Combinatorial antibody titrations for high-parameter flow cytometry.

The objective of titrating fluorochrome-labeled antibodies is to identify the optimal concentration for a given marker-fluorochrome pair that results in the best possible separation between the positive and negative cell populations, while minimizing the background within the negative population. Best practices in flow cytometry dictate that each new lot of antibody should be titrated on the sample of interest. However, many researchers routinely use large (30+) color panels due to recent technical advancements in fluorescence-based cytometry instrumentation which quickly leads to an unmanageable number of individual titrations. In this technical note, we provide evidence that antibodies can be effectively titrated in groups rather than individually, resulting in considerable time and cost savings. This approach streamlines the process, without compromising data quality, thereby enhancing the efficiency of setting up high-parameter cytometry experiments.

Optimal concentration of potassium iodide to reduce the black staining of silver diamine fluoride.

BACKGROUND/PURPOSE: Potassium iodide (KI) is used for reducing the degree of black staining occurred after silver diamine fluoride (SDF) application. However, the optimal KI concentration remains unknown. This study aimed to identify the optimal concentration of KI that effectively reduces black staining after SDF application. MATERIALS AND METHODS: Twenty-four extracted teeth with similar pattern of carious lesions were assigned into 6 groups as follows: 1) SDF only, 2) SDF+7.5%KI, 3) SDF+10%KI, 4) SDF+15%KI, 5) SDF+20%KI, and 6) SDF+saturated KI. The KI solution was applied immediately after SDF application. Tooth images were obtained for color measurement at different time points as followed; before SDF application, immediately after SDF application, immediately after KI application, 1, 3, 7 and 14 days after SDF+KI application. The photographs were analyzed for mean gray value using the ImageJ program. RESULTS: The KI groups demonstrated a dose-dependent significant immediate reduction in black staining after KI application, except the saturated KI group. The teeth in the 20% KI group had the highest Δ mean gray value compared with other groups immediately after KI application, whereas a reduction in black staining in the saturated KI group appeared 1 day after KI application. The Δ mean gray value in all groups decreased over time. After 7 and 14 days, the reduction in black staining was not clearly different between KI groups. CONCLUSION: KI application was able to reduce the degree of black staining in a dose-dependent manner, but the subsequent color change was minimal over the period of 14 days.

Optimal concentration of mesenchymal stem cells for fracture healing in a rat model with long bone fracture.

BACKGROUND: There is still no consensus on which concentration of mesenchymal stem cells (MSCs) to use for promoting fracture healing in a rat model of long bone fracture. AIM: To assess the optimal concentration of MSCs for promoting fracture healing in a rat model. METHODS: Wistar rats were divided into four groups according to MSC concentrations: Normal saline (C), 2.5 × 106 (L), 5.0 × 106 (M), and 10.0 × 106 (H) groups. The MSCs were injected directly into the fracture site. The rats were sacrificed at 2 and 6 wk post-fracture. New bone formation [bone volume (BV) and percentage BV (PBV)] was evaluated using micro-computed tomography (CT). Histological analysis was performed to evaluate fracture healing score. The protein expression of factors related to MSC migration [stromal cell-derived factor 1 (SDF-1), transforming growth factor-beta 1 (TGF-ß1)] and angiogenesis [vascular endothelial growth factor (VEGF)] was evaluated using western blot analysis. The expression of cytokines associated with osteogenesis [bone morphogenetic protein-2 (BMP-2), TGF-ß1 and VEGF] was evaluated using real-time polymerase chain reaction. RESULTS: Micro-CT showed that BV and PBV was significantly increased in groups M and H compared to that in group C at 6 wk post-fracture (P = 0.040, P = 0.009; P = 0.004, P = 0.001, respectively). Significantly more cartilaginous tissue and immature bone were formed in groups M and H than in group C at 2 and 6 wk post-fracture (P = 0.018, P = 0.010; P = 0.032, P = 0.050, respectively). At 2 wk post-fracture, SDF-1, TGF-ß1 and VEGF expression were significantly higher in groups M and H than in group L (P = 0.031, P = 0.014; P < 0.001, P < 0.001; P = 0.025, P < 0.001, respectively). BMP-2 and VEGF expression were significantly higher in groups M and H than in group C at 6 wk post-fracture (P = 0.037, P = 0.038; P = 0.021, P = 0.010). Compared to group L, TGF-ß1 expression was significantly higher in groups H (P = 0.016). There were no significant differences in expression levels of chemokines related to MSC migration, angiogenesis and cytokines associated with osteogenesis between M and H groups at 2 and 6 wk post-fracture. CONCLUSION: The administration of at least 5.0 × 106 MSCs was optimal to promote fracture healing in a rat model of long bone fractures.

Effect of Different Concentrations of Sodium Hypochlorite on the Compressive Strength of Endodontically Treated Roots.

INTRODUCTION: The aim was to compare the compressive strength of dentin after irrigation with different concentrations of sodium hypochlorite (NaOCl). METHODS: A total of 259 extracted human tooth roots with no root caries or previous endodontic treatment were collected. They were sorted by size and length, and assigned to 7 groups (n = 37): negative control (no instrumentation, no irrigant), positive control (saline irrigant), 1%, 2.5%, 4%, 5.25%, and 10% NaOCl. A total of 13 mL NaOCl or saline (in 1-mL increments) was used per canal while being instrumented to #35 files (approximately 13 minutes). EDTAC (15%) was then used to remove the smear layer (2 minutes), followed by NaOCl as a final rinse. The roots were sectioned into 2-mm-thick disks and subjected to compression testing. RESULTS: There was a significant difference between the negative control group and all instrumented groups (P < .05). The positive control group was significantly different from the 5.25% group (P < .05) and had a low P value when compared with the 2.5% group and the other higher concentrations (P â‰… .1). When the NaOCl groups were compared with each other, there was a significant difference between 1% NaOCl and all higher concentrations (P < .05), but no significant difference when 2.5% NaOCl was compared with higher concentrations. CONCLUSION: A concentration of 1% NaOCl had the least effect on the compressive strength of dentin and this was significantly different from all higher concentrations tested. Therefore, the use of concentrations of NaOCl above 1% should be reconsidered.

The optimal dose of oxidants in UV-based advanced oxidation processes with respect to primary radical concentrations.

UV-based advanced oxidation processes (AOPs) via photolysis of precursor chemical oxidants have been of interest to numerous researchers over the past several decades due to their capacity to generate highly active radical species and interesting radical chemistry. However, applications of UV-based AOPs have been commonly optimized case by case, due to the lack of theoretical investigations on process optimization, especially on oxidant doses. In this study, a simple equation for UV/H2O2 (•OH as the sole primary reactive species (PRS)) to obtain the theoretical optimal concentration (Copt-theoretical) for H2O2 was derived (Copt-theoretical=Ab·Scε·k). The equation was then validated for its accuracy in the calculation of Copt-theoretical for H2O2 in the UV/H2O2 AOP using a well-established comprehensive kinetic model. A competition kinetics method for the measurement of scavenging capacity (Sc, the unknown parameter for the simple equation) was designed, for which nitrobenzene was employed as the probe compound and tert­butyl alcohol was introduced as the standard compound. Based on this simple equation, we calculated the Copt-theoretical of 77 environmental water samples and introduced the concept of a practical optimal oxidants dose for the UV/H2O2 AOP, while minimizing the operation costs in engineering applications. Moreover, this study mathematically proved that the simple equation obtained from UV/H2O2 could be successfully extended to other UV-based AOPs, including UV/chlorine, UV/NH2Cl, UV/S2O82-, and UV/peracetic acid. The simple equation of Copt-theoretical derived in this study may not only help to provide instructions for engineering applications, but also point out the ultimate treatment capability of each UV-based AOPs.

Improving predictive capacity of the Amino acid Derivative Reactivity Assay test method for skin sensitization potential with an optimal molar concentration of test chemical solution.

The Amino acid Derivative Reactivity Assay (ADRA) is a convenient and effective in chemico test method for assessing covalent binding of test chemicals with protein-derived nucleophilic reagents as a means of predicting skin sensitization potential. Although the original molar-concentration approach to ADRA testing was not suitable for testing multiconstituent substances of an unknown composition, a weight-concentration approach that is suitable for such substances was developed, which also led to the realization that test chemical solutions prepared to molar concentrations higher than the original 1 mM would reduce false negative results as well as enhance predictive capacity. The present study determined an optimal molar-concentration that achieves even higher predictive capacity than the original ADRA. Eight chemicals that were false negatives when tested with 1 mM test chemical solutions were retested with test chemical solutions between 2 and 5 mM, which showed 4 mM to be the optimal molar-concentration for ADRA testing. When 82 chemicals used in the original development were retested with 4 mM test chemical solutions, false negative results were reduced by four. When an additional 85 chemicals used to evaluate the weight-concentration approach to ADRA were retested, the results essentially replicated those obtained with 0.5 mg/ml test chemical solutions and gave 10 fewer false negatives than original ADRA with 1 mM solutions. A comparison of these results for 136 chemicals showed that ADRA testing with 4 mM solutions achieved a four percentage point improvement in accuracy over original ADRA and a two percentage point improvement over DPRA testing.

Dosificación de vancomicina en pacientes sometidos a diálisis intermitente. Elaboración de un protocolo de dosificación inicial / Vancomycin dosage in patients undergoing intermittent dialysis. Preparation of an initial dosage protocol

Objetivo: Revisar la dosis inicial de vancomicina en pacientes en hemodiálisis intermitente, para establecer un protocolo de dosificación que permita alcanzar una concentración pre-diálisis óptima (15-20 µg/ml) tras la dosis de carga y primera dosis de mantenimiento.Método: Estudio observacional, retrospectivo, unicéntrico en el que se incluyeron todos los pacientes en hemodiálisis (HD) que recibieron vancomicina durante la última hora de la sesión entre septiembre de 2010 y enero de 2018.Resultados: Se incluyeron 87 pacientes, considerando el valor óptimo de Cmin=15-20 µg/ml, solo el 12,6% de los pacientes tras la dosis de carga y el 18,4% tras la dosis de mantenimiento alcanzaron el valor de referencia.Conclusiones: La pauta empírica inicial de vancomicina fue insuficiente en la mayoría de los pacientes para alcanzar concentraciones óptimas desde el inicio del tratamiento, por lo que se propone un nuevo protocolo de dosificación adaptado al peso del paciente. (AU)

Objective: To review the initial dosage of vancomycin in patients on intermittent hemodialysis, to establish a dosing protocol that allows reaching an optimal pre-dialysis concentration (15-20 µg/ml) after loading dose and first maintenance dose.Method: Observational, retrospective, unicentric study in which all hemodialysis (HD) patients who received vancomycin, during the last hour of the session, between September 2010 and January 2018, were included.Results: 87 patients were included, considering the value optimal Cmin=15-20 µg/ml, only 12.6% of the patients after the loading dose and 18.4% after the maintenance dose reached the reference value.Conclusions: The initial empirical regimen of vancomycin was insufficient in most patients to reach optimal concentrations from the beginning of treatment, so a new dosing protocol adapted to the patient’s weight is proposed. (AU)

Fluoride concentration of bottled water and public water in Lebanon.

BACKGROUND AND AIMS: People in Lebanon turned to bottle water consumption because of its poor public water quality. In general, fluoride is known as dental caries preventive. A study in 1986 and two other national surveys in 1994 and 2004 showed that the concentration of fluoride in public Lebanese water was insignificant for the prevention of dental caries (less than 0.3 mg/L). The aim of the study was to measure the fluoride concentration in the highest selling and known commercial brands for bottled water in Lebanon, as well as to assess their effectiveness regarding prevention of dental caries. RESULT: Fluoride has a notable therapeutic effect but in small doses that fluoride can be found in drinking water. Analysis using an absorptiometry of 625nm and another technique using an Orion electrode of 9609 BN have shown the low content of fluoride in the Lebanese waters (less than 0.3 mg/l). Strategies have recently been evolved based on fluroide supplementation (if the results turn negative) to reduce the index of caries in LEBANON.

Optimal concentration of the transversus abdominis plane block in enhanced recovery after surgery protocols for patients of advanced age undergoing laparoscopic rectal cancer surgery.

OBJECTIVE: The transversus abdominis plane (TAP) block ameliorates visual analogue scale scores and decreases morphine requirements, but its role remains unclear. Patients of advanced age are susceptible to local anesthetic intoxication. We aimed to identify an optimal concentration that can be used in enhanced recovery after surgery (ERAS) without compromising analgesic efficacy. METHODS: In total, 120 patients aged ≥65 years undergoing laparoscopic rectal cancer surgery received general anesthesia combined with a TAP block using 0.25% ropivacaine (Group A), 0.50% ropivacaine (Group B), or 0.75% ropivacaine (Group C) in a 40-mL volume. Group D only received general anesthesia. Epinephrine, plasma cortisol, interleukin-6, and tumor necrosis factor-α were measured at baseline, skin incision, celiac exploration, and tracheal extubation. The proportions of CD4+ and CD4+/CD8+ cells were measured at baseline and postoperative days 1 and 3. RESULTS: The TAP block relieved the stress response and accelerated intestinal functional recovery as shown by significant reductions in VAS scores and anesthetic requirements. However, there was no significant difference between Groups B and C. CONCLUSION: The TAP block plays an important role in ERAS in older patients undergoing laparoscopic rectal cancer surgery, and 0.5% ropivacaine is an optimal concentration that can reduce toxicity without undermining analgesia.

Assessment of non-linear combination effect terms for drug-drug interactions.

Drugs interact with their targets in different ways. A diversity of modeling approaches exists to describe the combination effects of two drugs. We investigate several combination effect terms (CET) regarding their underlying mechanism based on drug-receptor binding kinetics, empirical and statistical summation principles and indirect response models. A list with properties is provided and the interrelationship of the CETs is analyzed. A method is presented to calculate the optimal drug concentration pair to produce the half-maximal combination effect. This work provides a comprehensive overview of typically applied CETs and should shed light into the question as to which CET is appropriate for application in pharmacokinetic/pharmacodynamic models to describe a specific drug-drug interaction mechanism.

Optimal Concentration of 2,2,2-Trichloroacetic Acid for Protein Precipitation Based on Response Surface Methodology.

For low protein concentrations containing biological samples (in proteomics) and for non proteinaceous compound assays (in bioanalysis), there is a critical need for a simple, fast, and cost-effective protein enrichment or precipitation method. However, 2,2,2-trichloroacetic acid (TCA) is traditionally used for protein precipitation at ineffective concentrations for very low protein containing samples. It is hypothesized that response surface methodology, can be used to systematically identify the optimal TCA concentration for protein precipitation in a wider concentration range. To test this hypothesis, a central composite design is used to assess the effects of two factors (X1 = volume of aqueous solution of protein, and X2 = volume of TCA solution 6.1N) on the optical absorbance of the supernatant (Y1), and the percentage of protein precipitated (Y2). Using either bovine serum albumin (BSA) as a model protein or human urine (with 20 ppm protein content), 4% w/v (a saddle point) is the optimal concentration of the TCA solution for protein precipitation that is visualized by SDS-PAGE analysis. At this optimal concentration, the Y2-values range from 76.26 to 92.67% w/w for 0.016 to 2 mg/mL of BSA solution. It is also useful for protein enrichment and xenobiotic analysis in protein-free supernatant as applied to tenofovir (a model HIV microbicide). In these conditions, the limit of detection and limit of quantitation of tenofovir are respectively 0.0014 mg/mL and 0.0042 mg/mL. This optimal concentration of TCA provides optimal condition for protein purification and analysis of any xenobiotic compound like tenofovir.

Discussion on the Suitable Concentration of Potassium Permanganate Collyria in the Clinic / 中国药房

OBJECTIVE:To provide recommendation for the clinic on the optimal application concentration of potassium permanganate collyria.METHODS:3different concentrations of potassium permanganate collyria were prepared,the local ir-ritation of2of which on eyes of115conjunctivitis cases receiving conjunctival sac irrigation and the effects of the3different concentrations of potassium permanganate collyria on the comfort degree,corneal epithelium and bacterial colonies before and after eye irrigation of18pairs of oculus normalis receiving conjunctival sac irrigation were observed.RESULTS:Of the total115conjunctivitis cases,98(about85%)complained to have severe irritation;8cases(about7%)felt unbearable because of the strong irritation.Degree of comfort decreased with the elevation of concentration of collyria,while failed to have any effect on corneal epithelium and the numbers of bacterial colonies before and after irrigation.CONCLUSION:The recommended concentration of100ml∶1.5mg of potassium permanganate collyria for the irrigation of conjunctival sac in the clinic.