Applicability of Gene Expression in Saliva as an Alternative to Blood for Biodosimetry and Prediction of Radiation-induced Health Effects.

As the great majority of gene expression (GE) biodosimetry studies have been performed using blood as the preferred source of tissue, searching for simple and less-invasive sampling methods is important when considering biodosimetry approaches. Knowing that whole saliva contains an ultrafiltrate of blood and white blood cells, it is expected that the findings in blood can also be found in saliva. This human in vivo study aims to examine radiation-induced GE changes in saliva for biodosimetry purposes and to predict radiation-induced disease, which is yet poorly characterized. Furthermore, we examined whether transcriptional biomarkers in blood can also be found equivalently in saliva. Saliva and blood samples were collected in parallel from radiotherapy (RT) treated patients who suffered from head and neck cancer (n = 8) undergoing fractioned partial-body irradiations (1.8 Gy/fraction and 50-70 Gy total dose). Samples were taken 12-24 h before first irradiation and ideally 24 and 48 h, as well as 5 weeks after radiotherapy onset. Due to the low quality and quantity of isolated RNA samples from one patient, they had to be excluded from further analysis, leaving a total of 24 saliva and 24 blood samples from 7 patients eligible for analysis. Using qRT-PCR, 18S rRNA and 16S rRNA (the ratio being a surrogate for the relative human RNA/bacterial burden), four housekeeping genes and nine mRNAs previously identified as radiation responsive in blood-based studies were detected. Significant GE associations with absorbed dose were found for five genes and after the 2nd radiotherapy fraction, shown by, e.g., the increase of CDKN1A (2.0 fold, P = 0.017) and FDXR (1.9 fold increased, P = 0.002). After the 25th radiotherapy fraction, however, all four genes (FDXR, DDB2, POU2AF1, WNT3) predicting ARS (acute radiation syndrome) severity, as well as further genes (including CCNG1 [median-fold change (FC) = 0.3, P = 0.013], and GADD45A (median-FC = 0.3, P = 0.031)) appeared significantly downregulated (FC = 0.3, P = 0.01-0.03). A significant association of CCNG1, POU2AF1, HPRT1, and WNT3 (P = 0.006-0.04) with acute or late radiotoxicity could be shown before the onset of these clinical outcomes. In an established set of four genes predicting acute health effects in blood, the response in saliva samples was similar to the expected up- (FDXR, DDB2) or downregulation (POU2AF1, WNT3) in blood for up to 71% of the measurements. Comparing GE responses (PHPT1, CCNG1, CDKN1A, GADD45A, SESN1) in saliva and blood samples, there was a significant linear association between saliva and blood response of CDKN1A (R2 = 0.60, P = 0.0004). However, the GE pattern of other genes differed between saliva and blood. In summary, the current human in vivo study, (I) reveals significant radiation-induced GE associations of five transcriptional biomarkers in salivary samples, (II) suggests genes predicting diverse clinical outcomes such as acute and late radiotoxicity as well as ARS severity, and (III) supports the view that blood-based GE response can be reflected in saliva samples, indicating that saliva is a "mirror of the body" for certain but not all genes and, thus, studies for each gene of interest in blood are required for saliva.

The Effect of a Deproteinizing Pretreatment on the Bonding Performance and Acid Resistance of a Two-step Self-etch Adhesive on Eroded Dentin.

OBJECTIVES: This study evaluated how deproteinization using sodium hypochlorite (6% NaOCl) or hypochlorous acid (50 ppm HOCl) with or without the subsequent use of an arylsulfinate salt-containing agent (Clearfil DC Activator; DCA; Kuraray Noritake Dental) affects the micro-tensile bond strength (µTBS) and formation of an acid-base resistant zone (ABRZ) of a two-step self-etch adhesive on eroded dentin. METHODS: Coronal dentin surfaces of sound human molars were exposed to 48 cycles of demineralization (1% citric acid; 5 minutes) and remineralization (buffer solution with pH=6.4; 3.5 hours). They were then assigned to experimental groups according to the pretreatment used: none (negative control), NaOCl, NaOCl+DCA, HOCl, and HOCl+DCA. Sound dentin surfaces with no pretreatment were used as a positive control. The dentin surfaces were bonded with Clearfil SE Bond 2 (Kuraray Noritake Dental), and µTBS was measured either after 24 hours or 20,000 thermal cycles (TC). The µTBS data were statistically analyzed using a mixed-model analysis of variance (ANOVA) and t-tests with Bonferroni correction. Failure mode was determined with scanning electron microscopy (SEM), which was also used for the observation of ABRZ. RESULTS: Among experimental groups, there was no significant difference between the negative control, HOCl, and HOCl+DCA after 24 hours, but the HOCl-pretreated groups exhibited significantly higher µTBS than the negative control after TC (p<0.01). Pretreatment with NaOCl and NaOCl+DCA resulted in significantly higher µTBS (p<0.001), but the highest µTBS was measured on sound dentin (p<0.001). TC decreased µTBS significantly in all groups (p<0.001) except for sound dentin and NaOCl+DCA (p>0.05). Adhesive failures prevailed in eroded groups, whereas cohesive failures were predominant on sound dentin. ABRZ was recognized in all groups but marked morphological differences were observed. CONCLUSIONS: The combined use of 6% NaOCl and the arylsulfinate salt-containing agent partially reversed the compromised bonding performance on eroded dentin, while the effect of 50 ppm HOCl was negligible.

Management and housing factors associated with paratuberculosis-positive herds in small structured alpine cattle husbandry.

Paratuberculosis (Johne´s disease) is a world-wide cattle disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), associated with substantial economic losses. Purchase of subclinically infected animals or contact with animals and equipment of infected farms are known risk factors for disease transmission among herds. The aim of the present study was to identify specific management factors in Austria that triggered a MAP-positive herd status and to evaluate known risk factors for the transmission in cattle in small structured alpine agricultural systems. The agriculture in the Austrian province of Tyrol is characterized by smallholder structures, including shared alpine pastures and traditional barn management techniques. The data from an extensive survey with 50 questions in 2013/2014 and the development of the MAP herd status of 5592 cattle farms by taking feces and blood samples were examined and statistically evaluated. MAP herd status was determined by combining the results of boot swab samples, manure samples, pooled and individual feces samples as well as serological antibody testing by ELISA. The statistical analysis (odds ratio; OR) showed that the use of milk replacers for calf feeding (p = 0.047, OR=0.472) and the use of straw as bedding material for cows (p = 0.032, OR=0.625) were associated with a decreased chance of being a MAP-positive herd. Further, housing cows in deep litter systems (p = 0.028, OR=2.232), the presence of slurry channels (p = 0.028, OR=1.411) and the use of solid manure in young cattle (p = 0.041, OR=1.744) were associated with an increased OR for being MAP-positive. Surprisingly, sharing of lowland pastures (p = 0.564, OR=1.080), alpine pastures (p = 0.419, OR=1.143) or farm equipment (p = 0.733, OR=0.963) and farm size (p = 0.425) had no significant influence on the MAP herd status. The identified differences compared with previously published results in respect of MAP spread in cattle might be attributed to the traditional agricultural structures, including small family-based farms and common pasture during summer in alpine regions. Results of this study contribute to the understanding of the spread of MAP in cattle farming in alpine regions.

Restoration of a Microdont Using the Resin Composite Injection Technique With a Fully Digital Workflow: A Flexible 3D-printed Index With a Stabilization Holder.

Direct composite restorations are accepted as a treatment option for microdontia, which is a relatively prevalent condition that poses esthetic concerns. While free-hand composite placement is technique-sensitive and time-consuming, the resin composite injection technique is more straightforward and predictable. A fully digital workflow has been recently introduced, but the 3D-printed resin index is rigid and challenged by undercuts, as opposed to the silicone index. This case report presents a flexible 3D-printed resin index, which can accurately transfer the digitally simulated functional and esthetic form to the final restoration. In addition, a rigid stabilization holder was designed to stabilize the flexible index.

RENEB Inter-Laboratory Comparison 2021: The Dicentric Chromosome Assay.

After large-scale radiation accidents where many individuals are suspected to be exposed to ionizing radiation, biological and physical retrospective dosimetry assays are important tools to aid clinical decision making by categorizing individuals into unexposed/minimally, moderately or highly exposed groups. Quality-controlled inter-laboratory comparisons of simulated accident scenarios are regularly performed in the frame of the European legal association RENEB (Running the European Network of Biological and Physical retrospective Dosimetry) to optimize international networking and emergency readiness in case of large-scale radiation events. In total 33 laboratories from 22 countries around the world participated in the current RENEB inter-laboratory comparison 2021 for the dicentric chromosome assay. Blood was irradiated in vitro with X rays (240 kVp, 13 mA, ∼75 keV, 1 Gy/min) to simulate an acute, homogeneous whole-body exposure. Three blood samples (no. 1: 0 Gy, no. 2: 1.2 Gy, no. 3: 3.5 Gy) were sent to each participant and the task was to culture samples, to prepare slides and to assess radiation doses based on the observed dicentric yields from 50 manually or 150 semi-automatically scored metaphases (triage mode scoring). Approximately two-thirds of the participants applied calibration curves from irradiations with γ rays and about 1/3 from irradiations with X rays with varying energies. The categorization of the samples in clinically relevant groups corresponding to individuals that were unexposed/minimally (0-1 Gy), moderately (1-2 Gy) or highly exposed (>2 Gy) was successfully performed by all participants for sample no. 1 and no. 3 and by ≥74% for sample no. 2. However, while most participants estimated a dose of exactly 0 Gy for the sham-irradiated sample, the precise dose estimates of the samples irradiated with doses >0 Gy were systematically higher than the corresponding reference doses and showed a median deviation of 0.5 Gy (sample no. 2) and 0.95 Gy (sample no. 3) for manual scoring. By converting doses estimated based on γ-ray calibration curves to X-ray doses of a comparable mean photon energy as used in this exercise, the median deviation decreased to 0.27 Gy (sample no. 2) and 0.6 Gy (sample no. 3). The main aim of biological dosimetry in the case of a large-scale event is the categorization of individuals into clinically relevant groups, to aid clinical decision making. This task was successfully performed by all participants for the 0 Gy and 3.5 Gy samples and by 74% (manual scoring) and 80% (semiautomatic scoring) for the 1.2 Gy sample. Due to the accuracy of the dicentric chromosome assay and the high number of participating laboratories, a systematic shift of the dose estimates could be revealed. Differences in radiation quality (X ray vs. γ ray) between the test samples and the applied dose effect curves can partly explain the systematic shift. There might be several additional reasons for the observed bias (e.g., donor effects, transport, experimental conditions or the irradiation setup) and the analysis of these reasons provides great opportunities for future research. The participation of laboratories from countries around the world gave the opportunity to compare the results on an international level.

RENEB Inter-Laboratory Comparison 2021: The Cytokinesis-Block Micronucleus Assay.

The goal of the RENEB inter-laboratory comparison 2021 exercise was to simulate a large-scale radiation accident involving a network of biodosimetry labs. Labs were required to perform their analyses using different biodosimetric assays in triage mode scoring and to rapidly report estimated radiation doses to the organizing institution. This article reports the results obtained with the cytokinesis-block micronucleus assay. Three test samples were exposed to blinded doses of 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 13 mA, ∼75 keV, 1 Gy/min). These doses belong to 3 triage categories of clinical relevance: a low dose category, for no exposure or exposures inferior to 1 Gy, requiring no direct treatment of subjects; a medium dose category, with doses ranging from 1 to 2 Gy, and a high dose category, after exposure to doses higher than 2 Gy, with the two latter requiring increasing medical attention. After irradiation the test samples (no. 1, no. 2 and no. 3) were sent by the organizing laboratory to 14 centers participating in the micronucleus assay exercise. Laboratories were asked to setup micronucleus cultures and to perform the micronucleus assay in triage mode, scoring 500 binucleated cells manually, or 1,000 binucleated cells in automated/semi-automated mode. One laboratory received no blood samples, but scored pictures from another lab. Based on their calibration curves, laboratories had to provide estimates of the administered doses. The accuracy of the reported dose estimates was further analyzed by the micronucleus assay lead. The micronucleus assay allowed classification of samples in the corresponding clinical triage categories (low, medium, high dose category) in 88% of cases (manual scoring, 88%; semi-automated scoring, 100%; automated scoring, 73%). Agreement between scoring laboratories, assessed by calculating the Fleiss' kappa, was excellent (100%) for semi-automated scoring, good (83%) for manual scoring and poor (53%) for fully automated scoring. Correct classification into triage scoring dose intervals (reference dose ±0.5 Gy for doses ≤2.5 Gy, or reference dose ±1 Gy for doses >2.5 Gy), recommended for triage biodosimetry, was obtained in 79% of cases (manual scoring, 73%; semi-automated scoring, 100%; automated scoring, 67%). The percentage of dose estimates whose 95% confidence intervals included the reference dose was 58% (manual scoring, 48%; semiautomated scoring, 72%; automated scoring, 60%). For the irradiated samples no. 2 and no. 3, a systematic shift towards higher dose estimations was observed. This was also noticed with the other cytogenetic assays in this intercomparison exercise. Accuracy of the rapid triage modality could be maintained when the number of manually scored cells was scaled down to 200 binucleated cells. In conclusion, the micronucleus assay, preferably performed in a semi-automated or manual scoring mode, is a reliable technique to perform rapid biodosimetry analysis in large-scale radiation emergencies.

Dental fear/anxiety in Czech preschoolers: relationship to gender, age, location of residence, dental status and previous treatment experience.

AIM: Dental fear/anxiety (DFA) may considerably complicate treatment in paediatric dentistry. This cross-sectional study aimed to assess DFA in Czech preschoolers aged 3-6 years and its relationship to gender, age, location of residence, dental status and previous treatment. METHODS: The study involved 460 children from preschools in four location types selected according to the WHO criteria for oral health surveys. DFA was assessed using the Venham picture test (VPT, score range 0-8) prior to the evaluation of dental status using the dmfs (decayed, missing, filled surface) index. The significance level was set to 5%. CONCLUSION: DFA is common in Czech preschoolers. Higher levels of DFA were found in females, children aged 3-4 years, children from rural areas, children with a higher dmfs index and children who had a tooth extracted.

Dogs as carriers of virulent and resistant genotypes of Clostridioides difficile.

While previous research on zoonotic transmission of community-acquired Clostridioides difficile infection (CA-CDI) focused on food-producing animals, the present study aimed to investigate whether dogs are carriers of resistant and/or virulent C. difficile strains. Rectal swabs were collected from 323 dogs and 38 C. difficile isolates (11.8%) were obtained. Isolates were characterized by antimicrobial susceptibility testing, whole-genome sequencing (WGS) and a DNA hybridization assay. Multilocus sequence typing (MLST), core genome MLST (cgMLST) and screening for virulence and antimicrobial resistance genes were performed based on WGS. Minimum inhibitory concentrations for erythromycin, clindamycin, tetracycline, vancomycin and metronidazole were determined by E-test. Out of 38 C. difficile isolates, 28 (73.7%) carried genes for toxins. The majority of isolates belonged to MLST sequence types (STs) of clade I and one to clade V. Several isolates belonged to STs previously associated with human CA-CDI. However, cgMLST showed low genetic relatedness between the isolates of this study and C. difficile strains isolated from humans in Austria for which genome sequences were publicly available. Four isolates (10.5%) displayed resistance to three of the tested antimicrobial agents. Isolates exhibited resistance to erythromycin, clindamycin, tetracycline and metronidazole. These phenotypic resistances were supported by the presence of the resistance genes erm(B), cfr(C) and tet(M). All isolates were susceptible to vancomycin. Our results indicate that dogs may carry virulent and antimicrobial-resistant C. difficile strains.

Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes.

Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva.

Applicability of Exposure Reciprocity Law for Fast Polymerization of Restorative Composites Containing Various Photoinitiating Systems.

OBJECTIVES: The exposure reciprocity law (ERL) has been used to calculate the optimal irradiation time of dental composites. This study examined the applicability of ERL for fast polymerization of restorative composites containing various photoinitiating systems using a high-power multi-peak light-emitting diode (LED) lamp. METHODS: Three commercial composites differing in photoinitiating systems were tested: Filtek Ultimate Universal Restorative (FU) with a camphorquinone-amine (CQ-A) photoinitiating system, Tetric EvoCeram (TEC) with CQ-A and (2,4,6-trimethylbenzoyl)phosphine oxide (TPO), and Estelite Σ Quick (ESQ) with CQ and a radical amplified photopolymerization (RAP) initiator. Specimens 2-mm thick were polymerized using a high-power multipeak LED lamp (Valo) at 3 pairs of radiant exposures (referred to as low, moderate, and high) ranging from 15.8-26.7 J/cm2. They were achieved by different combinations of irradiation time (5-20 seconds) and irradiance (1300-2980 mW/cm2) as determined with a calibrated spectrometer. Knoop microhardness was measured 1, 24, and 168 hours after polymerization on specimen top (irradiated) and bottom surfaces to characterize the degree of polymerization. The results were statistically analyzed using a three-way analysis of variance and Tukey's post hoc tests, α = 0.05. RESULTS: Microhardness increased with radiant exposure and except for ESQ, top-surface microhardness was significantly higher than that on bottom surfaces. Combinations of high irradiance and short irradiation time significantly increased the top-surface microhardness of TEC at low and moderate radiant exposures, and the bottom-surface microhardness of FU at a low radiant exposure. In contrast, the microhardness of ESQ on both surfaces at high radiant exposure increased significantly when low irradiance and long irradiation time were used. With all tested composites, bottom-surface microhardness obtained at low radiant exposure was below 80% of the maximum top-surface microhardness, indicating insufficient polymerization. CONCLUSION: Combinations of irradiance and irradiation time had a significant effect on microhardness, which was affected by photoinitiators and the optical properties of composites as well as spectral characteristics of the polymerization lamp. Therefore, ERL cannot be universally applied for the calculation of optimal composite irradiation time. Despite high irradiance, fast polymerization led to insufficient bottom-surface microhardness, suggesting the necessity to also characterize the degree of polymerization on the bottom surfaces of composite increments when assessing the validity of ERL.

Ultrasonographic diagnosis of closed pedal bone fractures in bovine claws: An ex-vivo study in slaughterhouse specimens.

Pedal bone fractures are one of the most common fracture locations in adult cattle and can be diagnosed by radiographs in two planes. Most bovine practitioners do not have access to such X-ray machines, but many use ultrasound units on a daily basis, primarily for reproductive medicine. For this reason, in this double-masked, randomized controlled study, we aimed to investigate the suitability of ultrasonographic examination using a 5 MHz linear transducer for diagnosing closed fractures of the pedal bone in cattle. A total of 54 hindlimb claws from slaughtered cattle were prepared and approximately 50% of the claws were artificially fractured. All claws were ultrasonographically examined twice by two examiners to determine the presence or absence of fractures and their locations. Ultrasound results were confirmed using radiographs of the claws as the reference standard method. All fracture locations as determined by ultrasonography were situated within ±2 mm of the radiographically-determined fracture zone. Ultrasound examination yielded a calculated sensitivity of 93%, a specificity of 91% and an inter-rater reliability of 0.77. The intra-rater reliability for the examiners were 0.96 and 0.88. Examiner experience with ultrasound examination and using ultrasound images for diagnosis could have influenced diagnostic accuracy. We conclude that artificially-created pedal bone fractures in ex-vivo bovine claws can be diagnosed using ultrasonography; similar results are expected in live animals. These results should encourage veterinarians to use ultrasonography for diagnosing pedal bone fractures in cattle.

Dental composites - a low-dose source of bisphenol A?

Dental composite materials often contain monomers with bisphenol A (BPA) structure in their molecules, e.g. bisphenol-A glycidyl dimethacrylate (Bis-GMA). In this study, it was examined whether dental restorative composites could be a low-dose source of BPA or alternative bisphenols, which are known to have endocrine-disrupting effects. Bis-GMA-containing composites Charisma Classic (CC) and Filtek Ultimate Universal Restorative (FU) and "BPA-free" Charisma Diamond (CD) and Admira Fusion (AF) were examined. Specimens (diameter 6 mm, height 2 mm, n=5) were light-cured from one side for 20 s and stored at 37 °C in methanol which was periodically changed over 130 days to determine the kinetics of BPA release. BPA concentrations were measured using a dansyl chloride derivatization method with liquid chromatography - tandem mass spectrometry detection. The amounts of BPA were expressed in nanograms per gram of composite (ng/g). BPA release from Bis-GMA-containing CC and FU was significantly higher compared to "BPA-free" CD and AF. The highest 1-day release was detected with FU (15.4+/-0.8 ng/g), followed by CC (9.1+/-1.1 ng/g), AF (2.1+/-1.3 ng/g), and CD (1.6+/-0.8 ng/g), and the release gradually decreased over the examined period. Detected values were several orders of magnitude below the tolerable daily intake (4 microg/kg body weight/day). Alternative bisphenols were not detected. BPA was released even from "BPA-free" composites, although in significantly lower amounts than from Bis-GMA-containing composites. Despite incubation in methanol, detected amounts of BPA were substantially lower than current limits suggesting that dental composites should not pose a health risk if adequately polymerized.

Degree of Conversion Contributes to Dentin Bonding Durability of Contemporary Universal Adhesives.

CLINICAL RELEVANCE: The degree of conversion of contemporary universal adhesives positively correlates with the bond strength to dentin. The correlation is more marked after thermocycling, suggesting that a high degree of conversion is required for long-term dentin bonding durability. SUMMARY: Purpose: The objectives of this study were to evaluate the micro-tensile bond strength (µTBS) of five contemporary universal adhesives to dentin after 24 hours and thermocycling (TC), to measure their degrees of conversion (DC) and to test the correlation between µTBS and DC.Methods and Materials: Four commercially available universal adhesives, Prime&Bond universal (PBU), Ecosite Bond (EB), G-Premio Bond (GPB), and Clearfil Universal Bond Quick (UBQ), and one experimental adhesive, UBQ without an amide monomer (UBQ-A), were used in this study. For the µTBS test, midcoronal dentin of 50 human molars was exposed, ground using 600-grit SiC paper, and the adhesives were applied according to the manufacturers' instructions. After resin-composite buildup and 24-hour water storage, one-half of the specimens were subjected to 15,000 thermal cycles. The specimens were sectioned into beams and stressed in tension at a crosshead speed of 1 mm/min until failure. The DC of adhesives applied to dentin was evaluated using attenuated total reflectance Fourier-transform infrared spectroscopy immediately after light-curing. All data were statistically analyzed at a significance level of 0.05.Results: The highest µTBSs were obtained with UBQ, UBQ-A, and PBU, which were not significantly different from each other both after 24 hours and TC. The µTBS of GPB was lower compared with the aforementioned adhesives, but significantly only after TC, and the lowest µTBSs were obtained with EB. TC did not affect the µTBSs of UBQ, UBQ-A, and PBU significantly, but a significant decrease was observed with GPB and EB. The highest DC was obtained with PBU and UBQ, followed by 2-hydroxyethyl methacrylate-rich adhesives UBQ-A and EB, which exhibited significantly lower DCs. The DC of GPB could not be determined because the reference peak at 1608 cm-1 was not detected in its spectra. A significant positive correlation was shown between µTBS and DC after 24 hours (r=0.716) and TC (r=0.856).Conclusion: µTBS and DC were positively correlated, more markedly after TC, which suggests that DC may be an important factor for bond durability.

Establishing reference standards for the vasovagal tonus index in a cohort of healthy French Bulldogs.

INTRODUCTION: The vasovagal tonus index (VVTI), a time-domain indicator of heart rate variability, has been suggested as a diagnostic and prognostic tool in dogs with cardiac disease and heart failure. Brachycephalic breeds tend to have a higher VVTI than non-brachycephalic breeds.11 The objective of this study was to obtain standard VVTI reference values from a representative cohort of healthy French Bulldogs based on a single ECG recording. A second aim of this study was to evaluate the practicability of a VVTI screening in the clinical routine by evaluating repeatability of the VVTI across five successive measurements. In order to determine baseline factors potentially influencing the VVTI, the impact of stress, activity and character as well as sex and neutering status were evaluated. Out of 73 French Bulldogs screened, 60 dogs were considered healthy based on their medical history, physical examination, blood pressure and ECG, and no evidence of congenital or acquired heart disease on echocardiographic examination. The VVTI was calculated based on the variance of 20 R-R intervals and the corresponding HR extrapolated to one minute from the same 20 R-R intervals. In addition, a maximum of five consecutive VVTI values were calculated based on 100 successive R-R intervals recorded from each dog. VVTI values ranged from 5.66 to 11.3 with a mean (standard deviation [SD]) of 8.82 (1.43); the corresponding HR ranged from 78 to 173 bpm with a mean (SD) of 120 (23) bpm. Importantly, VVTI and HR values were negatively correlated (R = 0.689; adjusted R² = 0.466), which must be considered for clinical interpretation of the VVTI. The repeatability across five successive sets of 20 heartbeats was shown (mean intra-individual variability of 6.1%). Stress significantly influenced the VVTI and HR (p < 0.001). The VVTI range established in this study may be used as reference to assess the HRV of presumably healthy brachycephalic dogs at routine health checks. Deviations from the reference may permit the clinician to adapt the schedule and focus of subsequent follow-up investigations.

INTRODUCTION: L'indice de tonicité vaso-vagal (VVTI) est un indicateur temporel de la variabilité de la fréquence cardiaque; il est décrit comme un outil de diagnostic et de pronostic chez les chiens atteints de maladie cardiaque et d'insuffisance cardiaque. Les races brachycéphales ont tendance à avoir un VVTI plus élevé que les races non brachycéphales.11 Le but de cette étude était de déterminer les valeurs de référence VVTI à partir d'une cohorte représentative de bouledogues français en bonne santé à l'aide d'un enregistrement ECG. Un deuxième objectif de cette étude était d'évaluer la faisabilité du dépistage du VVTI dans la pratique clinique quotidienne en examinant la répétabilité du VVTI sur cinq mesures consécutives. Afin de déterminer les facteurs de base susceptibles d'affecter le VVTI, les effets du stress, de l'activité et du caractère, ainsi que le sexe et le statut de castration ont été évalués. Sur les 73 bouledogues français examinés, 60 chiens ont été jugés en bonne santé sur la base de leurs antécédents médicaux, de leur examen médical, de leur tension artérielle et de leur électrocardiogramme et aucun examen échocardiographique n'a révélé de maladie cardiaque congénitale ou acquise. Le VVTI a été calculé sur la variance de 20 intervalles R-R et sur la fréquence cardiaque extrapolée (HR) correspondante. De plus, un maximum de cinq valeurs VVTI consécutives ont été calculées pour chaque chien sur la base de 100 intervalles R-R consécutifs. Les valeurs de VVTI allaient de 5,66 à 11,3 avec une moyenne (écart type [SD] de 8,82 (1,43). La fréquence cardiaque correspondante était comprise entre 78 et 173 battements par minute avec une moyenne (SD) de 120 (23) battements par minute. Pour l'interprétation clinique de VVTI, la corrélation négative entre VVTI et HR (R = 0,689; R2 ajusté = 0,466) doit être prise en compte. La répétabilité a été vérifiée sur cinq séries consécutives de 20 pulsations (variabilité intra-sujet moyenne de 6,1%). Le stress a affecté de manière significative le VVTI et la HR (p.

Evaluation of a handheld device for the measurement of beta-hydroxybutyrate in capillary blood obtained by the puncture of the vulva as well as in venous whole blood in cattle.

A negative energy balance is a common condition in high yielding dairy cows causing the production of ketone bodies (KB), including beta-hydroxybutyrate (BHB), defined as subclinical ketosis (SCK) if clinical signs are missing. The aim of the present study was to evaluate a handheld electronic device for the detection of SCK (BHB-concentration > 1.2 mmol/l), in capillary blood and venous whole blood in cows (WellionVet BELUA, MED TRUST Handels GmbH, Marz, Austria) as well as the feasibility of the puncture of the external vulva with a single use lancet. For this purpose, the blood BHB-concentration was tested in 250 venous and capillary blood samples and compared to the results of a certified laboratory. The majority (76.3%) of the animals displayed no signs of discomfort related to the puncture and in 74.2% the procedure was successful on the first attempt. The BHB-concentrations detected in capillary blood showed good agreement with the reference method, both in capillary (correlation coefficient 0.94 (p⟨0.001), Kappa-value 0.89) and venous whole blood (correlation coefficient of 0.95 (p⟨0.001), Kappa-value 0.89). Altogether, 98% of all the samples were correctly classified as SCK or non-SCK by the handheld device in capillary blood (sensitivity 0.96, specificity 0.98) and 97.4% in venous whole blood (sensitivity 0.889, specificity 0.991), respectively. An increase in the correlation by the adaptation of the cut off level could not be achieved for both sampling sites.

The influence of trimming of the hoof wall on the damage of laminar tissue after loading: An in vitro study.

Laminitis is associated with failure of the suspensory apparatus of the distal phalanx (SADP) connecting the distal phalanx to the hoof wall. The specific aim of this study was to examine in vitro whether thinning of the hoof wall leading to increased deformability influences the damage of the laminar tissue created by loading of the hoof. Paired cadaver forelimbs from twelve horses were used. For each pair, the hoof wall from one hoof was thinned by 25%; this was ascertained by radiography. The contralateral hooves were used as controls. In a material testing machine, hooves were loaded in a proximodistal direction at 0.5mm/s until a cut-off value of 8kN or 14mm was reached. Afterwards, samples of the SADP were taken for histology. Image-based evaluation of the destruction of the SADP was performed using quantitative histogram analysis. Additionally, three examiners masked to treatment (trimmed/untrimmed) qualitatively evaluated SADP destruction. During hoof loading with forces from 0.5 to 1.8 times the body mass of the donor horses, hooves with thinned hoof wall underwent significantly more deformation (P<0.05). Quantitative histogram analysis detected a shift to higher brightness values and a higher pixel intensity in control hooves, representing disruption in the histologic analysis. Qualitative evaluation of histology sections showed significantly more disruption of the SADP in untrimmed hooves (P=0.03). These results confirm the hypothesis that reduced hoof wall thickness can decrease disruption of laminar tissue in vitro, thus supporting the evaluation of hoof wall reduction as a prophylactic measure in horses at imminent risk of SADP failure.

Comparison of vertical force redistribution in the pads of dogs with elbow osteoarthritis and healthy dogs.

Kinetic analyses are becoming increasingly important for biomechanical research in veterinary medicine and as a diagnostic tool for orthopaedic examinations in dogs. Such analysis enables accurate evaluation of the vertical force distribution (VFD) in canine paw pads. The aim of this study was to assess peak vertical force (PFz) as a percent of total force (%TF), vertical impulse (IFz, %TF) and time of occurrence of PFz (TPFz) as a percent of the stance phase (%SP) in the pads of all four limbs in 23 dogs with osteoarthritis in the elbow joint and 22 healthy dogs. Dogs walked over a pressure plate, and the pads were divided into four quadrants for VFD analysis. For statistical analysis, a general linear model was used to examine the difference in VFD between both groups, between fore- and hindlimbs, between body sides, and between medial/lateral and cranial/caudal quadrants. Lame dogs had lower PFz in the lame forelimb than in other limbs and transferred their weight to the caudal quadrants of the contralateral forelimb and the caudomedial quadrant of both hindlimbs. IFz was also lower in the affected forelimb and was compensated through higher loading of the caudal quadrants of the contralateral forelimb, the caudomedial quadrants of both hindlimbs and the caudolateral quadrant of the contralateral hindlimb. TPFz (%SP) occurred later in both forelimbs of the lame dogs than in those of healthy dogs. The analysis of force distribution over the paw quadrants can be used for further biomechanical studies of dogs with orthopaedic and neurological diseases.

Detection of selected viral pathogens in dogs with canine infectious respiratory disease in Austria.

OBJECTIVES: To assess the prevalence of canine parainfluenza virus, canine adenovirus type 2, canine distemper virus, canine respiratory coronavirus and influenza virus A infections in: (1) privately-owned or, (2) kennelled dogs showing signs consistent with canine infectious respiratory disease and, (3) clinically healthy dogs in Vienna, Austria. MATERIALS AND METHODS: Prospectively, nasal and tonsillar swabs from 214 dogs affected with infectious respiratory disease, and 50 healthy control dogs were tested for nucleic acids specific to the various viral infections. Concurrent bronchoalveolar lavage fluid from 31 dogs with chronic respiratory disease was investigated for the same viral pathogens. Additionally, anti-canine respiratory coronavirus antibody concentrations were measured in paired blood samples from 30 acutely diseased dogs. RESULTS: Canine respiratory coronavirus (7.5%) and canine parainfluenza virus (6.5%) were the most commonly detected viruses in samples from the upper airways of dogs with respiratory infections. Serological results showed a significant seroconversion in response to coronavirus in 50% of the examined cases. None of the samples was positive for influenza virus A-specific nucleic acid. Canine coronavirus-specific nucleic acid was detected in 4.0% of healthy dogs. CLINICAL SIGNIFICANCE: Canine coronavirus should be considered as a clinically relevant cause of infectious respiratory disease in crowded dog populations. For sample collection, the nasal mucosa can be recommended as the favoured site. Analysis of paired serum samples aids verification of canine coronavirus infection in respiratory disease.

Protein expression of KIT, BRAF, GNA11, GNAQ and RASSF1 in feline diffuse iris melanomas.

Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19-63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas. Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/µm2 and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.