This is expected to be used as basic data for the application of gene editing using CRISPR/Cas9 technology and for establishing a breeding strategy for high eating quality rice using molecular genetic technology.Ninety-five percent of the general nutrients in rice are concentrated in the rice bran and germ, and many nutrients such as vitamins, minerals, dietary fiber, and essential fatty acids, as well as antioxidants such as tocopherol, are lost during milling. In this study, we investigated the thickness of seed coat and aleurone layers using a 294 rice core collection, and found candidate genes related to thickness of seed coat and aleurone layers, by performing a genome wide association study (GWAS) analysis using whole genome resequencing data. Two primer pairs that can be used as high-resolution melting (HRM) markers were developed. As a result of genotyping BC Rice is one of the major grains in the world, especially in Asian food culture, and is used as a staple food . In geneGenome-wide association study (GWAS) is a method of searching the entire genome for phenotypes showing continuous variation and mutations representing associations in natural individuals, or for lines using whole genome sequences ,8. In orA core collection should be a group that can be used to analyze multiple traits, including as much genetic diversity as possible, with the minimum number of genetic resources in one crop, and should have high usability in research. The reduced size of a core collection is the key to manageability and represents the diversity of the entire collection, allowing analysis to be handled more efficiently and effectively. Zhao et al.  performeIn this study, we investigated the thickness of seed coat and aleurone layers using a 294 rice core collection, and candidate genes related to thickness of seed coat and aleurone layers were found by performing GWAS using SNPs in whole genome resequencing. In this way, SNP markers were developed to provide efficient molecular tools for improving the eating quality and processability of brown rice in breeding programs. This study will contribute to the understanding of the mechanisms of seed coat and aleurone layer formation in rice and will provide a basis for future research for breeding brown rice with an excellent taste and processability.Regarding the rice genetic resources used in this study, 166 core groups were first selected using introduced species and domestic breeding lines, weedy rice, and IRRI resources among 24,000 genetic resources collected from around the world. A total of 294 rice genome resequencing groups were finally constructed by secondarily selecting additional varieties that can be headed in the domestic climate and have a high utility value in Korea. Of the total 294 rice genome resequencing groups, the 225 domestic breeding lines consisted of 38 Indica, 178 Japonica, and 9 Tropical Japonica .Seeds of 294 brown rice genetic resources were cut into two halves using a razor blade, and then seeds were stained with Oil Red O dye used for lipid staining . Oil Redhttps://www.ncbi.nlm.nih.gov/assembly/GCF_001433935.1/ accessed on 30 July 2019), which is a standard genome sequence of rice [Genomic DNA of 294 rice accessions was extracted using DNeasy Plant Mini Kits , according to the kit manual instructions from 0.5 g of young leaf tissues. Quality of DNA was checked by diluting a sample extracted using a Quant-iT BR assay kit  and then measuring the concentration and quality of DNA. Tecan F200  was used to measure the optical density value, and the extracted DNA was confirmed by electrophoresis at 1% agarose gel. Genome re-sequencing was performed on an Illumina HiSeq 2500 platform , according to its standard sequencing protocol. Data analysis was compared with IRSGP1.0 downloaded from NCBI . Haplotypes were used for comparative phenotypic analysis.We used Haploview (v.4.2) for analysis of the LD block and haplotype analysis . We defihttps://shigen.nig.ac.jp/rice/oryzabase/ accessed on 30 July 2019) and the NCBI database (https://www.ncbi.nlm.nih.gov/ accessed on 30 July 2019) [Adjacent significant SNPs associated with target traits within a physical distance of 200 kb were screened for candidate genes. Genomic DNA sequence and cDNA region for the mRNA transcripts of each candidate genes were searched and extracted from the Rice Genome Annotation Project (ly 2019) .https://primer3plus.com accessed on 23 September 2020) [Molecular markers associated with the seed coat and aleurone layers were developed for high resolution melting (HRM) analysis. HRM specific primer sets based on target SNPs were designed using the web-based software Primer3plus (er 2020) . HRM anaFor the accuracy of seed coat and aleurone layers thickness, the cross-section of each seed was divided into five parts, namely the dorsal, upper lateral, lateral, lower lateral, and ventral part. The distribution of the seed coat and aleurone layer thickness of 294 rice core collection is as shown in p ≤ 0.001) were confirmed. PCA and kinship analyses were performed for population structure analysis using high-quality 1,842,515 SNPs, with a compressed mixed linear model (CMLM). A 294 rice core collection panel was determined by graphical representation of the K value on a screen plot, which indicated the presence of three sub-populations. This result was confirmed by the variation in the first 10 major components (PCs) showing inflection points in PC2, suggesting that the first two PCs dominated the population structure on the association analysis  SNPs with a read depth of 4× or more and 200× or less, MAF > 0.05, genotyping quality ≥ 20, and Hardy–Weinberg equilibrium  values of significant SNPs from the correlation analysis of the seed coat and aleurone layers thickness. It was confirmed that significant SNPs were detected based on −log10(p) > 5 in the Manhattan plot, according to the thickness of seed coat and aleurone layers. It was found that SNPs located on rice chromosomes 1, 3, 4, and 10 were more significant than other SNPs. In addition, the expected p-value of x-axis and observed p-value from significant SNPs showed a diagonal linear shape in the QQ plot, which means that the discovered SNPs expressed their characteristics well, with normality and significance. As a result of searching for SNPs that were correlated with aleurone layers thickness in this study, the top 10 most significant SNPs by region of aleurone layers are shown in 10(p) value of 5.94. The SNP related to the middle aleurone layers part was ‘S3_20312675’, which was highly associated with a –log10(p) value of 6.53. In addition, a significant SNP related to the thinnest aleurone layers part was ‘S1_6459856’, which showed a −log10 value of 6.00. The available haplotype analysis formats are ped and map files; the ped file requires individual information and individual genotype data for each marker, and the map file requires a marker name and marker location. LD is displayed as pairwise D’ values. Each color represents the magnitude and significance of pairs of LD, and the gradation from red to white is from a high LD value to a low value. Red diamonds without numbers correspond to a D’ value of 1.0. GWAS analysis using GAPIT usually uses about 1 million SNPs to simultaneously test for associations with traits . GWAS wahttps://rapdb.dna.affrc.go.jp/index.html accessed on 30 July 2019) and NCBI database (https://www.ncbi.nlm.nih.gov/gene/ accessed on 30 July 2019), based on the most significant SNP positions on each chromosome , THICK ALEURONE 2, which displayed functions as DNA demethylase and is related to endosperm development . Os01g022F2 , such as Samgwang, and eight individuals with a homozygous genotype (T/T), such as Seolgaeng, and 30 individuals with a heterozygous genotype (T/C) were identified. In addition, as a result of HRM analysis using the “TA2S1-148” marker, 26 individuals with a heterozygous genotype (C/A), out of 50 BC2F2 populations of Samgwang X Seolgaeng, were found to match their genotype and phenotype  located in rice chromosome 1 was similarly located to ROS1a (Os01g0218032), which is related to seed coat and aleurone layer thickness. Base changes in the OsROS1 gene cause mutations in the aleurone cell layer through inhibition of two putative transcription factor genes, RISBZ1 and RPBF, which increase aleurone layers due to lower levels of DNA hyper-methylation and expression . In null9856 locaAs a result of identifying the allelic mutations of the Os01g0218032 gene in the 294 rice core collection, two missense mutations were found in the first exon of the Os01g0218032 gene. S1_6444737 (C > T) is mutated from serine to phenylalanine at amino acid position 163. In addition, in the case of S1_6445148 (A > C), a SNP mutation was made, resulting in a lysine to threonine mutation at amino acid position 301. Three varieties with a thin seed coat and aleurone layers, and three varieties with thick ones, were selected to test genotypes, and their melting curve patterns were compared using the designed HRM markers. It was confirmed that the melting curve patterns were differentiated by genotypes, homo (P1): hetero: homo (P2) (1:2:1), so that they could be used as markers for selecting the thickness of the seed coat and aleurone layers of brown rice. In this study, two primer pairs that can be used as HRM markers were finally developed by GWAS analysis of the 294 rice core collection. As a result of genotyping with the 50 BC2F2 individuals derived from the cross between “Samgwang” and “Seolgaeng”, it was confirmed that different melting curve patterns are shown, depending on the thickness of seed coat and aleurone layers using a corresponding HRM marker. In conclusion, it was possible to develop the HRM markers for selecting seed coat and aleurone layer thickness. SNP-based HRM markers have been studied in various fields, such as disease resistance and susceptibility discrimination, with sequencing through cross breeding. Furthermore, markers for quantitative traits were developed. The markers developed in this study enable efficient testing of cross populations and provide materials that can be applied, not only to aleurone layers, but also in constructing breeding populations using various traits. In addition, gene mapping and QTL analysis are possible using the developed HRM molecular markers.This is expected to be used as basic data for the application of gene editing technology using CRISPR/Cas9 and for the establishment of a breeding strategy for high eating quality rice using molecular genetic technology.


To examine the incidence, location, and grade of blunt cerebrovascular injury (BCVI), as well as associated strokes in patients with ankylosis of the cervical spine, imaged with CT angiography (CTA) after blunt trauma. The related etiologies of ankylosis had an additional focus.Altogether of 5867 CTAs of the craniocervical arteries imaged after blunt trauma between October 2011 and March 2020 were manually reviewed for a threshold value of ankylosis of at least three consecutive cervical vertebrae. BCVI was the primary outcome and associated stroke as the secondary outcome. Variables were craniofacial and cervical spine fractures, etiology and levels of ankylosis, traumatic brain injury, spinal hematoma, spinal cord injury, and spinal cord impingement, for which correlations with BCVI were examined.n = 32) were in the vertebral arteries. Injuries were graded according to the Biffl scale: 17 grade II, 4 grade III, 14 grade IV, and 1 grade V. A ground-level fall was the most common trauma mechanism. Cervical spine fracture was the only statistically significant predictor for BCVI (OR 7.44). Degenerative spondylosis was the most prevalent etiology for ankylosis.Of the 153 patients with ankylosis and blunt trauma of the cervical spine, 29 had a total of 36 BCVIs, of whom two had anterior and 4 posterior circulation strokes. Most of the BCVIs (Ankylosis of the cervical spine increases the incidence of BCVI up to sevenfold compared to general blunt trauma populations, affecting especially the vertebral arteries. High-energy deceleration forces from blunt trauma resulting in hyperflexion, hyperextension, or rotation of the neck can cause cervical arteries to stretch over or shear against adjacent structures, in addition to the direct impact caused by fractured bones. The resulting intimal damage can affect both carotid and vertebral arteries and is defined as blunt cerebrovascular injury (BCVI) . BCVI haSpinal ankylosis carries a risk for unstable fractures, even from low-energy trauma, usually localized in the lower cervical spine . HLA B27Diffuse idiopathic skeletal hyperostosis (DISH) is defined as ankylosis of at least four adjacent vertebrae in the thoracic spine, usually presenting as anterior “flowing” syndesmophytes that tend to evade the pulsating aorta to the contralateral side –19. PrevWe hypothesize that ankylosis of the cervical spine leads to increased incidence of BCVI with less trauma energy compared to general blunt trauma populations. To our knowledge, no previous publications are focusing on the incidence of BCVI in the trauma of the ankylosed cervical spine. The purpose of this retrospective study was to examine the incidence, location, and Biffl grading of BCVI in the post-traumatic ankylosed cervical spine, with an additional focus on the related incidence of acute cerebral infarctions .The University Hospital’s institutional review board approved this retrospective study.Töölö Hospital, a part of Helsinki University Hospital, is the only level-1 trauma center for a catchment area of 1.67 million people, where emergency whole-body CTs for blunt trauma are routinely performed. Patients with serious trauma such as cervical spine fractures and injuries are frequently transferred to our institution from general hospitals both outside of and within the same health care district for further evaluation and treatment. All patients over the age of 15, both primary and referrals with any blunt trauma and ankylosis of at least three consecutive cervical spine vertebrae were included. To our knowledge, studies in the literature have not established a cut-off value for significant ankylosis that predisposes patients to fractures. Patients under 16 years or without ankylosis of at least three consecutive cervical vertebrae, as well as patients with penetrating trauma were excluded. The Impax Picture Archiving and Communications System  allowed manual retrieval and reassessment of all CTAs between October 2011 and March 2020.The trauma imaging protocol involves an initial CTA of the craniocervical arteries from the aortic arch to skull base followed by a split-bolus whole-body CT from clavicles to ischium in both the arterial and the venous phase and includes a nonenhanced head CT. We use contrast media with a concentration of 350 mg/ml  in the split-bolus technique. The first bolus consists of 80 ml of contrast media with a flow rate of 5 ml/s, followed by a second bolus of 50 ml with a flow rate of 4 ml/s injected with a 40 s delay. If diagnosed with a BCVI, a follow-up CTA is routinely performed after two weeks of anticoagulation treatment. CTA of craniocervical arteries and CT and MRI scans for cerebral ischemia or disorders of the spinal cord are performed upon request for topical ischemic insult. All CT images were obtained using a 64-slice CT scanner , while MRIs were performed on a 1.5 T closed-bore MRI scanner . For CTAs, coronal and sagittal reformatted series with 2 mm slice thickness, and, for head CTs, axial, coronal, and sagittal reformatted series with 3 mm slice thickness are made in addition to the basic volumetric dataset of 0.625 mm slice thickness for all patients. For CTAs of craniocervical arteries, maximal intensity projection (MIP) images are reconstructed using a vascular kernel, as well as bone window reformats of the cervical spine and skull using a bone kernel. The MRI protocol for BCVI consists of an unenhanced time of flight magnetic resonance angiography (TOF MRA), as well as a T1-weighted, fat-saturated three-dimensional turbo spin-echo (3D TSE SPACE) sequence of the neck. The MRI protocol for the trauma of the cervical spine includes T- and T2-weighted TSE sequences in both sagittal and transverse planes as well as STIR in the sagittal and a fat-saturated T2-weighted sequence of the craniocervical junction in the coronal plane.During the evening and night, images are interpreted by an on-call resident, while an attending radiologist who is specialized or a fellow in trauma radiology double-read images by day. A board-certified radiologist with 5 months of experience as a fellow in trauma radiology  reviewed all CTAs for ankylosis. Of the included patients, levels of fused segments, fractured cervical vertebrae, and intervertebral discs were recorded. A second board-certified trauma radiologist with 15 years of experience  was blinded to the original reports and reassessed all included CTAs for the etiology of ankylosis. A third board-certified trauma radiologist with 10 years of experience , along with Readers 1 and 2, reviewed all patients’ CTAs for BCVIs independently and blinded to one another, after which disagreements were settled by consensus. Documentation of BCVI comprised the affected artery, level of injury, and Biffl grading, as presented in Table The primary and secondary variables and outcomes were BCVI and associated acute stroke, respectively. The main predictive variables were fractures of the cervical spine, facial bones, and skull. Additional variables were age, gender, etiology and levels of ankylosis, trauma mechanism, and TBI. Spinal hematoma, spinal cord injury, and spinal cord impingement were recorded from MRIs, which were imaged upon request. TBI was defined as subdural, subarachnoideal, epidural, or intraparenchymal hemorrhage visible in head CTs or contusions visible only in MRIs. Furthermore, we recorded the development of BCVIs in follow-up CTAs and the 30-day mortality rate. Findings from follow-up CTAs regarding the progression of primarily detected BCVIs were evaluated Table . Originaχ2 test), skull fracture versus BCVI (Fisher’s test), gender versus BCVI (χ2 test), and age versus BCVI (Mann–Whitney U test). Only those with one type of predictive fracture variable were subjected to the calculation for association with BCVI. The logistic regression model analyzed predictive and other variables (except age) to explain the relative risk for BCVI. The number of ankylosed spinal segments and the number of fractured vertebrae and transversely fractured intervertebral discs were evaluated for correlation using Spearman’s ρ. Interobserver agreement was calculated using Cohen’s κ between Reader 1 and Reader 2 for analysis of etiology in ankylosis and for BCVI findings between readers’ results and consensus reading. If a patient was diagnosed with the correct number of BCVIs, the reading was considered valid. Strength of agreement was defined as follows: values < 0, no agreement; 0.00–0.20, slight; 0.21–0.40, fair; 0.41–0.60, substantial; 0.81–1.00, almost perfect .Fi.Fi25].Six patients (21%) developed strokes visible on five head CTs and one head MRI. Four patients had acute strokes, of which three were in CTs at presentation caused by a Biffl grade IV BCVI in the right common carotid artery Fig. , a BifflThe four patients with carotid injuries could not receive anticoagulation, and two of these developed anterior circulation strokes Fig.  and 5. COf the 26 patients with VAIs, four (15%) suffered posterior circulation strokes Fig. , all of The main trauma mechanism was a ground-level fall (82%), followed by motor vehicle (9%) and bicycle accidents (5%) Table . Of the In 153 patients with ankylosed cervical spines, 143 head CTs revealed 38 (25%) TBIs. Forty-five patients imaged with MRI revealed either one or a combination of the following: 25 spinal hematomas (16%), 17 spinal cord injuries (11%), and 25 spinal cord impingements (16%). Of the 29 patients with BCVI, six had spinal hematomas (21%).n = 8) or within 2 to 3 months (n = 2).Of the 29 patients with acute BCVIs, 13 had a total of 18 BCVIs on follow-up CTAs  that were missed by the attending trauma radiologist.The most common cause of ankylosis Table  was DS , the two most cranial vertebrae , and the two most caudal vertebrae . In patients with ankylosis of four or fewer vertebrae, most concomitant vertebral body and intervertebral disc fractures were located superior to the level of ankylosis. In 10 patients with ankylosis of four adjacent segments to completely ankylosed cervical spine, multiple fractures were seen within the ankylosed segment.Ninety-three fractured vertebrae and 25 fractured intervertebral discs were found in the 153 patients with ankylosis of the cervical spine. Forty-three fractured vertebrae or fractures running through the intervertebral discs (36%) were located above the ankylosed segment, and only six (5%) were located beneath. Sixty-nine were located within the ankylosed segments and distributed evenly to the most central vertebrae , followed by DISH , surgical fusion , and one with AS (3%).Of the 82 patients with cervical spine fractures, 45 (55%) were diagnosed with the ankylosed cervical spine by a radiologist, and 37 (45%) had no mentions of ankylosis. Of these 37, four had mentions of unstable fractures, and eight were noticed to have ankylosis by the treating orthopedic surgeon. The majority of patients with insufficient reports of ankylosis had DS  nor gender (P = 0.40) was associated with BCVIs, while the association between cervical spine fracture and BCVI showed statistical significance (P < 0.001). In the logistic regression model (Table P < 0.001). The incidence of fractured vertebrae and intervertebral discs correlated with the number of ankylosed intervertebral discs . Interobserver agreement on the etiology of ankylosis between Reader 1 and 2 measured by Cohen’s κ was substantial , reflecting the discrepancy among 20 patients of either DS or DISH. Reader 1 and 2 agreed on all 18 patients with AS or seronegative spondyloarthropathy. Among all readers and consensus reading on BCVIs, agreements were almost perfect .Neither age P = 0.0 nor genPatients in our study with an ankylosed cervical spine of at least three consecutive vertebrae had an incidence of 19% for BCVI, more than seven times as high as non-specified blunt trauma patient populations, and twice as high as those with TBI , 5, 28. Most BCVIs were on the level of the upper cervical spine with a strong association to cervical spine fractures, as previously documented –31, 33. Vascular atherosclerosis, especially in the elderly, presents as uneven intimal surfaces and luminal narrowing and may cause difficulties in interpretation. Soft atherosclerotic plaques are not easy to differentiate from a Biffl grade I BCVI in trauma patients. This likely contributed to five VAIs out of 36 BCVIs being missed by attending trauma radiologists. Overly sensitive assessment resulting in unnecessary treatment should be avoided, which most likely explains the lack of grade I BCVIs in our observations. Owing to a narrower lumen, especially with atherosclerosis, vertebral arteries are more prone to progress to grade IV BCVI compared to the carotid arteries , 29, 32.Upon suspicion of an acute BCVI, patients had received anticoagulation treatment where possible. A BCVI in a carotid artery is considered more severe than VAI because of the larger vascular territory, and, without collateral circulation, this location results in a higher stroke rate, morbidity, and mortality , 25. AltTwo patients with TBIs and one with a skull fracture did not receive anticoagulation treatment because their BCVIs were initially interpreted as non-acute findings, which often poses a challenge in the absence of a previous CTA for comparison. Post-traumatic patients with AS show an increased incidence of spinal hematoma, which can cause neurological deficits . In the The most common trauma mechanism was a ground-level fall (82%), which inflicted multiple BCVIs and fractures with considerably less trauma energy than those in general blunt-trauma populations –30, 33. Lebl et al. found a total of five patients with ankylosis caused by either AS or DISH in a cohort of 253 patients with CTA after blunt cervical spine trauma, of whom three had VAIs . This suThe limitations of this study were the retrospective design and the sample size. Our hospital, as a level-1 trauma center, receives a higher amount of severely injured patients than other local centers, which probably contributed to the high incidence of concomitant injuries. Partly because of the retrospective design, only 14 out of 29 patients had follow-up imaging. The small sample size prevented reliable statistical analysis between age and ankylosis.In conclusion, ankylosis of the cervical spine increases the incidence of BCVI up to sevenfold compared to general blunt trauma populations, affecting especially the vertebral arteries. DS was the most prevalent etiology for ankylosis, which is nevertheless less consistently included in reports than AS or DISH.


Annual tooth rasping is integral to prophylaxis work in equine dentistry. The procedure begins with a thorough examination of the oral cavity, including assessment of incisor occlusal surface angles. Any dental misalignment should be corrected or reduced to prevent abnormal forces from acting on teeth and supporting tissues and to maximize functionality of the masticatory apparatus. Thus far, clinical methods for dental angle assessment are scarce and not validated in terms of data reproducibility and comparability. These parameters are vital to objectively assess treatment needs and outcome, obtain population data, and establish data comparability among studies. However, this is the first study systematically validating a commercial dental angle measuring device. The data indicate high device performance and its applicability in practice.MaPHorse1). Five examiners  measured six cadaver heads on 2 consecutive days in a blinded block-randomization design, resulting in 16 measures per examiner*head. Sagittal and transversal angle measures revealed low intraexaminer variability at scale-level independent mean SDs of α 0.58° and α 0.69°, respectively. Sagittal angle measures associate with low interexaminer variability, showing small mean angle differences (max. α 0.51° ± 0.35°), small scatter, and more consistent data reproducibility. Despite comparable mean interexaminer differences, the spread of transversal angle measures was relevantly higher using the proposed landmarks (average 2.2-fold higher interquartile range). The measurement performance of experienced and inexperienced examiners did not systematically differ. The time required for individual measurements was already comparable after 24/96 repetitions. This instrument may help deciphering sagittal angle normocclusal range and orthognathic dynamics and, with a proposed procedural amendment, transversal angle as well.Incisor malocclusions are common in horses. As yet, an evidence-based understanding of incisor occlusal surface angle dynamics and normocclusal range is missing. Orthodontic measuring devices could help unravel this information objectively but imply measurement validation. We evaluated intra- and interexaminer variability of repeated sagittal and transversal incisor occlusal surface angle measures using a commercial orthodontic gauge device ( Occlusal surface angles in equine pre- and post-canine dentition receive increasing attention as a certain range of inclination is accepted as crucial for physiological mastication and, thus, feed conversion ,2,3,4. IIncisors can be overlong, which is favored by less abrasive forage and resulting lack of wear and can reduce functional cheek tooth occlusion ,15. TherSA indicates the rostro-caudal inclination of the occlusal surface in reference to a global or subordinate transversal plane ,3,20. ThTA indicates the latero-lateral inclination of the occlusal surface in reference to a global or subordinate sagittal plane ,3,20. DeMaPHorse1. However, in their research, the orthodontic gauge device was utilized for sole determination of DIM direction, i.e., DGL-3/-4. According to the authors, no metric angle values were reported as measurement repeatability and reproducibility had not yet been investigated for the device [Malocclusions can impair dental and periodontal function and require individualized treatment to maximize functional occlusion ,30,31. Ce device .MaPHorse1. The study was conducted following a blinded two-day block-randomization design where five examiners of different experience status repeatedly measured SA and TA on six cadaver heads.This cadaveric study aims to assess intraexaminer variability (repeatability) and interexaminer variability (reproducibility) of both SA and TA measurements using the orthodontic gauge device n = 5) or fresh (n = 1) heads of horses from the anatomical collection of the University of Veterinary Medicine Vienna were used. Horses were euthanized at the Equine Clinic of the university for reasons unrelated to the study or any orodental, maxillofacial, or cranial disease. Heads were resected at the level of the atlas. Details on the animals and cadaver heads are listed in Six fresh-frozen . According to the study design, no ethical approval for animal experimentation was required.Five examiners performed the measurements . Two of them  already had extensive experience with the measuring device over a period of several years before the trial started. Three examiners  had no experience using the instrument at the beginning of the experiment.MaPHorse1 that was designed for clinical determination of equine incisor occlusal surface angles, i.e., SA and TA. The instrument was designed and developed by one of the coauthors (M.P.) and patented by the United States Patent and Trademark Office under patent no.: US 8,793,888 B2 [We used the commercially available orthodontic gauge device 3,888 B2 .The device consists of two elongated interconnected bars, i.e., lateral bar and frontal bar a, each cThe experiment was carried out on two consecutive days in the dissection room of the Institute of Morphology of the University of Veterinary Medicine Vienna. One of the authors (S.K.), who did not perform measurements, organized preparation and coordination of the experiment.Heads were mounted on custom-made headrests a 12 h beIn each passage, the examiners moved from box to box following a separate examination protocol . The proA total of 3 measuring devices were used parallel in the data collection. This allowed 3 examiners to take measurements at the same time. Before the experiment started, all devices were calibrated by the manufacturer according to a standard procedure. One of the experienced examiners (M.P.) demonstrated the handling of the device and landmarks to be used on a separated cadaver head before the experiment started. The bite plate was inserted between incisor arcades until the ventral stop ridge (lower side of bite plate) touched the lower central incisors. The central marking line of the bite plate was aligned to the median interdental space. Then, the frontal bar was positioned in plane with a line connecting the medial canthi of the eyes and the TA was read off the measuring scale. The measurement was performed sitting on a height adjustable seat. The SA was first measured on the right and then on the left side. The unfolded side bar was adjusted to the most lateral palpable part of the TMJ gap. For measuring the SA on the left side, the central retainer was loosened and the frontal bar turned α 180° so that the side bar was positioned on the left side. The retainer was closed and the previously measured TA value was reset on the frontal angle scale. Then, the left SA was assessed as described for the right side. Measured values were immediately recorded in the examination protocol. After the left SA measure, the device was removed from the head and the examiner went to the next box, continuing measurements in the respective passage. Non-measuring examiners had to wait off the experimental setup.To identify time budget differences between experienced and inexperienced examiners and to investigate whether repeated use of the measuring device has a quickly recognizable handling learning effect, the time required for the measuring process was assessed for each examiner in different measurement blocks. Time measurement began with the insertion of the biteplate between the incisors and ended with the removal of the device.Analyses and data visualization were carried out using GraphPad Prism  and Excel  software. All data were analyzed using descriptive statistics and checked for normal distribution using Shapiro–Wilk test. Homogeneity of variance was determined using Levene’s test. Intraexaminer variability (repeatability) was determined using coefficient of variation (COV) calculations as ((SD/mean)*100). The respective scale level of angle measures was considered for interpretation of COVs. Used inferential statistical methods are shown in the respective results section.MaPHorse1. Data collection was performed on 2 consecutive days, each divided into two measurement blocks and four respective measurement passages. Each head was measured once per passage, resulting in a total of 192 SA  and 96 TA measures per examiner and 32 SA  and 16 TA measures per examiner*head. Thus, all examiners collected 960 SA  and 480 TA values. Examiners 1 and 2 entered passage values for heads 1, 3, and 5 (ex1), and head 4 (ex2) in the list once, illegibly. These data clusters were completely removed from the analyses, resulting in 30 SA  and 15 TA measures per examiner*head.We used a blinded block-randomization study design to evaluate intra- and interexaminer variability for repeated SA and TA measures using orthodontic gauge device Five frozen-thawed heads (heads 1–5) and one fresh head (head 6) were available for study purposes. Identification of bony landmarks to measure SAs was reported as easy by all examiners. As by evaluation of the study coordinator (S.K.), no changes in the cadaver material were observed in the TMJ regions over the period of both study days. The bulbi of previously frozen heads showed slightly progressive enophthalmos and distortion of the lateral canthi. The medial canthi remained largely unaltered. However, all the examiners individually reported difficulties in identifying the latter for TA measures. This was not reported for the fresh head, in which anatomical structures appeared unaltered. Both experienced investigators quoted that frozen-thawed specimens were not ideal for measuring the TA. Experienced examiners further stated that this was not comparable to live horses. No difference to live horses was reported for SA measures or the fresh head in general.The SA was recorded twice for each measurement using the same anatomical reference structures on the right and left side of the head. The obtained values indicate no marked difference of right and left SA measures among all the examiners . In 52% Mean right vs. left SA difference, integrating measures of all heads and examination blocks, was below α 1° and only differed between examiner 2 (experienced) and examiner 5 (inexperienced) b. This wMaPHorse1. In a total of 476 repeated TA measures, 1.1% (n = 5) revealed a DGL-4, 88.2% (n = 420) a DGL-3, and 10.7% (n = 51) no TA deviation (TA α 0°) (We determined the coefficient of variation (COV) for each examiner and head as a measure of intraexaminer variability (repeatability) of SA and TA measurements using TA α 0°) . Due to The mean relative COV was 0.09 for SA and 0.239 for TA across all examiners and heads, implying an average 9% and 23.9% dispersion of individual measures around the mean, respectively a,b. At tAlthough SA COV (except for examiner 2) was highest in head 1 a, the abTo display consistency and scale-level independency of SA and TA measurement dispersion, we rearranged heads according to increasing angle values c,e and oThe within examiner SA COVs did not decisively differ between examiners, although the mean COVs of experienced examiners were slightly lower  a. ExaminBased on the results, we assume that criteria for positive instrument validation for intraexaminer variability of SA and TA measures, using specified reference points, could be met.MaPHorse1 interexaminer variability (reproducibility) comparing single examiner SA and TA measurements on different heads. Although statistical analyses showed that the measured SA values in some heads differed significantly for certain examiner combinations  and span (mean span: α 1.90° ± 1.10°) across different examiners and heads  and higher span (mean span: α 2.33° ± 1.30°) across different examiners and heads , whereas experienced examiners performed faster a. Both enverging a. The vaThe overall time required for measurements did not systematically differ between experienced and inexperienced examiners on either day of the experiment. However, the calculated difference was slightly higher on day 1. Inexperienced examiners improved their performance, requiring markedly less time for measurements on day 2 of the experiment b.MaPHorse1. The results indicate scale-level-independent high repeatability for both SA and TA measures. Referring to bony TMJ landmarks, the SA measures also revealed high reproducibility. Despite high methodical repeatability, the TA measures shared lower reproducibility using soft tissue landmarks. The general measurement performance was not substantially impacted by the previous experience of the examiners.Standardized static and dynamic orthodontic measurements during routine dental prophylaxis can provide essential population data for more accurate diagnostics and effective treatment planning but also evaluation of treatment success, detailed patient follow-up, and client communication. In equine dentistry, it is common to subjectively appraise occlusal angles and the need for odontoplasty, as well as treatment outcomes, without applying objective metric analyses. We, therefore, conducted this blinded block-randomized cadaver study evaluating intra- and interexaminer variability in repeated SA and TA measures using the orthodontic gauge device MaPHorse1, a clinical gauge device that can be used by equine dental physicians to measure incisor occlusal surface angles. Although the results show examiner variability largely in the sub-angular range, this study does not consider other measuring methods, is confined to one anatomical landmark per measured angle, and is limited to cadaver specimens. Thus, affirmation of method performance requires further methods and landmarks to be validated, in vivo investigations, and subsequent data comparison. Even though experienced study participants reported parts of angle measurements being more easily performed in live horses, standards for head positioning and used landmarks have yet to be established.Gathering reliable population data on any dental or dentofacial angles in the horse requires validation of orthodontic measuring methods, which is currently largely missing. For validation of clinical measures, intra- and interexaminer variability are key parameters to assess measurement repeatability and reproducibility, respectively . Both arDespite missing SA and TA population data from validated measures, incisor shortening and occlusal surface alignment are recommended in horses to restore functional occlusion after cheek tooth equilibration or dependent from incisor occlusal surface abnormalities ,16,17. NThe investigated rostro-caudal inclination of the incisor table is referred to as the SA . VariousThe investigated latero-lateral inclination of the incisor table is referred to as the TA . LiteratSchlicher et al. (2012), in their study on precision and consistency of cephalometric landmark identification, opine that error distribution for most landmarks reflects the difficulty in locating the landmarks . AlthougMaPHorse1 and equal landmarks. The vast majority of cases (>90%) showed SAs above the TMJ level [Our study did not primarily intend on delivering population data on either angle. However, mean SAs  deviated α 0–18° in reference to bony TMJ landmarks, which is similar to previous studies. Pellachin 2013) described SAs ranging between α -5°  and 20° in most of the 2700 examined cases using 13 descriMaPHorse1, the results suggest that even inexperienced examiners can perform rapid and valid clinical orthodontic measurements without requiring much training.Landmark identification errors are considered a major source of error in cephalometric studies . TraininThe investigated device could help deciphering incisor table angle normocclusal range and angle dynamics and may help to determine individual angle correction need and treatment success at high repeatability and reproducibility levels. In this cadaver study, the SA and TA measures were equally repeatable, but, compared to the high reproducibility of the SA measures using bony TMJ reference landmarks, the TA measures using soft tissue landmarks of the eye region performed slightly inferiorly at the interexaminer level. With a proposed procedural amendment, TA measures may also perform with higher reproducibility and consistency. Since the general measurement performance was not substantially impacted by the previous experience of the examiners, this device can be easily implemented into clinical practice.


Colloidal semiconductor quantum dots (QD), as well as other nanoparticles, are useful in cell studies as fluorescent labels. They may also be used as more active components in various cellular assays, serving as sensors or effectors. However, not all QDs are biocompatible. One of the main problems is their outer coat, which needs to be stable and to sustain hydrophilicity. Here we show that purpose-designed CdSe QDs, covered with a Puf protein, can be efficiently accumulated by HeLa cells. The uptake was measurable after a few hours of incubation with nanoparticles and most of the fluorescence was localised in the internal membrane system of the cell, including the endoplasmic reticulum and the Golgi apparatus. The fluorescence properties of QDs were mostly preserved, although the maximum emission wavelength was slightly shifted, and the fluorescence lifetime was shortened, indicating partial sensitivity of the QDs to the cell microenvironment. QD accumulation resulted in a decrease in cell viability, which was attributed to disturbance of endoplasmic reticulum performance. Quantum dots (QD) are colloidal, semiconductor nanocrystals, widely known due to their bright fluorescence. Most QDs are approximately nanospheres (1 to 10 nm diameter). The size of the sphere imposes spatial confinement effects and affects the extent of the energy gap. Consequently, the diameter of QDs defines their emission maxima. The most useful are QDs which emit in the visible light range , although there are some QDs able to emit in the infrared window  [The spectroscopic properties of QDs, namely their narrow emission band, broad absorption generating huge Stokes shifts, and resistance to photobleaching, make them perfect tools for labeling and tracking in cell studies. On the other hand, QDs built of cadmium and other heavy metals show high cytotoxicity. This is unsurprising given the possibility of nanocrystal decomposition and the release of toxic ions. A further problem is the actual delivery of QDs to cells. Many QD synthesis routes result in the formation of hydrophobic nanocrystals, which are non-mixable with the cellular medium and, therefore, are not accessible to cells. Hydrophobic QDs must be converted into water-dispersible QDs if intended for cellular studies. There are several strategies for such conversion, starting from the simple substitution of a hydrophobic coat by small hydrophilic molecules, such as thiocarboxylates or, less often, thioamines. QDs may also be covered with long amphiphilic polymers  or embedThe CdSe QDs used in the present study are produced as hydrophobic nanocrystals covered with trioctylphosphine oxide (TOPO). To convert them into water-dispersible particles, we used derivatives of apolipoprotein A  or a Puf2 20 X-TWIN microscope . As-prepared QDs were dispersed in chloroform, with an emission maximum of 560 nm. Puf protein was expressed in E.coli Bl21 and purified from inclusion bodies, as described in [Quantum dots (2.8 nm core diameter) were prepared by a thermal decomposition method, as described in . The mor553 = 117,431 M−1cm−1 (for QD) and ε280 = 33,640 M−1cm−1 for Puf, respectively [QD, Puf and QD:Puf concentrations were determined on the basis of absorption spectra, with extinction coefficients of ε2.HeLa and A375 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 u/mL), streptomycin (100 u/mL) and L-glutamine (1 mM). The cells were cultured in a humidified atmosphere at 37 °C, with 5% CO2 and subjected to further analysis. To determine the influence of QD:Puf on HeLa cells, the culture medium was removed and replaced by fresh complete DMEM containing an appropriate QDs dilution. The cells were then incubated for 24 h at 37 °C, in 5% COFor endocytosis inhibition tests, the temperature of incubation with QD:Puf was lowered to 22 °C, while the other parameters of cell incubation were kept unchanged. The CLSM images collected for this experiment were analyzed with Fiji software . At leas3 cells/well or 1 × 103 cells/well and cultured overnight in a complete DMEM medium at 37 °C, with 5% CO2. The medium was then removed and replaced by a QD:Puf preparation diluted in fresh culture medium (10–0.3125 nM). The cells were incubated for 24 h at 37 °C, with 5% CO2. After the incubation, the mixture was discarded, and the cells were washed before the addition of 100 µL of MTT reagent  in a complete DMEM medium to each well. The cells were then incubated for 3 h at 37 °C, with 5% CO2. Finally, the solution was carefully removed and the formazan crystals were dissolved by adding 100 µL of DMSO. The plates were shaken for 10 min and the absorbance was measured using a UVM 340 microplate reader  at 550 nm with a reference wavelength of 630 nm. The values obtained for the untreated controls were normalized to 100% and the viability of treated cells was estimated as the percentage of the control. The toxicity of Puf-QDs towards HeLa cells was assessed by MTT assay. For this purpose, the cells were seeded in triplicate in 96-well plates at 5 × 10Absorption spectra were determined with a DU 800 spectrophotometer . Steady-state emission and time-resolved fluorescence decays were recorded with an FS5 spectrofluorometer , using excitation with a xenon continuous lamp or a pulse laser (471 nm), respectively. Data were processed with Fluoracle software .CLSM and FLIM images were obtained using the Stellaris (Leica) platform. For most cases, a white pulse laser was used, and the precise excitation wavelength was given in the image description. For Laurdan-stained cells, a 405 nm diode laser was used. All cell images were obtained directly in growing chambers of borosilicate glass (LabTek system). The collected data were processed and analyzed with LasX and FLIM LasX software .Colocalization (Spearman and Manders’ coefficients) was determined using the Fiji software Coloc2 plugin.4 cells/well. The cells were cultured overnight and fluorescently stained immediately before the observations. For this purpose, the cells were washed with PBS buffer and fixed with 4% formaldehyde for 20 min. at room temperature. Then the cells were washed with PBS buffer and stained simultaneously with 2.5 µg/mL of propidium iodide and 10 µg/mL of Laurdan in PBS buffer . Finally, the cells were rinsed several times and maintained in PBS buffer while imaging was performed.For microscopy imaging, HeLa cells were grown in 8-well LabTek chambers (Nunc) and seeded at 2 × 10For immunofluorescence, the formaldehyde-fixed cells were permeablized for 15 min with 0.05% Triton in PBS, followed by washing with PBS (three times). Then cells were blocked by 1%BSA in PBS (1 h) and incubated for 3 h with primary antibodies diluted (1:100) in 1% BSA in PBS. Here, we used anti-calnexin  for staining ER and anti-golgin 97  for visualization of the Golgi apparatus. After washing , cells were incubated (1 h) with secondary antibodies  diluted (1:2000) in PBS with 1% BSA. Finally, cells were washed again three times with PBST and once (5 min) with PBS containing DAPI (2-(4-Amidinophenyl)-6-indolecarbamidine 5 µg/mL). The last was added to visualize the nucleus, as propidium iodide emission overlaps with Alexa Fluor 647.® ZOOM system . HeLa cells were seeded in 24-well plates at 5 × 104 cells/well and cultured overnight to allow the cells to attach to the plate. The next day, the culture medium was replaced by a complete DMEM medium (no phenol red) containing the appropriate concentration of QD:Puf. The cells were incubated for 24 h at 37 °C, and 5% CO2 and image sets were collected every 2 h. The obtained data were analyzed using Fiji software [The penetration of QD:Puf into HeLa cells was monitored using the IncuCytesoftware . The anaIncuCyte images were also used for the analysis of cell number changes upon QD:Puf treatment. For this, images series regions with 30–50 cells and relatively low starting confluence (20–30%) were selected. Cells were counted using the Fiji software CellCounter plugin.Statistical analysis was performed using one-way ANOVA with Tukey test.To check whether QD:Puf may have entered the HeLa cells, nanohybrid preparation was added to the cell medium directly and incubated under the previous growth conditions. We tested several dilutions of the QD:Puf preparation (1–20 nM). Additionally, we investigated whether the size of the nanohybrid mattered for in-cell delivery. For this purpose, we chose a fraction, corresponding to a single QD embraced by Puf , and a fraction of larger particles . Both O and M preparations were stable in the cell medium. This was demonstrated by fluorescence emission monitoring of QD:Puf mixed with the cell medium. No intensity changes were noted during the first 6 h incubation at 37 °C and only a 10–15% reduction in fluorescence intensity was noted after 24 h of incubation . For theLocalization of QD:Puf inside a cell was confirmed by detailed Z-stack projection analysis . On orth2+ toxicity. The preservation of QD:Puf nanohybrids was confirmed by fluorescence spectroscopy and gel electrophoresis. However, this verification cannot exclude the possibility that some parts of nanocrystals were destroyed in the process, even in the presence of glycerol as a cry-protectant. This highlights that a fresh preparation, or one stored at 4 °C, should be used in further studies.The use of higher QD:Puf concentrations resulted in higher fluorescence accumulation inside cells, both for M and O fractions. When nanohybrid fractions containing extremely large aggregates (without a gel filtration pass) were used, no fluorescence corresponding to QDs was found inside cells (not shown). Administration of QD:Puf preparation, stored at −20 °C, resulted in significantly increased cell death, probably due to released CdThe morphology of treated cells did not change, and their viability was further estimated quantitatively by MTT test (see paragraph 4). The dead cells contained a higher amount of QD:Puf, as evaluated from the total fluorescence intensity associated with a particular cell.As the distribution of QD:Puf-related fluorescence was not equal across the cell body, we attempted more precisely to identify the actual cellular compartments accumulating the nanohybrids. In the cell, QDs may be accumulated by lysosomes , the endTherefore, to identify QD:Puf target compartments, we stained the nucleus (with propidium iodide or DAPI) and membranes (plasma membrane by Laurdan stain as well as the ER and Golgi apparatus by specific immunostaining), and we compared the obtained pictures with separate images of QD fluorescence. QD:Puf emission was only clearly detected with immunostaining—the presence of other fluophores interfered with this measurement.Stained cells (control and treated with QD:Puf) are shown in the QD:Puf with a maximal diameter of about 15 nm (monomeric fraction) or 20–30 nm (aggregates fraction) were localized inside the ER cisternae (diameter of about 20–60 nm ). The acTo learn more about the kinetics of the process, we analyzed QD:Puf nanohybrid absorption by HeLa cells using an automated live cell imaging system (IncuCyte). The cells were imaged every 2 h for 24 h of incubation. Several images were analyzed for QD:Puf accumulation as measured by cell-associated fluorescence increase. Examples of the data obtained are shown in We investigated this issue further by comparing QD:Puf accumulation in the HeLa cells at 37 °C and at 22 °C. The lowering of temperature is a simple but efficient endocytosis inhibitor [The nanohybrids tended to aggregate in the medium, which was visible as clusters of grains. This may cause an impression of heterogeneity of distribution and lead to the conclusion that cells are differently susceptible to QD:Puf treatment. These clouds were mostly, but not only, associated with the cells. Their presence indicates that at least part of the “cell-associated” fluorescence measured by this method may be related to external fluorophores (QD:Puf not incorporated into the cell or only loosely associated with the plasma membrane). Part of the effect, however, may have resulted from slightly different focal planes of the cells. In CLSM, such clouds were not noted as the medium was exchanged before imaging. CLSM also did not confirm different sensitivity to QD:Puf among the cells.For a few cases, we were able to track the inheritance of green fluorescence by dividing cells E–H. As tVisual inspection of cell cultures suggested that, in the presence of nanohybrids. the number of dead cells increased. Therefore, we performed quantitative toxicity tests. The results indicated a decrease in viability of about 20–60% of the control , dependi2+ ions released during QD decomposition. Such a process is more likely when the QD surface is directly accessible to the cytoplasm and other cell fluids, especially at low pH in lysosomes. Moreover, the free cadmium ions may originate from the synthesis of QDs if not properly purified [3 cells per well). Counterintuitively, the resulting toxicity was slightly lower . Therefore, it is possible that the observed toxicity was not (or not only) due to CdThe fluorescence emission maximum was red-shifted inside the cells and the fluorescence lifetime was shortened, indicating some influence of the cell microenvironment on the QD surface. These observations might be an indirect effect, related to changes in the covering protein, Puf, and to amino acid ionization.In sum, we have demonstrated that QD cover, composed of long peptides, assures good nanoparticle performance in cell studies. This makes Puf and its derivatives the next option in fluorescent probes and sensor development. In such applications, a Puf-based (or other peptide-based) cover offers places for secondary chemistry to occur on the nanoparticle surface. A protein-based cover may provide protection of the nanoparticle surface from the reactive cellular interior, resulting in resistance to decomposition and release of toxic cadmium ions. In addition, a peptide might be modified by the addition of special tags using protein engineering before nanohybrid assembly.


Salivary gland pleomorphic adenoma (SPA) is a benign neoplasm that can still recur even after radical surgery. To investigate its underlying pathogenesis, here, we examined the significance of lncRNA ENST00000603829 in the proliferation and invasion of SPA. n = 30) and adjacent normal tissues  were collected from SPA patients treated at our hospital from June 2017 to December 2019. The human normal salivary gland epithelial cell line (HSG) and SPA cells  were cultured. qRT-PCR was used for detecting the expression of cyclin D1 and lncRNA ENST00000603829 in tissues and cells. lncRNA ENST00000603829/cyclin D1 was knocked down or overexpressed in PA116 cells. The expression of cyclin D1 and lncRNA ENST00000603829 in different cell lines was examined using qRT-PCR. Transwell assays and cell counting kit-8 (CCK-8) were used to assess cellular invasion and proliferation. The testing result regarding the apoptosis rate and cell cycle was obtained via flow cytometry. Western blot provided the measurement of cyclin D1 expression in cells.  SPA tissues ( We observed an upregulation of lncRNA ENST00000603829 and cyclin D1 expression in SPA tissues and cells. Knockdown of lncRNA ENST00000603829 inhibited cell invasion and proliferation, promoting apoptosis and retaining the cells during the G0/G1 phase. However, such effects of lncRNA ENST00000603829 knockdown were inhibited when cyclin D1 was overexpressed.  lncRNA ENST00000603829 can promote the occurrence and development of SPA through increasing cyclin D1 expression. Salivary gland pleomorphic adenoma (SPA) comprises of 40–70% of all salivary gland tumors . In saliCyclins have important roles in the development of PA. Further, studies have shown the possibility of an involvement of cell cycle markers cyclin D1, p-16, E2F, and CDK4 in PA's relapse and malignant transformation . Some stStudies have shown that in spite of the fact that from the human genome, many RNA species have been transcribed, the total transcripts include only a fraction of protein-coding sequences, with the remaining part being occupied by noncoding RNAs (ncRNAs) . The latlncRNA ENST00000603829 is a newly discovered lncRNA of 541 nt in length, located on chr19: 58379338-58380202. However, its function and mechanism on PA have been reported minimally. This presents research attempts to investigate lncRNA ENST00000603829's mechanism and its effects on SPA cells through in vitro assays, aiming at finding new targets for the treatment of SPA, as well as at providing an effective experimental basis.n = 30) and adjacent normal tissues  were obtained from SPA patients who were treated at Taizhou First People's Hospital during the period of time that starts in June 2017 and ends in December 2019. The identification of SPA tissues was made by three independent pathologists. The study protocol gained consent from the ethics committee of our hospital and strictly followed the Declaration of Helsinki. Informed consent was obtained from all patients for their participation in this research. This study was approved by the medical ethics committee of Taizhou First People's Hospital.The tumor tissue  and SPA cells  were provided by the American Type Culture Collection (ATCC). Culture of the entire cell lines was performed in Dulbecco's modified Eagle's medium (DMEM) containing 10 mg/mL streptomycin , 100 U/mL penicillin, and 10% fetal bovine serum (FBS) . Next, an incubator that was with 5% CO5 cells/well were used for cell transfection. Upon attaining cell confluence of 70–80%, using the Lipo 3000 Kit , the cells were transfected with negative siRNA plasmid (siNC), lncRNA ENST00000603829 siRNA plasmid (si-lncRNA ENST00000603829), negative pcDNA (pcNC), lncRNA ENST00000603829 pcDNA (pc-lncRNA ENST00000603829), and cyclin D1 pcDNA (cyclin D1). The media were replaced after transfection of 6 hours, and then, the cells were cultured for a further 48 hours and collected for subsequent experiments.Briefly, 6-well plates at a density of 2 × 10Ct−ΔΔ method was employed for the purpose of calculating the target gene's relative expression. Tissues and cells were treated with the TRIzol method, which was performed to extract their total RNA. Subsequently, NanoDrop was used to examine total RNA's purity besides concentration and cDNA  was prepared according to the random primer reverse transcription kit. The expression levels of lncRNA ENST00000603829 and cyclin D1 mRNA were detected following the manufacturer's protocol of the SYBR GREEN Kit , with GAPDH as the internal reference and 6 replicates of this experiment. The 24 cells) with 200 μL fresh DMEM complete after 24 hours, 48 hours, and 72 hours of culture. Then, 10 μL of CCK-8 detection solution was added to each well and cultured for 1.5 hours in an incubator, after which their absorbance was measured at 450 nm.First, 96-well plates were utilized for seeding the transfected PA116 cells (5 × 10μL of propidium iodide (PI) (20 mg/L) following the manufacturer's instructions. The apoptotic index was estimated using the following equation: apoptotic index = number of apoptotic cells/ × 100%.Cell apoptosis was measured by flow cytometry using Annexin V-FITC and 10 μg/mL of PI. Then, the stained cells were analyzed by flow cytometry.Briefly, transfected cells were trypsinized, washed twice with prechilled sterile PBS, fixed in 70% ethanol, and stained with 50 μL of cell suspension in serum-free media was added to the upper chamber, while 700 μL of medium containing 10% FBS was added to the lower chamber. After 24 hours of incubation at 37°C with 5% CO2, the invaded cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. Lastly, images from 3 fields were randomly captured with an inverted microscope and the invaded cells were counted.For this experiment, a Matrigel  was inserted at the bottom of the upper chamber. Briefly, 100 μg of protein with 1x loading buffer was boiled for denaturation. SDS-PAGE was then used for protein electrophoresis, and the proteins were transferred onto PVDF membranes. After one hour of blocking the membranes using 5% skim milk powder, the membranes were incubated with primary antibodies ; CDK6 , and p21 ) overnight at a temperature of 4°C. Next, the membranes were washed thrice and incubated for one hour with secondary antibodies at ambient temperature, followed by three washing. The membranes were then developed using an enhanced chemiluminescence reagent, and the blots were detected using an enhanced chemiluminescence system. The ImageJ software was used to analyze the grayscale values of bands. GAPDH  was utilized as an internal reference for calculating the relative protein expression.We first collected the cell lines and tissues for total protein extraction using cell lysate. Then, the supernatant proteins were collected after centrifugation at 12,000 rpm for 5 minutes at 4°C. Next, the BCA protein assay was used to determine the protein concentration. Subsequently, 20 t-test. The results of measurement data were expressed as mean ± standard deviation (SD). Pearson correlation analysis was performed to analyze the correlation between lncRNA ENST00000603829 and cyclin D1 expression in SPA tissues. p < 0.05 refers to a statistical difference.SPSS (version 24.0) was used for statistical analysis. The one-way analysis of variance was used for making comparisons among multiple groups, while the comparison between two groups was made by utilizing an independent We detected lncRNA ENST00000603829 expression in SPA. The results revealed a notable increase of lncRNA ENST00000603829 expression in SPA tissues compared to the normal ones . MeanwhiFor the purpose of verifying the effect that lncRNA ENST00000603829 had on the growth performance of SPA cells, proliferation, invasion, apoptosis, and cell cycle of PA116 cells were examined after knockdown or overexpression of lncRNA ENST00000603829. The results showed a significant reduction in lncRNA ENST00000603829 expression after knockdown of this lncRNA, while showing an opposite trend after overexpression of this lncRNA , indicatWhen exploring lncRNA ENST00000603829's mechanism, we found that mRNA and protein expression of cyclin D1 (a key factor of SPA) were notably increased in SPA tissues and cells Figures . SubsequWe further analyzed the role of cyclin D1 and lncRNA ENST00000603829 in SPA. Compared with the siNC group, si-lncRNA ENST00000603829 notably reduced the invasion and proliferation of cells and improved the apoptosis rate. Besides, the latter notably increased the percentage of cells in the G0/G1 phase while decreasing that in the S phase. CyclinD1 and CDK6 decreased considerably, and p21 increased after lncRNA ENST00000603829 knockdown. However, the effects were opposite after lncRNA ENST00000603829 overexpression Figures . The menSPA mainly attacks patients over 60 years old, with no difference in sex . The occThe change of lncRNA expression is a medical sign of diseases but also affects a series of signal transmissions in cells, subsequently resulting in changes in cellular physiological functions as well as metabolic status. Some scholars believe that the function of lncRNAs to regulate signals affects the occurrence and development of diseases. Loss of lncRNA HOTAIR has the pivotal effect of not only inducing apoptosis but also regulating proliferation, invasion, and migration of cells in breast cancer . KnockdoCyclin D1, on the other hand, is considered to be a protein encoded by the CCND1 gene, acting as a significant regulator of cell cycle in addition to a prognostic and predictive factor for cancer. According to previous evidence, it is feasible to take cyclin D1 as a biomarker to forecast the prognosis of esophageal squamous cell carcinoma . By analIn summary, lncRNA ENST00000603829 expression was found upregulated in SPA and affected the growth performance and development of SPA cells by positively regulating cyclin D1 expression. These results suggest lncRNA ENST00000603829 as a promising diagnostic biomarker and could be a therapeutic target for SPA.


Sarcophaga sp. Larvae) via morphological and microscopic studies compared with control, providing important biological information could aid in the environmental impact of the toxic level of MOF materials.Metal–organic frameworks (MOFs) are promising materials for several applications. Thus, they have been intensively reported and commercialized by several international companies. However, little is known about the fate and risk of MOFs to living organisms. Here, the toxic effect of two Zinc (Zn)-based MOFs; zeolitic imidazolate frameworks (ZIF-8) and leaf-like ZIF (ZIF-L), was tested to investigate the impact of the postmortem period of mice carrions and arthropods which found in decomposing carrions. The data analysis revealed an increase in zinc content over time. Toxicology in forensics studies biological materials for the presence of poisons, such as pharmaceuticals. The toxicology report can provide important details about the types of chemicals present in a person and whether the amount of those substances is in line with a therapeutic dose or exceeds a dangerous level. These findings conclude the possible fate and impact after mortality. This study presents the first study of the toxic effect of ZIFs materials using mice carrions and arthropods ( Establishing the cause of death is one of the most critical aspects of a fatality investigation. This feature may be difficult to determine when the corpse is retrieved and decomposed; this feature may be difficult to determine2. Carcasses or carrions are natural and vital nutrients for arthropods. So, insects (Phylum: Arthropoda) have considerable importance in forensic5. Insects could perform as reliable evidence for toxicological analysis when body fluids and tissues are absent or unsuitable for analysis 6. These insects will also be nutrients for earth-raising environmental aspects.The postmortem period is an essential task for forensic science and environmental concerns7. Decomposition caused by insect activity in and on the body is a continual process that may be quantified, allowing for accurate minimum Postmortem interval (PMI) estimations up to several months after the death, depending on the circumstances. Thus, numerous publications have reported that drugs and toxins can change the development rates of insects feeding on decomposing Carcasses8.The principal aims of forensic toxicology are to institute the presence and detection of toxicants and establish whether these substances give adverse results. Careful exploration of entomological findings affecting the insects dwelling in carrion can aid in discovering a lot of information critical for investigation concerned with the death of an individual that would otherwise be lost in the absence of awareness on the part of forensic science staffs9. These arthropods are not entirely reliant on people. Synanthropic insects such as filth flies, biting midges, punkies, and mosquitoes (Diptera) feed on humans either directly by blood-sucking or indirectly through waste and garbage. Some of these insects have developed an endophilic lifestyle, which means they feed and rest in our homes. Like some stored goods pests, some hematophagous insect species have lost their wild or peridomestic environment and have become utterly reliant on domestic harborage and people. So, Forensic arthropodology is getting much awareness9. Sarcophagidae flies are important forensic markers in assessing human decomposition because they can detect a body within minutes of death, even at great distances. This is also linked to human corpses. Miltogrammatinae, Paramacronychiinae, and Sarcophaginae are the three subfamilies of the Sarcophagidae. Over 3100 species are described in this family, divided into 400 genera. Representatives of this family have a worldwide geographic distribution, with most species residing in tropical or mild temperate regions.Nowadays, forensic science entails using any science to study legal issues. Items, facts, or opinions submitted in a criminal case developed or supported utilizing a body of forensic science commonly employed in criminal trials are forensic evidence. With such a wide variety of arthropods, it’s not surprising that some have evolved to take advantage of the haven we provide or to exploit us and our products. The common Anthropophilic arthropods include cockroaches (Blattodea), silverfish (Thysanura), house flies (Diptera), and house and dust mites (Acari)18. They were applied for several applications, including gas storage, separation, catalysis, and biomedicine applications28. They advanced biomedical applications such as drug delivery, phototherapy, and bone/tissue engineering38. However, little is known about the potential of these materials for health or environmental hazards. Before considering a MOF-based material for biological systems, assessing the material toxicity is necessary. There are currently few relevant studies on ZIF-8 toxicity, and more research is needed in the following areas to evaluate its bioavailability and direct its appropriate applications accurately.Porous materials such as metal–organic frameworks (MOFs), including zeolitic imidazolate frameworks (ZIFs), are the most appealing porous materials in the research and marketSarcophaga sp. Larvae) by the number of insects collected from mice carrions of different groups, by morphological characterization using electron microscopies , and transmission (TEM)). To achieve the relationship between combined chemistry and their toxicological impact on arthropods found on carrions, address issues associated with the toxic effects of substances on humans that are germane to legal events. Based on our knowledge, this study is the first time using mice carrions to evaluate the toxic effect of ZIF-8 and ZIF-L.Forensic toxicology is the branch of science that resolves problems in this field of law. Forensic entomotoxicology in death by poisoning investigations has grown in favor. When traditional toxicological samples have decayed or are no longer accessible, insects might be used as an alternative specimen (evidence). Herein, we investigated the toxic effect of ZIF-8 and leaf-like ZIF (ZIF-L) using arthropods as evidence for decomposing carrions. Various analyses have proved the successful detection, identification, and quantification of ZIF-8 and ZIF-L compounds from insects (3)2•6H2O, triethylamine (TEA), and 2-methylimidazole (Hmim) were purchased from Sigma-Aldrich (Germany). Stock solutions of Zn(NO3)2•6H2O and  Hmim were prepared by dissolving 25 g and 62.5 g in 100 and 250 mL of H2O.Zn was added to a solution of Zn(NO3)2•6H2O  during stirring. Then, a Hmim (8 mL) solution was added, and the solution was stirred for one hour. The materials were then separated using centrifugation and washed using water (2 × 50 mL) and ethanol (2 × 50 mL).ZIF-8 and ZIF-L were synthesized at room temperature using water as solvent and Hmim: Zn molar ratio of 25 and 8, respectivelyFifteen male mice Sprague–Dawley mice (aged 21–35 days), with a weight of (20–23 g) average body weight, were used. Mice were delivered from the 4 Animal House, Faculty of Medicine, Assiut University (Egypt). The study was conducted at Assiut University, Faculty of Medicine, from 1 July to 31 August 2021. This location was poor vegetation, and the environment was arid, with typical temperatures of (40 °C / 37 °C: 36 °C / 34 °C). Each mouse was placed on a plate and enclosed by a perforated plastic box to keep birds, cats, and animal customers out. The site's substrate is primarily made up of sand and dust. All methods were performed following Care and Use of Laboratory animals guidelines.50) of ZIF-8 (2800 mg/kg) once orally by gastric gavage which caused their death; Group III (n = 5): received double LD50 of ZIF-L (2800 mg/kg) once orally by gastric gavage which caused their death.Mice were employed in this investigation to mimic the decomposition of a human body. Each mouse was placed on an outdoor plate. Mice were classified randomly into three groups; Group I (Control groups): negative control n = 5), received nothing, neither saline nor drugs, rats were anesthetized with 50 mg/kg intraperitoneal thiopental and sacrificed; Group II (n = 5): received double Lethal Dose 50  were collected by measuring the concentration of zinc, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and ultrastructural studies as shown below:-From all of the decomposition stages, Sarcophaga sp. were digested in nitric acid  after 1, 2, 3, and 5 days. The solution was sonicated for 30 min in an ultrasonic bath to dissolve Sarcophaga sp completely. The residual was removed via filtration to obtain a clear solution for the analysis using Atomic Flame Absorbance .The collected Sarcophaga sp. Larvae were fixed on cover slides and coated with gold before measurments. Whole Larvaefrom in three groups were selected for 1.5 h in 5 percent glutaraldehyde in sodium cacodylate buffer, washed in distilled water, and dehydrated in ethanol. The drying of the critical spot was completed. Samples were placed on stubs, coated with carbon or gold, and analyzed at 20 kV using a Scanning Electron Microscope .Sarcophaga sp. Larvae were used to prepare semithin sections before using transmission electron microscopy . The cross-sections were examined under TEM (JEOL 100-CXII) operated at 80 kV. The results were presented as micrographs.P-value of ≤ 0.05 was accepted as statistically significant.Data represented observed numbers of Arthropods stage. Data obtained were subjected to statistical analysis of variance (ANOVA) test using Open Epi version 2.3.1. A This study was approved by the Ethics Committee affiliated with Assiut University Hospital. All procedures performed in the study involving animal participants were following ARRIVE guidelines.Protocols approved by the Laboratory Animal Care and Use Committee of the Affiliated Assiut University and Faculty of Medicine.2 and Zn(mim)2•(Hmim)1/2•(H2O)3/2, respectively. The synthesized materials were characterized using XRD . The particle size and morphology were determined using TEM images , all the decomposition period was 70 days, Sarcophaga sp. (flesh fly) were collected from decomposition stages   Fig. .Figure 4Sarcophaga sp. (flesh fly) larvae of the different stages of decomposition was reported, as shown in Fig. ZIF-8 nanoparticles (NPs) have a wide range of cytotoxic effects. Initially, ZIF-8 NPs have a higher bioavailability than typical zinc-containing nanoparticles such as ZnO NPs, allowing more cells to absorb more zinc. The elevated zinc concentration may then inhibit the GR enzyme, reducing the amount of GSH generated by the cells. ZIF-8 NPs will also raise the level of cellular ROS. GSH depletion would raise the level of ROS in the cells even more. As a result, the cell's inflammatory response will rise, causing inflammation-related genes such as CCL4 and IL6 to be upregulated. Cell necrosis will occur as a result. The concentration of Zn inside the At the fresh stage, a group I, there were only five families and six species of arthropods without the appearance of pupa or larva 2. While in group II, there were only three families and 3species without the appearance of pupa and larva 2, and in group III, there were only four families and 4species without pupa formation Table .Table 1CChrysomya albiceps, Musca domestica, and Sarcophaga sp., were the first invaders. Numerous isopods, spiders, and beetles were found beneath the carrions. Group II showed less number of eggs and larva than other groups without the appearance of pupa in all groups , and there is no visible demarcation between the thorax and the abdomen. There is a change in the elongated, truncated posteriorly, and tapering toward the anterior extreme curved downward; they are cylindrical, segmented , The body is divided into pseudocephalon (pc), three thoracic segments (t1–t3), seven abdominal segments (a1–a7), and an anal division (ad), which carries the posterior spiracle (ps) intestine (i), ted Fig. a. In groted Fig. b, and inted Fig. c.Figure For scanning electron micrographs showing: Group I, the whole body, ventral view showing pseudocephalon (PC), anterior spiracles (as) the posterior spiracles (Ps.), well developed annular bands (ab) of intersegment spines (s) is in 4–6 rows of pinted spines, the grooves of the body are apparent, anterior portion showing three thoracic segments with anterior spiracular, maxillary palpus consists of several sensillae, In the frontal and inner margins, a group of small spines delimiting the atrial cavity, the array (R) of the cuticular ridges (CR) were found associated between the mouth  and the prominent hooks. (cs) Cephalo-pharyngeal skeleton., anal segment, the posterior spiracles are enclosed within the deep stigmas cavity, the spiracular openings (caves), pores, perispiracular glands, and sensilla, rima, and rays are also visible, and the fleshy projections (fp)  larvae. The groups that received the ZIF-8 and ZIF-L received apparent damage as visible on TEM micrographs. These findings are also consistent with our recent studies, which reveal that this ZIF-8 and ZIF-L toxin causes many cell death in vitro, with apoptosis being the most common death mechanism activated.In this study, various arthropods were collected on the mice carrions belonging to eleven orders; and have been implicated by other researchers of forensic45. Five decomposition phases were identified; the dry decay stage took the longest to decompose, the advanced decay stage, and the fresh stage took the shortest. This result follows the current development.Emeka et al. worked on three pig carrions for 60 days; arthropods were gathered twice a day for the first week and once daily for subsequent weeksMorphological Sarcophaga sp. (flesh fly) larvae damage index was used for evaluation of the degree of larvae damage; it depends mainly on the percentage of ZIF-8 and ZIF-L in the tail, so it is considered an accurate measure for the degree of toxin when we compare with normal as reported by Szpila et al. (2015).46. Conversely, work by Kharbouche et al. revealed an increased rate of development for larvae of Lucilia sericata and Boettchersica peregrine reared on minced pig liver containing different codeine concentrations47. Thus, the same species could react differently toward two molecules belonging to the same family. Keshavarzi et al. pointed out that insect succession became comparable among the dealt with and untreated rabbit carcasses. However, the styles of the sequence of Chrysomya albiceps and Calliphora vicina differed barely among dealt with and untreated carcasses.Salim et al. established potential underestimation after death based on the damaged interpretation of the development of C. albiceps larvae reared on the tissues of rabbits carrionss containing morphineThe present study discovered that larvae reared on decomposing tissue-containing ZIF-L had retardation of development compared with the ZIF-8 groups and control group as the means of weights, lengths significantly, and widths of the ZIF-L and ZIF-8 groups were considerably lower than those of the control larvae at different time intervals.In the present study, Sarcophagasp. (flesh fly) larvae were of great importance in forensic medicine, as they appeared in the five stages of decomposition, so we used the flesh fly larvae to estimate zinc inside the larvae. On the other hand, study the morphological, SEM, and TEM electronic microscopy to review the changes that occurred in control and treated groups because it is substantial evidence in forensic medicine. This investigation presented for the first time the use the morphological changes, electronic microscopy studies, and Zn estimate of arthropods as evidence of toxins that are used in suicide or death, even by mistake.The decomposition of carcasses and carrions arthropods (larvae of Sarcophaga sp.) significantly changes terrestrial ecosystems. The present work has shown that a careful study of two chemical compounds, ZIF-8 and ZIF-L, and their interaction with the decomposition of carcasses and entomology has profound effects on the arthropods (larvae) Sarcophaga sp.) forensically significant invertebrates. Our study reveals that ZIF-8 and ZIF-L can be present in the investigated anthropods feed on mice carriers. It will aid in improving the evidential usefulness of entomology to the Forensic Science and Policing communities in criminal investigations. Thus, further research is highly required in the future to determine the factors that govern the toxicology and the changes in the soil content after the decomposition of carrion anthropods.


The conditions and management practices imposed during mating can influence the success of a sheep production enterprise. However, the impact of mating practices across southern Australian sheep production systems are relatively unknown. Mating liveweight and body condition score data were collected at mating from four sheep breeds and during three seasons of mating across southern Australia. Further, the seasonal conditions during mating were ranked by the producers and ram percentage and region were also recorded. Bayesian Network analysis was used to explore the relationships between these variables and pregnancy and fetal number at scanning. The results of this survey study emphasize the interrelatedness of the explored mating conditions and practices and the importance of understanding their interactions for optimizing sheep reproduction and nutrition from mating.n = 58) were recruited across southern Australia to take part in a survey and mating variables were collected from over 30,000 ewes between October 2020 and August 2021. A Bayesian Network (BN) was developed to identify the interrelatedness and most influential variable on pregnancy and fetal number (of pregnant ewes) outcomes under different scenarios. The BN analysis indicated a low association between the variables explored, however, were breed dependent. In wool-based breeds a mating liveweight of 60–69.5 kg predicted the lowest non-pregnant and greatest number of fetuses, and in shedding ewes 70–79.5 kg predicted the lowest non-pregnant rate and 90–99.5 kg the greatest number of fetuses. Pregnancy rate and fetuses per ewe were optimized at ram percentages of 1.5% for Composite and Merino ewes and 2% for Maternal ewes. A mating BCS 4 resulted in greatest pregnancy rate and number of fetuses across all breeds. Curvilinear relationships between mating liveweight, BCS and ram percentage were observed with pregnancy rate and fetal number. Practically, reproductive potential is best managed on a breed basis and with consideration of all variables explored.Sheep production in southern Australia may vary by breed, time of year, production output , region and seasonal influence. Sheep producers with flocks of approximately 300–500 ewes ( Sheep have a seasonal reproductive cycle, becoming most active as photoperiod shortens and experience anestrus or low ovarian activity as photoperiod lengthens ,2. FurthThe management of different sheep breeds provided with the same resources may result in different liveweight gain and productivity due to the different energy requirements of larger versus smaller animals ,17. ThisComputer modelling of whole sheep enterprises across different locations indicated the optimum lambing time was dictated by the average length of the pasture growing season and production output (wool and/or lamb target weight) . This waManagement conditions and practices leading up to and at mating, including ewe breed, mating season, and management of liveweight and BCS may influence reproductive performance. While guidelines have been developed, the impact of mating conditions and practices in a commercial setting is relatively unknown. The objective of this study was to examine relationships between mating liveweight and BCS on pregnancy and fetal number at pregnancy scanning across different breeds and seasons of mating in sheep enterprises in southern Australia. It was hypothesized that the relationship between mating liveweight and BCS and pregnancy and fetal number would vary between ewe breed and mating season. The impact of other variables and practices were also explored.Commercial farms within New South Wales (NSW), Victoria (VIC) and South Australia (SA) were established as experimental sites between October 2020 and May 2021, totaling 56 sites, 58 ewe flocks and 30,030 individual ewe records. Producers were recruited to the project through professional networks across a broad range of regions. These regions represent Central NSW, Northern NSW/Queensland (QLD), the Wimmera Malley Murray, East VIC and the SA Peninsula.Each site joined flocks of either Composite, Maternal, Merino or shedding ewes during either spring, summer, or autumn (2020 to 2021) according to individual routine management practices. Ewes were individually identifiable by electronic identification tags and between 300 and 500 mature (having had at least one previous mating event) ewes were mated in each flock. The Composite hybrid animal, the result of crosses of multiple breeds, has low wool value and are primarily meat-producing animals, excluding shedding breeds. Maternal ewes are defined as meat-producing crossbred ewes which retain some wool value. Merino ewes are defined as those of various Merino genotypes with high wool value. Shedding breeds have no wool value due to a shedding hair-based coat and are solely meat-producing animals, and included Dorper and Australian White breeds.®, Ceva Animal Health Pty Ltd., Glenorie, NSW, Australia) to enhance reproductive performance [The liveweight and BCS measurements were taken when rams were introduced to each flock to ensure accurate mating information was collected across the study. For spring mating, one Composite flock and one Merino flock used a melatonin-based hormone product , single (one) or multiple (two or more) fetuses. In two instances the data for multiple fetuses  was removed from the data set due to inaccurate scanning performance or because of a delayed date of scanning greater than 100 days from the start of mating. For these two sites, pregnancy status (pregnant or non-pregnant) was retained.n = 29,270) and BCS  was recorded at the commencement of mating. Seven-hundred and sixty liveweight records were missing from the dataset due to unavoidable recording errors while collecting data on commercial farms. Liveweight was recorded to 0.5 kg accuracy level, with a mean liveweight of 67.7 ± 13.4 kg across the survey. Body condition score was performed by a single assessor across the survey and recorded at quarter intervals, according to Jefferies [Ewe liveweight  and Pr (B) are the marginal probability distributions of A and B, respectively; Pr (B|A) is the conditional probability distribution of B given A; Pr (A|B) is the conditional probability of A given B; and Pr  is the joint probability distribution for A and B. Since a BN model represents a joint probability of the variables, inferential analysis may be performed by fixing the values of a set of selected variables , then the values of remaining variables  in the BN model can be predicted/estimated [Bayesian Network (BN) analysis was used to explore the interrelationships of the variables. The BN model was developed using the Netica software package . A BN mostimated .The most influential variable in determining pregnancy outcome (pregnant or non-pregnant) and fetal number (single or multiple fetus), respectively, could be investigated using a BN model with respect to the potential variables of breed, mating BCS, mating liveweight (weight), mating season (season), region, seasonal conditions during mating (during) and ram percentage (ram) . Mating 2, the goodness-of-fit measure from a regression analysis that represent the proportion of the variation explained in the selected variable by fixing the value in one of the other variables. A sensitivity analysis was performed for pregnancy outcome and number of fetuses (of positive (‘1’) pregnancy outcomes), respectively, to quantify the strength of the association between the interrelated variables breed, mating BCS, mating liveweight, mating season, region, seasonal conditions during mating and ram percentage. The sensitivity analysis output percentage (%) values are similar measures to the goodness of fit measure adjusted R2 used in a regression model, which represents the proportion of variation in the response variable being explained by those predictor variables.Sensitivity to findings is an inbuilt Netica function that enables the ranking of impacts of other variables (‘evidence variables’) on a selected target variable . The senn = 18,293) region followed by Northern NSW/QLD (n = 4481), Wimmera Malley Murray (n = 3816), East VIC (n = 1984) and SA Peninsula (n = 1456) regions. A total of 58 ewe flocks were recorded across three mating seasons; spring, summer and autumn. The majority of flocks were Merino followed by Composite, Maternal and shedding ewes (http://www.multimeat.com.au/ (accessed on 21 August 2022)).The majority of data was collected in the Central NSW , followed by Composite (71.0 ± 11.4 kg), shedding (68.8 ± 13.2 kg) then Merino (62.6 ± 14.0 kg) ewes. The highest average mating BCS by breed was shedding ewes (BCS 3.72 ± 0.7) followed by Maternal (BCS 3.62 ± 0.9), Composite (BCS 3.51 ± 0.8) and Merino (BCS 3.07 ± 0.7) ewes. The number of ewes surveyed by breed and season, and pregnancy scanning information is reported in Based on the sample data, by first excluding the pregnancy outcome variable and designating the fetal number as the target variable, the Netica inbuilt Tree Augmented Naïve Bayes Net algorithm was employed to specify a BN model structure. The pregnancy outcome was then manually added into the model as the parent node of the fetal number node. Finally, the BN model was completed by employing the inbuild Expectation-Maximization algorithm for model parameter estimation. Thus, the resultant BN model should have the best prediction performance given the sample data. However, the BN model was further refined based on a priori knowledge about the data by manually adding three links between associated variables: (1) link between ‘season’ and ‘breed’; (2) link between ‘breed’ and ‘region’; and (3) link between ‘breed’ and ‘ram’. The resultant BN model is presented in The sensitivity analyses (sensitivity to findings) for pregnancy outcome and fetal number of pregnant ewes (pregnancy outcome ‘1’ selected) allowed a quantitative comparison of the variables breed, mating BCS, mating weight, mating season, region, seasonal conditions during mating and ram percentage to determine the most influential variable . OverallThe model predicted relationship between Composite ewe fetal number and each level within each node/variable is displayed in The model predicted relationship between Maternal ewe fetal number and each level within each node/variable is displayed in The model predicted relationship between Merino ewe fetal number and each level within each node/variable is displayed in The model predicted relationship between shedding ewe fetal number and each level within each node/variable is displayed in Across breeds, predicted multiple fetus rates decreased when ewe body condition at mating exceeded BCS 4 and the corresponding non-pregnant and single bearing ewe proportions increased. Similar observations were made for liveweight and ram percentage for each breed (except shedders where there was no variation in ram percentage). Taken together, the data suggests curvilinear relationships between reproduction outcomes and ewe liveweight, ewe BCS and ram percentage.The dataset contained little variation in pregnancy rate 90.1%) and the number of multiple fetuses (53.5%), being similar to a previous pregnancy scanning report . This wa0.1% and The pregnancy rates of spring mated flocks 85%) across the survey, albeit lower than summer (90%) and autumn (93%) mated flocks, were greater than expected given the cyclic nature of the ovine reproductive cycle . The len% across Reports of ovulation and pregnancy rates outside of the breeding season are varied. The number of ovulating Merino ewes may be as low as 30% during spring in the absence of rams , howeverFurther, producers ranked the season in terms of pasture availability during the mating period between ‘well below average’ to ‘well above average’, a total of five possible ranks. Over 65% of producers with spring mated flocks reported ‘above average’ to ‘well above average’ seasons. As observed previously ,41, imprThe pregnancy rates for autumn-mated Maternal and Merino ewes in the current survey are very similar to previous reports ,38,39,42Pregnancy and fetal number outcomes were predicted to peak in the BN analysis at 60–69.5 kg in Composite, Maternal and Merino ewes. At liveweights lower than 60 kg and greater than 69.9 kg, pregnancy and fetal number decreased in Composite, Maternal and Merino ewes. The percentage of non-pregnant shedding ewes was least at a mating liveweight of 90–99.5 kg. Fetal number in shedding ewes, however, was greatest at a mating liveweight of 70–79.5 kg. The selection for greater fleece weight, due to the positive genetic correlation between fleece weight and bodyweight, has resulted in heavier sheep that have higher feed requirements ,44. HoweThe influence of mating liveweight and BCS on conception and ovulation rate varies by breed. No improvement in conception or ovulation rate was achieved above a mating liveweight or BCS threshold in Romney, Composite and Cheviot ewes ,14, howeThe predicted impact of ram percentage on pregnancy rate and number of fetuses in the BN analysis was unexpected and was also curvilinear. Guidelines for producers recommend a ram percentage for mature ewes of 1.0–1.5% plus one additional ram ,57. HoweThis survey had several limitations. Capturing data across two (or multiple) years would allow the consistency of these effects to be explored as reproductive performance in the current year may be influenced by performance in the previous year . FurtherThe objective of this study, to examine the relationships between mating weight and BCS on reproductive success, respectively, and secondly, to explore the influence of seasonal conditions during mating and ram percentage on pregnancy and fetal number outcomes across a variety of sheep breeds and mating seasons was achieved. The BN developed is specific to the data of the sheep surveyed in this study, and, as such, extrapolating conclusions more broadly should be approached with caution. Overall, all variables explored were lowly associated with pregnancy and fetal number outcomes across this dataset and highlight the importance of understanding the interrelationships between the different mating practices and variables explored. Curvilinear relationships were observed between fetal number outcomes and mating liveweight and BCS and ram percentage. Practically, pregnancy rate and number of fetuses are predicted to vary between breeds, peaking between 60–69.5 kg in the wool-based breeds explored and between 70–79.5 and 90–99.5 kg, respectively, in shedding breeds. Further, summer-mated Composite (with no hormone manipulation) and Merino ewes with 1.3–1.75% rams and autumn mated Maternal ewes with 1.8–2.3% rams and shedding ewes  resulted in optimum conception rate and fetal number. However, further nuances within and between breeds may be found from a larger dataset with greater flock numbers during each of the explored mating seasons and greater variation of seasonal conditions during mating. This survey study emphasizes the importance of understanding the interaction between breed, mating season, ram percentage and the influence of seasonal conditions, while ensuring an appropriate liveweight and BCS at mating.