Determination of Sex Ratio In Bovine Semen Using SYBR Green Real-Time PCR

A SYBR green real-time PCR assay was developed to find out the sex skewness in bovine sex-sorted semen samples. The qPCR assay of PLP and SRY genes revealed the mean values of X- and Y-bearing spermatozoa as 50.24 ± 0.65 and 49.75 ± 0.62 per cent in unsorted, and 91.80 ± 0.79 and 8.20 ± 0.73 per cent in X-enriched semen samples respectively.. The amplification efficiencies of the PLP and SRY primers were 99.25 and 98.03 per cent respectively. The method was validated by a series of repeatability and reproducibility assays which revealed low co-efficients of variations as 2.19 and 3.12 per cent respectively Thus becoming a reliable and inexpensive tool to evaluate the sorted semen on routine basis and validation of other sperm sexing technologies.

Comparison of the kinematics, repeatability, and reproducibility of five different multi-segment foot models

Background Multi-segment foot models (MFMs) for assessing three-dimensional segmental foot motions are calculated via various analytical methods. Although validation studies have already been conducted, we cannot compare their results because the experimental environments in previous studies were different from each other. This study aims to compare the kinematics, repeatability, and reproducibility of five MFMs in the same experimental conditions. Methods Eleven healthy males with a mean age of 26.5 years participated in this study. We created a merged 29-marker set including five MFMs: Oxford (OFM), modified Rizzoli (mRFM), DuPont (DFM), Milwaukee (MiFM), and modified Shriners Hospital for Children Greenville (mSHCG). Two operators applied the merged model to participants twice, and then we analysed two relative angles of three segments: shank-hindfoot (HF) and hindfoot-forefoot (FF). Coefficients of multiple correlation (CMC) and mean standard errors were used to assess repeatability and reproducibility, and statistical parametric mapping (SPM) of the t-value was employed to compare kinematics. Results HF varus/valgus of the MiFM and mSHCG models, which rotated the segment according to radiographic or goniometric measurements during the reference frame construction, were significantly more repeatable and reproducible, compared to other models. They showed significantly more dorsiflexed HF and plantarflexed FF due to their static offset angles. DFM and mSHCG showed a greater range of motion (ROM), and some models had significantly different FF points of peak angle. Conclusions Under the same conditions, rotating the segment according to the appropriate offset angle obtained from radiographic or goniometric measurement increased reliability, but all MFMs had clinically acceptable reliability compared to previous studies. Moreover, in some models, especially HF varus/valgus, there were differences in ROM and points of peak angle even with no statistical difference in SPM curves. Therefore, based on the results of this study, clinicians and researchers involved in the evaluation of foot and ankle dysfunction need an understanding of the specific features of each MFM to make accurate decisions.

Management of validation of HPLC method for determination of acetylsalicylic acid impurities in a new pharmaceutical product

The work mainly focused on a validation of the method for determining the content of salicylic acid and individual unknown impurities in new pharmaceutical product—tablets containing: 75, 100 or 150 mg of acetylsalicylic acid and glycine in the amount of 40 mg for each dosage. The separation of the components was carried out by means of HPLC, using a Waters Symmetry C18 column (4.6 × 250 mm, 5 μm) as the stationary phase. The mobile phase consisted of a mixture of 85% orthophosphoric acid, acetonitrile and purified water (2:400:600 V/V/V). Detection was carried out at a wavelength of 237 nm, with a constant flow rate of 1.0 ml min−1. In order to verify the method, linearity, precision (repeatability and reproducibility), accuracy, specificity, range, robustness, system precision, stability of the test and standard solution, limit of quantification and forced degradation were determined. Validation tests were performed in accordance with ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) guidelines. The method was validated successfully. It was confirmed that the method in a tested range of 0.005–0.40% salicylic acid with respect to acetylsalicylic acid content is linear, precise and accurate.

Repeatability and reproducibility of corneal higher-order aberrations measurements after small incision lenticule extraction using the Scheimpflug-Placido topographer

Background To evaluate the precision of corneal higher-order aberrations measurements after small incision lenticule extraction (SMILE) using the Sirius Scheimpflug-Placido topographer (CSO, Italy). Methods Seventy-five eyes from 75 postoperative subjects were included in this prospective study. Three consecutive corneal aberrometric measurements were obtained with the Scheimpflug-Placido topographer by two experienced operators to assess intra- and inter-observer reproducibility. The within-subject standard deviation (Sw), test-retest repeatability (TRT) and the intraclass correlation coefficient (ICC) were calculated. Results For intraobserver repeatability of anterior and total corneal aberrations, all ICCs were more than 0.922, except for trefoil (0.722 to 0.768). The ICCs of total root mean square (RMS), coma Z (3, ± 1), and spherical aberration Z (4, 0) were over 0.810 while higher-order RMS, trefoil Z (3, ± 3), and astigmatism II Z (4, ± 2) were below 0.634 for posterior corneal surface aberrations. All Sw values for all types of aberrations were equal to or below 0.07 μm. Regarding interobserver reproducibility, all TRT values were no more than 0.12 μm, 0.05 μm, and 0.11 μm for anterior, posterior, and total corneal aberrations, respectively. The ICC values ranged from 0.875 to 0.989, from 0.686 to 0.976 and over 0.834 for anterior, posterior, and total corneal aberrations, respectively. Conclusions The repeatability of measurements of anterior and total corneal aberrations with the Sirius system in corneas after SMILE surgery was high, except for trefoil. There was some variability in posterior corneal aberrometric measurements. High reproducibility of corneal aberrometric measurements was observed between measurements of both examiners, except for trefoil, with poor to moderate reproducibility.

Evaluation of the Validity of Digital Optical Microscopy in the Assessment of Marginal Adaptation of Dental Adhesive Interfaces

Analysis of marginal adaptation of dental adhesive interfaces using scanning electron microscopy has proven to be a powerful nondestructive method to evaluate the quality of adhesion. This methodology is, however, time-consuming and needs expensive equipment. The purpose of this study was to evaluate the possibility and efficiency of using a digital optical microscope (DOM) to perform marginal analysis and to compare it with the scanning electron microscope (SEM) analysis. Fifteen defect-free molars were selected for this study. Class V cavities were prepared and restored with resin composite, and epoxy replicas were obtained from silicone impressions of the restored teeth. Custom-made image analysis software was then used to measure the percentage of the noncontinuous margins (NCM) of each sample. To compare the DOM to the gold standard, SEM, each sample was analyzed 10 times using the DOM and three times using the SEM, by the same experienced operator. The repeatability coefficient and concordance were evaluated, and a Bland and Altman analysis was used for comparison of the two methods of measurements. To validate the DOM analysis method, an ANOVA approach (Gage R R) was used. Repeatability and reproducibility, which are two components of precision to validate the DOM analysis system, were calculated. For this, the same restorations were analyzed by two additional operators three times with the DOM. The duration of each step of the analysis using both methods was also recorded as a secondary outcome. Regarding the repeatability of each method, the Friedman test showed no statistically significant difference within the repetitions of measurements by SEM and DOM (p = 0.523 and p = 0.123, respectively). Moreover, the Bland-Altman analysis revealed a bias of 0.86 and concluded no statistically significant difference between the two methods, DOM and SEM. ANOVA evaluated DOM measurement system variation including the variances of repeatability and reproducibility that represented, respectively, 0.3% and 4% of the variance components. Reproducibility or inter-operator variability represented the principal source of variability with a statistically significant difference (p = 0.024). The time required for analysis with SEM was almost double that of DOM. The use of digital optical microscopy appears to be a valid alternative to the SEM for the analysis of marginal adaptation of dental adhesive interfaces. Further studies to evaluate the effect of training of operators in digital optical microscopy could reveal higher accuracy for this method and inter-operator agreement when experience is gained.

Assessing hemodynamics from the photoplethysmogram to gain insights into vascular age: A review from VascAgeNet

The photoplethysmogram (PPG) signal is widely measured by clinical and consumer devices, and it is emerging as a potential tool for assessing vascular age. The shape and timing of the PPG pulse wave are both influenced by normal vascular ageing, changes in arterial stiffness and blood pressure, and atherosclerosis. This review summarises research into assessing vascular age from the PPG. Three categories of approaches are described: (i) those which use a single PPG signal (based on pulse wave analysis); (ii) those which use multiple PPG signals (such as pulse transit time measurement); and (iii) those which use PPG and other signals (such as pulse arrival time measurement). Evidence is then presented on the performance, repeatability and reproducibility, and clinical utility of PPG-derived parameters of vascular age. Finally, the review outlines key directions for future research to realise the full potential of photoplethysmography for assessing vascular age.

Development and Multi-Site Assessment of a Novel Shoulder Motion Joint Simulator

Background: Multiple biomechanical shoulder simulators have been described in the literature, with a trend toward increasing complexity to better simulate clinical scenarios. Our objective was to develop an advanced, novel shoulder joint simulator and compare outcomes at two separate institutions, for a typical shoulder joint motion simulation. Methods: Identical shoulder simulators were developed & deployed at both institutions. Eight cadaveric upper extremities were tested by simulating actively controlled, arm elevation in the plane of the scapula for two sequential test conditions (intact and non-destructive simulated cuff-tear), each repeated for a total of five trials. Muscle forces and joint translations were recorded for both conditions. The intact condition was repeated following simulated cuff-tear to assess effect of testing order. Statistical analyses were aimed at assessing repeatability and reproducibility of results within specimens, between specimens, and between institutions. Results: The highest average forces were observed for the middle deltoid (233N or 32.5% body weight), followed by infraspinatus (99.0N), and posterior deltoid (93.7N) muscles. Differentiation between test conditions was unhindered by variability between repeated trials. Data from testing repeated over time, and between the two institutions were not significantly different. Conclusions: The novel shoulder simulator produced repeatable results with low trial-to-trial variation and outcomes were comparable between the two institutions. The results demonstrated a consistent response in muscle forces and humeral translation for the simulated rotator cuff tear condition. Such advanced shoulder simulators could thus be used for evaluating and optimizing surgical interventions and implant strategies.

Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information

The study and manipulation of T cell receptors (TCRs) is central to multiple fields across basic and translational immunology research. Produced by V(D)J recombination, TCRs are often only recorded in the literature and data repositories as a combination of their V and J gene symbols, plus their hypervariable CDR3 amino acid sequence. However, numerous applications require full-length coding nucleotide sequences. Here we present Stitchr, a software tool developed to specifically address this limitation. Given minimal V/J/CDR3 information, Stitchr produces complete coding sequences representing a fully spliced TCR cDNA. Due to its modular design, Stitchr can be used for TCR engineering using either published germline or novel/modified variable and constant region sequences. Sequences produced by Stitchr were validated by synthesizing and transducing TCR sequences into Jurkat cells, recapitulating the expected antigen specificity of the parental TCR. Using a companion script, Thimble, we demonstrate that Stitchr can process a million TCRs in under ten minutes using a standard desktop personal computer. By systemizing the production and modification of TCR sequences, we propose that Stitchr will increase the speed, repeatability, and reproducibility of TCR research. Stitchr is available on GitHub.