed procedure explaining how to develop and format such a database is currently missing. In consequence, this work is aimed at presenting a detailed workflow explaining from start to finish how to develop such a curated reference database for any barcode sequence. RESULTS: We developed DB4Q2, a detailed workflow that allowed development of plant reference databases dedicated to ITS2 and rbcL, two commonly used barcode sequences in plant metabarcoding studies. This workflow addresses several of the main bottlenecks connected with the development of a curated reference database. The detailed and commented structure of DB4Q2 offers the possibility of developing reference databases even without extensive bioinformatics skills, and avoids 'black box' systems that are sometimes encountered. Some filtering steps have been included to discard presumably fungal and misidentified sequences. The flexible character of DB4Q2 allows several key sequence processing steps to be included or not, and downloading issues can be avoided. Benchmarking the databases developed using DB4Q2 revealed that they performed well compared to previously published reference datasets. CONCLUSION: This study presents DB4Q2, a detailed procedure to develop custom reference databases in order to carry out taxonomic analyses with QIIME2, but also with other bioinformatics platforms if desired. This work also provides ready-to-use plant ITS2 and rbcL databases for which the prediction accuracy has been assessed and compared to that of other published databases.

Detection of Alphitobius diaperinus by Real-Time Polymerase Chain Reaction With a Single-Copy Gene Target.

Use of edible insects as an alternative source of proteins in food and feed is increasing. These last years, numerous companies in Europe have started producing insects for food and feed purposes. In the European Union, the use of edible insects for human consumption falls within Regulation (EU) No. 2015/2283 on novel foods. For feed, Commission Regulation (EU) 2017/893 authorizes seven insect species as processed animal proteins for aquaculture. Methods of authentication are required to check the conformity of the products. In this study, we propose a real-time polymerase chain reaction (PCR) method for the specific detection of the lesser mealworm (Alphitobius diaperinus), one of the species included in the shortlist of authorized insects. The selected target is the cadherin gene with a single-copy (per haploid genome) illustrated by our experimental evidence. The PCR test amplified a 134-bp fragment of the cadherin gene. The qualitative method was assessed toward several performance criteria. Specificity was checked against 54 insect species next to other animal and plant species. The sensitivity, efficiency, robustness, and transferability of the PCR assay were also successfully tested. Finally, the applicability of the test was assessed on real-life processed samples (industrial meals) of A. diaperinus. The study also showed that there seems to be a huge confusion on the correct labeling of the marketed mealworms. We did not succeed to get Alphitobius laevigatus samples. They all appeared to belong to the A. diaperinus taxon.

Assessment of clinical and computed tomographic findings for association with the outcome of intraoral cheek tooth extraction in horses and ponies.

OBJECTIVE: To describe clinical and CT findings for horses and ponies undergoing intraoral cheek tooth extraction and assess potential associations between these features and outcome of the procedure. ANIMALS: 74 horses and 7 ponies. PROCEDURES: Medical records were searched to identify horses and ponies that underwent CT and intraoral extraction of ≥ 1 cheek tooth with standing sedation. Signalment and clinical variables were recorded, and CT scans were reviewed. Anatomic location and measurements of affected teeth; abnormalities of the periodontium, pulp, infundibula, roots, and tooth shape; fracture presence and type; presence of sinusitis; and affected sinus cavities were assessed by a surgeon and a radiologist. Intraoral extraction outcome was recorded as successful (complete removal of the tooth in 1 intraoral extraction procedure) or unsuccessful. Univariable and multivariable logistic regression analyses were performed to assess associations between clinical or CT findings and outcome. RESULTS: 89 cheek teeth (80 maxillary and 9 mandibular) were included in the analyses. Sixty of 89 (67%) cheek teeth were extracted successfully (56/80 [70%] maxillary and 4/9 [44%] mandibular cheek teeth). Only presence of a simple fracture (vs no fracture) was associated with outcome on multivariable regression analysis; odds of successful intraoral extraction were significantly lower when this feature was present. CONCLUSIONS AND CLINICAL RELEVANCE: Most extractions of cheek teeth in the study sample were successful, and results may be useful for practitioners in refining cheek tooth extraction plans for horses and ponies. Further studies are required to assess whether specific CT findings can be used to predict the outcome of intraoral extraction in equids.

Reactivity of gluten proteins from spelt and bread wheat accessions towards A1 and G12 antibodies in the framework of celiac disease.

In the framework of celiac disease, this research aims at evaluating the reactivity of 195 wheat accessions and 240 spelt accessions to A1 and G12 monoclonal antibodies. A great variability in reactivity was found among the accessions of both subspecies. On average, spelt showed very slightly higher reactivity than wheat but accessions with low reactivity were encountered in both subspecies. In both wheat and spelt, there was no significant difference in the level of reactivity between varieties and landraces. Similarly, there was no significant difference in reactivity between old, mid and new varieties of wheat. In contrast, new spelt varieties showed lower levels of reactivity than old and mid ones. No relationship could be established between level of reactivity, protein content and the Zeleny index. This research did not establish a link between the breeding strategies for baking quality improvement and A1-G12 antibodies reactivity.

Interim PET Response-adapted Strategy in Untreated Advanced Stage Hodgkin Lymphoma: Results of GOELAMS LH 2007 Phase 2 Multicentric Trial.

BACKGROUND: Patients with advanced stage Hodgkin lymphoma still present unsatisfactory outcomes. PATIENTS AND METHODS: The Groupe d'étude des Leucémies Aigues et des Maladies du Sang (GOELAMS) group conducted a prospective multicentric trial (NCT00920153) for advanced stage Hodgkin lymphoma to evaluate a positron emission tomography (PET)-adapted strategy. Patients received an intensive regimen (VABEM [vindesine, doxorubicin, carmustine, etoposide, and methylprednisolone]) in front-line and interim 18FFDG-PET evaluation after 2 courses (PET-2). Patients with negative PET-2 findings received 1 additional course. Patients with positive PET-2 findings underwent early salvage therapy followed by high-dose therapy/autologous stem cell transplantation. RESULTS: Fifty-one patients were included. The final complete remission rate was 88%. With a median follow up of 5.3 years, 5-year event-free survival and overall survival rates were 75.3% and 85.3%, respectively, for the whole cohort. Patients who were PET-2-negative had 5-year event-free survival and overall survival rates of, respectively, 77.8% and 88.2% versus 85.1% and 91.7% for patients who were PET-2-positive. CONCLUSION: A PET-guided strategy with early salvage therapy and high-dose therapy/autologous stem cell transplantation for patients with interim PET-2-positive findings is safe and feasible and provide similar outcome as patients with a negative PET-2.

Development of TaqMan probes targeting the four major celiac disease epitopes found in α-gliadin sequences of spelt (Triticum aestivum ssp. spelta) and bread wheat (Triticum aestivum ssp. aestivum).

BACKGROUND: Celiac disease (CD) is caused by specific sequences of gluten proteins found in cereals such as bread wheat (Triticum aestivum ssp. aestivum) and spelt (T. aestivum ssp. spelta). Among them, the α-gliadins display the highest immunogenicity, with four T-cell stimulatory epitopes. The toxicity of each epitope sequence can be reduced or even suppressed according to the allelic form of each sequence. One way to address the CD problem would be to make use of this allelic variability in breeding programs to develop safe varieties, but tools to track the presence of toxic epitopes are required. The objective of this study was to develop a tool to accurately detect and quantify the immunogenic content of expressed α-gliadins of spelt and bread wheat. RESULTS: Four TaqMan probes that only hybridize to the canonical-i.e. toxic-form of each of the four epitopes were developed and their specificity was demonstrated. Six TaqMan probes targeting stable reference genes were also developed and constitute a tool to normalize qPCR data. The probes were used to measure the epitope expression levels of 11 contrasted spelt accessions and three ancestral diploid accessions of bread wheat and spelt. A high expression variability was highlighted among epitopes and among accessions, especially in Asian spelts, which showed lower epitope expression levels than the other spelts. Some discrepancies were identified between the canonical epitope expression level and the global amount of expressed α-gliadins, which makes the designed TaqMan probes a useful tool to quantify the immunogenic potential independently of the global amount of expressed α-gliadins. CONCLUSIONS: The results obtained in this study provide useful tools to study the immunogenic potential of expressed α-gliadin sequences from Triticeae accessions such as spelt and bread wheat. The application of the designed probes to contrasted spelt accessions revealed a high variability and interesting low canonical epitope expression levels in the Asian spelt accessions studied.

Molecular diversity of α-gliadin expressed genes in genetically contrasted spelt (Triticum aestivum ssp. spelta) accessions and comparison with bread wheat (T. aestivum ssp. aestivum) and related diploid Triticum and Aegilops species.

The gluten proteins of cereals such as bread wheat (Triticum aestivum ssp. aestivum) and spelt (T. aestivum ssp. spelta) are responsible for celiac disease (CD). The α-gliadins constitute the most immunogenic class of gluten proteins as they include four main T-cell stimulatory epitopes that affect CD patients. Spelt has been less studied than bread wheat and could constitute a source of valuable diversity. The objective of this work was to study the genetic diversity of spelt α-gliadin transcripts and to compare it with those of bread wheat. Genotyping data from 85 spelt accessions obtained with 19 simple sequence repeat (SSR) markers were used to select 11 contrasted accessions, from which 446 full open reading frame α-gliadin genes were cloned and sequenced, which revealed a high allelic diversity. High variations among the accessions were highlighted, in terms of the proportion of α-gliadin sequences from each of the three genomes (A, B and D), and their composition in the four T-cell stimulatory epitopes. An accession from Tajikistan stood out, having a particularly high proportion of α-gliadins from the B genome and a low immunogenic content. Even if no clear separation between spelt and bread wheat sequences was shown, spelt α-gliadins displayed specific features concerning e.g. the frequencies of some amino acid substitutions. Given this observation and the variations in toxicity revealed in the spelt accessions in this study, the high genetic diversity held in spelt germplasm collections could be a valuable resource in the development of safer varieties for CD patients.

Magnitude of force perception errors during static contractions of the knee extensors in healthy young and elderly individuals.

The accuracy of force perception during muscular contraction has not been studied extensively, despite its importance in rehabilitation and training. The purpose of this study was to quantify the errors made by healthy young and elderly individuals in their perceptions of force produced at the knee. Four different tasks were used to evaluate the perception of force and the effect of a sensory-motor reference and simultaneous contraction on the accuracy of perception. The absolute errors were similar between groups, with values of 11.9% to 16.3%, depending on the task. The raw perception errors were greater for high levels of force (>50% of the maximal voluntary contraction, or MVC), indicating an overestimation of the forces produced for both groups. At 70% MVC, the sensory-motor reference reduced raw perception errors, and the simultaneous contraction improved the accuracy of force production. Healthy young and elderly individuals had about the same capacity to judge the muscular force of their knee extensors. Therapists involved in the training of active elderly individuals should be aware that the accuracy of force perception is not perfect and that these clients have the same ability as young individuals to perceive their knee extension strength.