Assessment of different diagnostic methods for the identification of subclinical endometritis in dairy cows with pathological puerperium and their reliability to conceive

The aim of study was to evaluate different methods for subclinical endometritis (SCE) diagnoses and their accuracy to predict pregnancy. The study was performed on 51 postpartum cross breed dairy cows with pathological puerperium. Artificial insemination (AI) was done in spontaneous estrus. Endometrium samples were taken by cytotape (CT), cervical mucus (CM) samples by Metricheck and smears for crystallization degree were obtained at AI day. Blood serum samples for progesterone (P4) concentration determination were taken at AI day and 17 days later. Cows were examined by ultrasound 35 days after AI to confirm pregnancy. Percent (%) of polymorphnuclear leukocytes (PMNs) taken by CT were expressed by ROC curve (with cut-off point set at 1%). Out of the total of 51 cows involved in the study, 13.73% true positive and 41.17% true negative cows were found (54.90%). The content of CM sampled with Metricheck was altered in 11 cows (21.57%), and one of them was pregnant (1.96%). An average macroscopic CM value for pregnant cows was 0.06±0.24 vs. 0.39±0.79 for non-pregnant (p<0.05). An average value of CM crystallization in pregnant cows was 2.50±1.04 vs. 1.85±1.15 in non-pregnant (p<0.05). P4 concentration was not statistically different at AI day, but was statistically significantly higher 17 days later in pregnant cows (p = 0.002). CT alterations may be a reliable diagnostic tool for SCE diagnosis, while macroscopic CM alterations may be used for CE detection. P4 determination is beneficial in pregnancy diagnosis while CM crystallization degree is helpful in determination of best time for AI.

Influence of the chirality of carbon nanodots on their interaction with proteins and cells

Carbon nanodots with opposite chirality possess the same major physicochemical properties such as optical features, hydrodynamic diameter, and colloidal stability. Here, a detailed analysis about the comparison of the concentration of both carbon nanodots is carried out, putting a threshold to when differences in biological behavior may be related to chirality and may exclude effects based merely on differences in exposure concentrations due to uncertainties in concentration determination. The present study approaches this comparative analysis evaluating two basic biological phenomena, the protein adsorption and cell internalization. We find how a meticulous concentration error estimation enables the evaluation of the differences in biological effects related to chirality.

FPCount protocol - Full protocol v1

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression/purification using Thermo's HisPur Cobalt Resin. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 5. Gel1: Verification of Expression/Fractions 6. Purification 7. Gel2: Verification of Purification 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 11. Quantification of FP concentration (part2) 12. Protein Storage 13. Calibration of Plate Reader

FPCount protocol - in-lysate (purification free) protocol v1

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression and production of cell lysates. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. This in-lysate version of the protocol uses the ECmax protein quantification protocol of FPs in lysates and does not require His-tag purification of the FPs. Note that it is only suitable for FPs with entries in FPbase. If you want to verify or validate results, it's recommended you follow the 'short' protocol, which requires FP purification, or the 'complete' protocol, which requires FP purification and compares three protein quantification methods. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 12. Protein storage 13. Calibration of Plate Reader

FPCount protocol - Short protocol v1

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression/purification using Thermo's HisPur Cobalt Resin. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. This short version uses the ECmax protein quantification protocol, and is only suitable for FPs with entries in FPbase. If you want to verify or validate results, it's recommended you follow the complete protocol, which describes three protein quantification methods. The short protocol also skips the SDS-PAGE steps. If you require these, please see the complete protocol. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 6. Purification 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 12. Protein storage 13. Calibration of Plate Reader

System Suitability Testing of the Method Developed to Measure the Active Concentration of a Fab Fragment Via Surface Plasmon Resonance

SPR methods are increasingly accepted by the biopharmaceutical industry in quality control (QC), R&D and clinical research. However, there are few reports on method validation and system suitability testing (SST), in particular on concentration determination methods related to the potency of the drug. System compliance must be demonstrated with certain tests in order, for the method validation to be ensured, as agreed by the scientific community and the industry, and specified in the guidelines. To contribute to the alleviation of the deficiency in this matter, we developed a system suitability testing of an SPR-based active concentration measurement method using Fab-fragment as a model. High amounts of anti-FAB1 were captured onto sensor chip surfaces via biotin/neutravidin interaction. The unknown concentration of FAB1 measured via calibration curve dependent concentration measurement. Controls were run between unknown samples to check the reliability of the method. The standard four-parameter logistic equation was used to determine the unknown concentration. CV % of samples and Difference % of controls form standards were calculated from obtained data. The CV % and Diff. % values were less than or equal to 10 % at each parameter. The 100 % level of unknown sample measured as 105 %. Obtained CV % values and repeated analyses showed that the system and method are suitable for the concentration analysis of the FAB1 fragment. The system suitability parameters evaluated in the study can be applied for all SPR analyzes. Resumen. Los métodos SPR (Surface Plamon Resonance) son cada vez más aceptados por la industria biofarmacéutica en el control de calidad (QC), investigación y desarrollo (R & D) e investigación clínica. Sin embargo, existen pocos informes sobre la validación de los métodos y las pruebas de idoneidad del sistema (SST), en particular sobre los métodos de determinación de la concentración relacionados con la potencia del fármaco. La idoneidad del sistema debe demostrarse para garantizar la validación del método, según la normatividad acordada por la comunidad científica y la industria. Con el fin de contribuir al alivio de la deficiencia en este asunto, se desarrolló una prueba de idoneidad del sistema con un método de medición de concentración activa basado en SPR utilizando el fragmento Fab como modelo. Se capturaron grandes cantidades de anti-FAB1 en las superficies del chip sensor mediante la interacción biotina/neutravidina. Se estableció la concentración desconocida de FAB1 a través de la determinación de la concentración dependiente de la curva de calibración. Se ejecutaron controles entre muestras desconocidas para comprobar la fiabilidad del método. Se utilizó una ecuación estándar de cuatro parámetros para determinar la concentración desconocida. A partir de los datos obtenidos se determinó el porcentaje de CV de las muestras y la diferencia respecto a los controles. Los valores de % CV y % Diff. fueron menores o iguales al 10 % en cada parámetro. Los valores de % CV obtenidos y los análisis repetidos mostraron que el sistema y el método son adecuados para el análisis de concentración del fragmento FAB1. Los parámetros de idoneidad del sistema evaluados en el estudio se pueden aplicar a todos los análisis SPR.

Accumulation of the Major Components from Polygoni Multiflori Radix in Liver and Kidney after Its Long-Term Oral Administrations in Rats

Although Polygoni Multiflori Radix (PMR) has been widely used as a tonic and an anti-aging remedy for centuries, the extensively reported hepatotoxicity and potential kidney toxicity hindered its safe use in clinical practice. To better understand its toxicokinetics, the current study was proposed, aiming to evaluate the biodistributions of the major PMR components including 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside (TSG), emodin, emodin-8-O-β-D-glucopyranoside (EMG) and physcion as well as their corresponding glucuronides following bolus and multiple oral administrations of PMR to rats. Male Sprague-Dawley rats received a bolus dose or 21 days of oral administrations of PMR concentrated granules at 4.12 g/kg (equivalent to 20.6 g/kg raw material). Fifteen minutes after bolus dose or the last dose on day 21, rats were sacrificed and the blood, liver, and kidney were collected for the concentration determination of both parent form and glucuronides of TSG, emodin, EMG, and physcion by HPLC-MS/MS. Among all the tested analytes, TSG, EMG, EMG glucuronides in liver and TSG, EMG, as well as all the glucuronides of these analytes in the kidney demonstrated the most significant accumulation after multiple doses. Moreover, the levels of the parent analytes were all significantly higher in liver and kidney in comparison to their plasma levels. Strong tissue binding of all four analytes and accumulation of TSG, EMG, and EMG glucuronides in the liver and TSG, EMG, as well as the glucuronides of all four analytes in the kidney after multiple dosing of PMR were considered to be associated with its toxicity.