Agilent Technologies Research Articles

Study on content of flavonoids and antioxidant activity of the raw materials of Parthenocissus quinquefolia (L.) Planch.

The aim of this work was to determine the component composition and quantitative content of flavonoids, including catechins, in the leaves, shoots and fruits of Virginia creeper (Parthenocissus quinquefolia (L.) Planch.) for further standardization of raw materials and establishing antioxidant activity of their water-ethanol extracts. Materials and methods. The leaves, shoots of Parthenocissus quinquefolia were collected during the phase of mass flowering in July 2020 and fruits were collected during full ripeness (September – October 2020) in Lisnyky village of Obukhiv district of Kyiv Oblast (Ukraine). Determination of the component composition and quantitative content of flavonoids, including catechins, was carried out by the method of high-performance liquid chromatography (HPLC) on an Agilent Technologies 1200 liquid chromatograph. Identification and quantitative analysis were carried out using standard solutions of flavonoids (rutin, quercetin-3-b-glycoside, naringin, neohesperidin, quercetin, naringenin, kaempferol, luteolin, apigenin) and catechins (pyrocatechin, catechin, epicatechin, epicatechin gallate and halocatechin). Antioxidant activity was determined by the spectrophotometric method at 347 nm by the ability to inhibit the autooxidation of adrenaline in vitro.Results. As a result of the study, 9 phenolic compounds were identified: rutin, quercetin, quercetin-3-b-glycoside, naringin, epicatechin, catechin, gallocatechin, epicatechin gallate. It was determined that rutin, quercetin, epicatechin, and catechin predominate in the leaves, shoots, and fruits of Virginia creeper.It was established that the extracts of leaves with shoots and fruits of Virginia creeper show a pronounced antioxidant activity due to their ability to inhibit autooxidation of adrenaline in vitro.Conclusions. The high content of rutin, quercetin, epicatechin and catechin in the leaves, shoots and fruits of Virginia creeper has scientific interest, due to discovered compounds have a valuable pharmacological effect. The obtained results can be used for the standardization of raw materials of Virginia creeper, and differences in the quantitative content of halocatechin in different types of raw materials, as well as the presence of naringin in leaves and shoots and luteolin in the fruits of Virginia creeper as diagnostic features of this species raw material.The results of the study of antioxidant activity confirm the prospects of using this raw material for the creation of new drugs with antioxidant activity

Studies on High-Resolution Airborne Synthetic Aperture Radar Image Formation with Pseudo-Random Agility of Interpulse Waveform Parameters

By means of alteration of the transmitted linear frequency modulation (LFM) signal waveform parameters, such as pulse width or chirp rate, initial phase, pulse repetition interval (PRI), and chirp rate polarity at every position of synthetic apertures, the pseudo-random agility technology of interpulse waveform parameters in airborne Synthetic Aperture Radar (SAR) actively increases the complexity and uncertainty of radar waveforms. This technology confuses jamming interception receivers, thus improving its anti-interference ability for active coherent jamming, which is one of the main research interests of airborne SAR technology. But the pseudo-random agility technology for interpulse waveform parameters faces certain challenges of large computation and complex system design, which need to be further studied and solved. To address these issues, a processing scheme of high-resolution SAR image formation which is appropriate for agile interpulse waveform parameters is proposed in this paper. This method can deal with multiple agile parameters, not only single ones as in most existing literature. Its computation load is nearly comparable to that of traditional SAR image formation with constant waveform parameters. The high-resolution SAR imaging results obtained by processing SAR raw data with agile interpulse waveform parameters demonstrate the effectiveness of the proposed method. In addition, real SAR images with resolutions of 0.5 m and 0.15 m, which are rarely found in the public literature, are shown under the circumstance of randomly changing the transmitted wideband LFM signal pulse parameters one by one.

Method for the molecular and quantitative identification of oocytes and their developmental stage in teleost fish gonads

Fish display diverse reproductive strategies and their gametogenesis is influenced by numerous genetic, physiological and environmental factors. The analysis of 5S rRNA expression levels in gonads has been proposed as useful method for the molecular identification of the presence of oocytes in fish tissues. The present method provides an easy and unbiased approach to analyse the expression of tRNAs and 5S rRNA in teleost gonads and stablish the presence and developmental stage of oocytes. Total RNA extracted from gonads is analysed through capillary electrophoresis in a Bioanalyzer 2100 (Agilent Technologies) using Small RNA Assays. Electropherograms allow quantifying the concentrations of tRNAs, 5S rRNA and 5.8S rRNA per sample and calculate their tRNA/5.8S rRNA and 5S/5.8S rRNA indices. Both indices clearly differentiate ovaries from testes and can be used to identify testes that present oocytes due to exposure to environmental xenoestrogens. The tRNA/5.8S and 5S/5.8S indices show the highest values in ovaries in previtellogenic stage, values decreasing as they advance towards maturity.•Detailed molecular method to sex fish and quantitatively identify the maturity stage of females.•tRNA levels in gonads can help in the study of teleost reproduction (female fecundity assessment, molecular gonad sexing) and environmental health assessment.

GC-MS and FTIR analyses of bioactive compounds present in ethanol leaf extract of Sida acuta from Imo State, Nigeri

Medicinal plants are vital to human and community health. This work aimed at using gas chromatography-mass spectrometry (GC-MS) and Fourier transform infrared (FTIR) to determine the phytochemical contents of ethanol leaf extract of Sida acuta from Imo State, Nigeria. Leaves of Sida acuta were collected, washed, air-dried, and pulverized. Pulverized leaves were extracted in 100% ethanol at 70°C in a water bath. Bioactive compounds from the extract were analyzed using Agilent Technologies GC equipment and Buck Scientific M530 USA FTIR. The GC-MS Screening of the plant’s extract revealed the presence of seventy-one (71) phytocompounds including gamma-terpinene (1.11%), p-cymene (0.29%), linalool (2.45%), alpha-cubebene (0.11%), alpha-copaene (1.34%), aromadendrene (6.21%), humulene (1.90%), alpha-selinine, guaiol (0.58%), phytol (0.11%), butyl-2-ethylhexylphthalate (0.32%), 1,2-benzenedicarboxylic acid (0.40%), 1-docosene (0.36%), 1,4-dichlorobenzene (0.22%), (e)-.beta.-farnesene (10.87%), and piperine (0.61%), which are reported to exhibit various biological activities. The FTIR analysis of the plant extract revealed functional groups such as Primary (1°) and Secondary (2°) amines, alkanes, alkenes, alkyl halides, carboxylic acids, allenes, aromatics, aliphatic amines, esters, ethers, phenols, and aldehydes. Cross-referencing the phytocompounds with literature revealed a variety of biological functions, including antibacterial, antifungal, anti-inflammatory, anticancer, antimutagenic, antioxidant, antitumor, and others. It may be concluded that Sida acuta contains bioactive compounds with anti-microbial, anti-inflammatory, anti-oxidant, hepatoprotective, and anti-carcinogenic properties, with (E). beta.-farnesene being the most abundant (10.87%).

Error-Corrected Next-Generation Sequencing Provides a Comprehensive Overview of the Subclonal Mutation Landscape and Its Prognostic Implications in Juvenile Myelomonocytic Leukemia

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myelodysplastic/myeloproliferative neoplasm characterized by mutations in genes activating the RAS pathway ( PTPN11, NF1, NRAS, KRAS, and CBL). Approximately 20% of patients with JMML have secondary mutations in SETBP1 and/or JAK3, and recent droplet digital PCR (ddPCR) analyses revealed that mutations in these two genes, including very rare mutations (variant allele frequency [VAF] < 0.05) that are difficult to detect by conventional next-generation sequencing (NGS), are associated with poor prognoses. Meanwhile, the landscape of low VAF subclonal mutations of other genes in patients with JMML has not been adequately evaluated. Therefore, we utilized error-corrected NGS, which can reduce the systematic sequencing error rate of conventional NGS by incorporating molecular barcodes (MBCs). Methods: We performed error-corrected NGS in 93 patients with JMML and 11 patients with JMML-like myeloproliferative disorder with Noonan syndrome (NS/MPD) using a small panel associated with myeloid malignancies. Samples for error-corrected NGS were prepared using a SureSelect XT HS2 DNA Library Preparation Kit (Agilent Technologies), including unique dual index and duplex MBCs. A unique MBC is incorporated into each DNA fragment, and the incorporated MBC can differentiate true and false duplications, allowing for highly accurate sequencing that can detect mutations with VAF > 0.001. We used the HiSeq X platform (Illumina) and SureCall v.4.2 application (Agilent Technologies) for analysis. In addition, DNA methylation analysis was performed on the same cohort using the Infinium HumanMethylation 450K BeadChip and/or Digital Restriction Enzyme Analysis of Methylation as previously described (Murakami et al. 2018;131:1576-1586, Kitazawa et al. Blood Adv. 2021;5:5507-5518). This study was approved by the Ethics Committee of the Nagoya University Graduate School of Medicine. Results: In the cohort of 104 patients with JMML or NS/MPD, 160 mutations were identified, of which 30 (19%) had VAF < 0.05 (eight in JAK3, seven in NF1, five in ASXL1, three in NRAS, two each in PTPN11 and SOS1, and one each in SETBP1, SH2B3, and ZRSR2). Subclonal mutations in SETBP1 p.D868N and JAK3 p.R657Q identified by error-corrected NGS were confirmed using ddPCR, and consistent results were obtained for both assays (R 2 = 0.9977 and 0.9919 respectively). Of 93 patients with JMML, 30 (32%) had subclonal or clonal secondary mutations, whereas no such mutations were detected in patients with NS/MPD ( P = 0.031). Of 104 patients, 23 (22%) had secondary mutations with VAF ≥ 0.05 (Major group), 7 (7%) only had secondary mutations with VAF < 0.05 (Minor group), and the remaining 74 (71%) had no secondary mutations (Negative group). We assessed the associations of subclonal mutations detected by error-corrected NGS with the DNA methylation profile, the most important prognostic factor in JMML. Whereas 22 of 23 (96%) and 5 of 7 (71%) patients in the Major and Minor groups, respectively, had high methylation (HM) profiles, only 20 of 74 (27%) patients in the Negative group featured HM profiles ( P < 0.001). The 4-year overall survival (OS) rates were significantly lower in the Major (52.2% [95% confidence interval {CI} = 30.5%-70.0%], P = 0.01) and Minor groups (42.9% [95% CI = 9.8%-73.4%], P = 0.03) than in the Negative group (76.3% [95% CI = 64.6%-84.5%]). In a subgroup analysis of 30 patients with secondary mutations, patients with secondary mutations in RAS pathway genes (n = 18) had significantly lower 4-year OS rates than those with secondary mutations in other genes (n = 12; 32.4% [95% CI = 12.7%-54.0%] vs. 75.0% [95% CI: 40.8%-91.2%], P = 0.047). Conclusions: We successfully performed error-corrected NGS to assess comprehensive subclonal secondary mutational profiles, including very low VAF variants. The presence of subclonal mutations, particularly in RAS pathway genes, was associated with poor OS. These findings provide important information for appropriate risk stratification, which will contribute to the implementation of precision medicine for patients with JMML.

A Novel, Rapid, and Accurate Quantitative Hydroxyurea Assay

Introduction: Hydroxyurea is a highly effective treatment for children and adults with sickle cell anemia (SCA) and is now considered standard-of-care therapy in high-resource countries. There is wide interpatient variability in the optimal dose, which is defined as the daily dose that provides maximal induction of protective fetal hemoglobin to decrease erythrocyte sickling while causing only mild marrow suppression. The time and cost of hydroxyurea dose titration are barriers to its widespread use in low-resource settings. The titration process required to identify an optimal dose can be abbreviated by measuring individualized hydroxyurea pharmacokinetics (PK) to guide personalized precision dosing, which could be especially useful in low-resources settings with limited laboratory monitoring. However, measurement of hydroxyurea in serum and other physiological fluids is not commonly performed during routine clinical treatment. Current methodology to quantify hydroxyurea concentrations requires complex and time-consuming chemical derivatization followed by detection by colorimetric spectrophotometry, high performance liquid chromatography (HPLC), or liquid chromatography mass spectrometry (LC-MS). To consider performing PK-guided dosing in low-resource settings, a simple, accurate, and efficient hydroxyurea assay is needed. Methods: A commercially available open-system HPLC unit for hemoglobin separation (SmartLifeLC, PolyLC Inc, Columbia MD) was equipped with a 3.5-micron, 4.6 mm × 50 mm Zorbax Eclipse XDB-C18 column with matching guard column (Agilent Technologies, Santa Clara, CA); mobile phases of 46% methanol and 100% acetonitrile; and a 200-600nm multi-wavelength detector. The chemical derivatization process was modified from previously published techniques that use xanthydrol (Sigma-Aldrich, St. Louis MO) for detection of primary amides. N-methylurea (Sigma-Aldrich) was included as an internal standard. Hydroxyurea (Sigma-Aldrich) was diluted in whole blood collected from healthy controls to create a calibration curve encompassing the typical pharmacological range of hydroxyurea at 80, 40, 20, 10, and 5 mg/mL. Quality Control (QC) samples were prepared at 50, 25, 12.5, and 6.25 mg/mL to validate the calibration curve before testing patient samples. Blood was applied to 20mL volumetric absorptive microsampling devices (Mitra, Trajan Scientific and Medical, Torrance CA) and dried for future elution and analysis. HPLC results were compared to LC-MS results to assess accuracy. Results: Blood was eluted from the Mitra microsampling device and derivatized in a xanthydrol solution before HPLC analysis. Using a 10-minute run with 210nm wavelength detection, the xanthydrol-modified hydroxyurea reproducibly yielded a linear calibration curve with r 2 >0.95 (Figure), and the actual concentrations were within 20% of the theoretical values. Repeated testing of control and patient samples revealed a typical intra-day coefficient of variation <15% and inter-day coefficient of variation <20%. The agreement between nominal values and assay results determined for a wide range of QC samples from both methods exceeded the FDA-recommended threshold of 67% for chromatographic assay validation with 86% agreement on LC-MS and 85% with the new method. The entire elution and derivatization procedure could be completed using dried Mitra blood samples in less than 60 minutes. Conclusions: Quantitative hydroxyurea measurement of blood or serum can now be accomplished using a novel, rapid, HPLC technique that features xanthydrol-based derivatization of hydroxyurea, an internal standard, and UV wavelength detection. This technique is accurate, reproducible, and relatively simple, and thus deemed suitable for hydroxyurea concentration measurements needed for PK-guided dosing calculations for children with SCA living in low-resource settings. This novel technique will be added to the multi-country Realizing Effectiveness Across Continents with Hydroxyurea (REACH, NCT01966731) trial that will feature PK-guided precision dosing for young children with SCA starting hydroxyurea treatment.

Analysis of Metabolic Mechanism of CD19CAR-T with CD79A/CD40 Co-Stimulatory Domain Based on Comparison with Established CAR-Ts

Introduction: Anti-CD19 chimeric antigen receptor T cell (CD19CAR-T) therapy has achieved significant clinical outcomes and changed treatment strategy in B-cell malignancies. However, recurrence after CAR-T therapy remains a major obstacle due to poor peak expansion and short persistence of CAR-T cells. We have reported that CD19CAR-T incorporating a co-stimulatory domain consisting of CD79A and CD40 (79A40CAR) provided superior anti-tumor efficacy and survival compared to CD19CAR-T with CD28 or 4-1BB (CD28CAR/4-1BBCAR) in mice xenograft models. 79A40CAR exhibited sustained NF-κB phosphorylation and superior in vivo efficacy compared to the CD28CAR or 4-1BBCAR. (Molecular Therapy 2021, Julamanee J et al.) In this study, we aimed to clarify the molecular mechanism of 79A40CAR by comparing with CD28/4-1BBCAR in terms of T-cell metabolism. Methods: CD8+T cells were isolated from PBMCs of healthy donors and retrovirally transduced with either CD28IC-, 4-1BBIC-, or 79A40IC-CD19CARs. CD28CAR, 4-1BBCAR, or 79A40CAR were cocultured with 100 Gy irradiated NALM6 at E:T ratio = 1:5, respectively. Repeated antigen stimulations (Ag-stim) were performed, and day 7 after each stimulation was subjected to analysis. RNA-seq, expansion capacity, T-cell phenotype, and metabolic evaluation using a seahorse flux analyzer (Agilent Technologies) were performed. After Ag-stim, CD45RA negative CD62L positive and CD45RA negative CD62L positive CAR-T cell fractions were sorted as central memory (T CM) and effector memory (T EM), respectively. T CM and T EM received Ag-stim. After Ag-stim, we performed evaluation of mitochondrial functionality with MitoTracker based on mitochondrial mass and membrane potential. Results: All CD19CAR genes were successfully transduced into primary CD8+ T-cells. To understand the differences between co-stimulatory domains, we first confirmed bulk RNA-seq, expansion capacity, and T-cell phenotype. After Ag-stim, 79A40CAR showed enhancement of the NF-κB pathway. We detected few DEGs when 79A40CAR compared to 4-1BBCAR, however, we detected 162 upregulated DEGs when 79A40CAR compared to CD28CAR. Principal component analysis of gene expression demonstrated similarity between 4-1BBCAR and 79A40CAR. We confirmed that 79A40CAR had superior expansion capacity than CD28, or 4-1BBCAR after repeated Ag-stim. PD-1 and Tim-3 expression in 79A40CAR was lowest after repeated Ag-stim. In the evaluation of T-cell differentiation, 4-1BB and 79A40CAR showed an earlier transition to the effector phenotype compared to CD28CAR after Ag-stim. To evaluate differences in terms of T-cell metabolism, we performed metabolic analysis using a seahorse flux analyzer. After repeated Ag-stim, 4-1BB and 79A40CAR showed a predominant oxidative phosphorylation metabolic pathway (Fig1A) and higher spare respiratory capacity (SRC), which has been correlated with mitochondrial functionality (Fig1B), than CD28CAR. After repeated Ag-stim, 4-1BB and 79A40CAR demonstrated higher ECAR than CD28CAR. Because we observed differences in the expression of T-cell differentiation phenotype, we performed cell sorting to evaluate T-cell metabolism of each differentiation phenotype. After applying repeated Ag-stim to T CM and T EM-derived CD19CAR-T, mitochondrial functionality was evaluated using Mitotracker. T CM- (Fig1C) and T EM- (Fig1D) derived 4-1BB and 79A40CAR had more functional mitochondria and fewer dysfunctional mitochondria compared to CD28CAR. Conclusion: 79A40CAR seems to preserve the mitochondrial oxidative phosphorylation-dominant metabolic pathway and supply energy demands for a longer period, especially when compared to CD28CAR. 79A40CAR showed functionally very similar to 4-1BBCAR. We showed that CD19CAR-Ts had different metabolic pathway according to the different co-stimulatory domains. To reveal additional metabolic pathway differences between 4-1BBCAR and 79A40CAR, we are now conducting proteomics analysis.

A New Method for Studying the Kinetics of the Release of Poorly Soluble API from Solid Oral Dosage Forms on the Example of Quertin®

In this paper, it is proposed to consider a new method developed for studying the kinetics of release of substances that are poorly soluble in aqueous media on the example of quercetin. The study object was the drug containing plant bioactive components – Quertin® chewable tablets, 40 mg, 3 blisters, 10 pcs – produced by PJSC SIC “Borshchahivskiy CPP”. An Agilent 1290 Infinity II LC System liquid chromatograph with an Agilent 6530 mass selective detector (Agilent Technologies) was used for the analysis. Solubility profiles were studied in accordance with the requirements of the Biopharmaceutical Classification System (BCS). The solubility limit of the substance in the media studied has been determined. A method for the quantitative determination of quercetin in test media in the range of specified concentrations with high sensitivity and selectivity has been developed. The dissolution of Quertin® chewable tablets in 3 different aqueous dissolution media with pH 1.2, pH 4.5 and pH 6.8 was studied, the dissolution profiles were compared, and the f2 factor was calculated. This factor is a criterion for evaluating the study by comparing dissolution kinetics with in vivo results. The results obtained indicate that the approach proposed to studying the kinetics of the release of substances that are sparingly soluble in aqueous solutions allows us to correctly assess the release of such substances in accordance with the requirements of the BCS. The method developed has been validated.

PSIII-4 Effect of Photobiomodulation Treatment on Atp Concentrations in Bovine Oocytes During Maturation

Abstract Oocyte cytoplasmic maturation is critical in determining the success of subsequent embryo development. It has been proposed that light in the red-to-near infrared optical region (~600–1000 nm) enhances cellular metabolic activity through activation of the mitochondrial respiratory chain, increasing intracellular ATP concentrations, and has the potential to modify the oocyte metabolome. Previous research indicated a greater cleavage rate and similar blastocyst rates for bovine oocytes treated with photobiomodulation at hours 16 and 20 from the start of maturation compared with control. For the present study, two separate experiments were conducted to determine ATP concentration in oocytes during maturation after photobiomodulation treatment. A standardized in vitro maturation (IVM) protocol and commercial media (IVF Bioscience) were used. Briefly, bovine ovaries were obtained at a local slaughterhouse, cumulus oocyte complexes (COCs) were collected by aspiration of small (2 to 6 mm) follicles. For experiment 1, compact COCs were cultured in in vitro maturation media (n = 30/well) and treated with no light (control); light (660-665nm) for 10 minutes at hour 16 from start of maturation (L-16) and light for 10 minutes at hour 20 from the start of maturation (L-20). At hour 24 of in vitro maturation, COCs were vortexed to remove cumulus, stained with BioTracker ATP-Red Live Cell Dye (Sigma-Aldrich SCT045), imaged with Cytation-1 (Agilent Technologies Inc.) in 6 replicates. For experiment 2, COCs were cultured in in vitro maturation media (n = 40/well) and treated with no light (control) and light (660-665nm) for 10 minutes at hour 16 from start of maturation (L-16). At hours 18, 20, 22 and 24 of in vitro maturation, COCs were vortexed to remove cumulus, stained with BioTracker ATP-Red Live Cell Dye, and imaged with Cytation-1 in 4 replicates. For experiment 1, one-way ANOVA and Student’s T-Test were performed (JMP software 16.0); in experiment 2, each well was considered as an experimental unit in a model of repeated measures, main effects of time and treatment, blocked by replicate (Mixed procedure, SAS Institute Inc.). In experiment 1, there was a significant (P < 0.05) effect of treatment; ATP concentrations were greater in L-16 than control, and L-20 was intermediate. ATP concentrations were 19,579.3a ± 596.5, 21,652.0b ± 602.7 and 19,710.9ab ± 570.6 relative fluorescence units (RFU) for control, L-16, and L-20 respectively. In experiment 2, there was a significant (P < 0.05) effect of treatment and no significant effect of time. Overall ATP concentrations were 25,668 ± 894.6 and 28,332 ± 894.6 RFU for control and L-16 respectively. In conclusion, photobiomodulation treatment with LED 660-665nm for 10 minutes at hour 16 from start of oocyte maturation increases intracellular ATP concentrations from hour 18 to 24 of maturation.

Quantitative determination of cyclic guanosine monoposphate (c-GMP) in rat tissues using liquid chromatography and tandem mass spectrometry

Relevance. Cyclic 3′,5′-guanosine monophosphate is a secondary intracellular messenger that plays a key role in many physiological processes.Quantitative determination of the level of c-GMP in the tissues of laboratory animals is an urgent task of experimental pharmacology and physiology.Purpose of the study. Development of a method for the quantitative determination of cyclic guanosine monophosphate in various tissues of rats using high performance liquid chromatography with mass spectrometric detection.Methods. The biomaterial was homogenized with deionized water. Extraction of c-GMP from homogenates was performed with methanol, acyclovir was used as an internal standard. Detection of c-GMP and acyclovir was performed using a Sciex QTrap 3200MD mass spectrometer, chromatographic separation was performed using an Agilent Technologies 1260 Infinity II HPLC. The mobile phase was methanol and deionized water.Results. Detection of c-GMP was performed by MRM transitions m/z 346.2/152.1; 346.2/135.1, chromatographic determination of c-GMP was performed in reverse phase mode on an Agilent InfinityLab Poroshell 120 EC-C18 4.6×100 mm, 2.7 µm column. The retention time of c-GMP and acyclovir was 7.85 and 7.45 minutes, respectively, the total duration of the chromatographic analysis was 12 minutes. The analytical range of the procedure for determining c-GMP in homogenates was 0.5–1000.0 pmol/ml. The content of c-GMP in the tissues of intact Wistar rats was analyzed using the developed method.Conclusion. The developed bioanalytical HPLC-MS/MS method for the quantitative determination of c-GMP fully complies with the validation requirements. The metrological characteristics of the method make it possible to estimate the content of c-GMP in various tissues of rats with high accuracy.

Telomere Length in Survivors of Childhood Hematologic Malignancies

Background: Telomeres are involved in maintenance of genomic stability and DNA repair. Telomeres of hematopoietic cells shorten with age, and accelerated telomere shortening is seen with replicative stress, such as during administration of chemotherapy for the treatment of cancer. We aimed to analyze leukocyte relative telomere length (RTL) among survivors of childhood hematologic malignancies, and to evaluate the associations of RTL with disease characteristics, treatment, and chronic health conditions (CHCs). Method: This study was conducted at a single center in a retrospective cohort of survivors of childhood cancer diagnosed between 2000 and 2021 with a longitudinal follow-up of ≥1 year after cessation of treatment. The peripheral blood samples were collected between June 2022 and January 2023. This study enrolled survivors of childhood hematologic malignancies with the following criteria: diagnosis before the age of 19 years, continuous maintenance of clinical remission, compliance with regular clinical follow-up, and cessation of treatment more than one year before the start of the study. Considering that telomeres shorten with age, this study limited participants to those between 18 and 35 years of age at blood sampling. The telomere PNA Kit/Fluorescein isothiocyanate (FITC) for flow cytometry (Agilent Technologies, Singapore) was used for flow FISH for the measurement of leukocyte RTL. Result: Forty-three survivors of childhood hematologic malignancies with a median follow-up period of 73 months were recruited. Median age at diagnosis and at DNA sampling were 13.8 years (range, 6-18) and 22.6 years (range, 15-34), respectively. Survivors had leukemia (57.1%), lymphoma (31.4%), and myelodysplastic syndrome (11.4%). RTL in survivors of pediatric hematologic malignancies was not significantly shorter than the predicted value for age-matched references. There was no difference in RTL according to disease subtypes ( p=0.35). Survivors who experienced relapse had significantly shorter RTL than those who did not (7.6 ± 1.0 vs. 12.1 ± 5.0, p=0.009). Those who underwent allogeneic hematopoietic stem cell transplantation (HSCT) showed significant shortening of the RTL compared with those who did not (9.4 ± 4.1 vs. 12.6 ± 5.1, p=0.039). Total body irradiation (TBI) during pre-transplant conditioning did not influence the RTL (9.5 ± 3.8 with TBI vs. 12.1 ± 5.1 without TBI, p=0.117). Among the 15 patients who underwent allogeneic HSCT, those who developed acute graft-versus-host disease (GVHD) of grade II or higher had significantly shorter RTL than those who did not (6.8 ± 1.9 vs. 11.7 ± 4.1, p=0.013). Patients with chronic GVHD tended to have shorter RTL than those without GVHD, but the p-value was not statistically significant (7.1 ± 2.8 vs. 10.9 ± 4.2, p=0.088). Seventeen patients had CHCs, with 13 having grade I and 4 having grade II. Patients with grade II CHCs had significantly shorter RTL than those without CHCs or with grade I CHCs (7.1 ± 1.6 vs. 11.5 ± 5.3, p=0.041). When the influence of multiple CHCs on RTL was examined, survivors with ≥2 CHCs had shorter RTL, but this difference was not significant ( p=0.062). Conclusion: The overall RTL in survivors of childhood hematologic malignancy was not significantly shorter than the predicted values for age-matched references. However, patients who received allogeneic HSCT showed RTL attrition compared to those who did not receive HSCT. In addition, those who had GVHD showed augmented RTL attrition. Patients with grade II CHCs had shorter RTL compared to patients without or with grade I CHCs. Although there is currently no consensus on the extent and clinical significance of accelerated shortening of the RTL, the RTL may be a potential biomarker for determining CHCs in childhood hematologic malignancies.

PD-L1 IHC 22C3 pharmDx Demonstrates Precision and Reproducibility in Triple-negative Breast Cancer

In the United States, the PD-L1 IHC 22C3 pharmDx indications for use have expanded to include the assay’s use as an aid in identifying triple-negative breast cancer (TNBC) patients for treatment with pembrolizumab (KEYTRUDA®). Analytical validation for TNBC was conducted by Agilent Technologies in support of the device performance and FDA-approval. Analytical validation studies included device precision (inter-instrument, inter-operator, inter-day, inter-lot, and intra-run) and external reproducibility (inter- and intra-site, inter- and intra-observer) using the Combined Positive Score (CPS) ≥ 10 cutoff. All precision and external reproducibility studies achieved confidence interval lower bound values of greater than 85% for positive, negative, and overall percent agreement. These analytical validation studies demonstrate that PD-L1 IHC 22C3 pharmDx is precise and reproducible in evaluating PD-L1 protein expression at a CPS ≥ 10 cutoff when used for testing TNBC tissues.

Chemicals in Cigarette Flavor Capsules From Guatemala and Mexico.

The global market share for cigarettes with flavor capsules has grown exponentially over the last decade, particularly in Latin America. When smoking, consumers crush a liquid capsule in the filter that flavors the smoke. Little is known about the chemical constituents of the liquids in capsules or their potential health risks. Based on consumer data and availability in Mexico and Guatemala, 31 flavor capsule brands were purchased (19 and 12, respectively) in 2020. Since some cigarettes included multiple capsules in a stick (up to 2) or pack (up to 4), the final analytic sample included 50 capsules. We conducted qualitative and quantitative analyses using gas chromatography with mass spectrometry (GC-MS, Agilent Technologies, Inc). The qualitative analysis detected 296 compounds (range = 9-67 per capsule), and all capsules contained menthol. Among the compounds detected in more than half the sample were limonene, menthone, benzaldehyde, eucalyptol, and triacetin. Traces of nicotine were found in 22% of the capsules. In the quantitative analysis, menthol concentrations were 33 times greater than the next most common compound (limonene). Benzyl alcohol and vanillin were also found in high concentrations. Comparing same-brand varieties across countries showed substantial variability in the concentration of menthol and other compounds. Menthol is an omnipresent constituent in capsule cigarettes, perhaps because of its anesthetizing and reinforcing addictive properties. Other compounds found are toxic, potentially carcinogenic, and may enhance addictiveness. Variance in the presence and concentrations of such compounds highlights the importance of product standards to regulate capsule content. This study evaluated the chemical content of capsule cigarettes from two Latin American countries that have two of the highest market shares for capsule cigarettes worldwide. Compared to other studies, our assessment included brand varieties from two countries to compare the differences in chemical content by country. Our results yield that menthol is found in all capsules, and that other chemicals found may prolong nicotine exposure and therefore reinforce the addictive properties of cigarettes.

Features of the implementation of corporate tools to optimize personnel activities

The article presents the experience of a law firm in drawing up and implementing corporate Instructions for employees. The manager used SMART, Agile and SCRUM business technologies in his work. For four years, the Employee Violation Log was used to record findings. When their number began to directly impede business development, the manager analyzed the violations and noticed certain patterns. Regardless of qualifications and work experience, employees make the same mistakes, which are of an “everyday” nature. It has been established that employees do not know how to act in situations of uncertainty. Taking the most repeated mistakes as a basis, the manager created “Instruction No. 0” — a set of rules that describe the way out of possible controversial situations. It is noted that employees take a more responsible approach to completing tasks, and the manager began to devote more time to strategic business development. These rules have become the company's corporate culture.

Key Trace Elements Zinc and Magnesium Levels Decline After Fracture in a Rat Model

Category: Basic Sciences/Biologics; Basic Sciences/Biologics Introduction/Purpose: Zinc and magnesium are crucial for bone health, and their deficiencies can lead to impaired bone metabolism and repair. Although enhancing their bioavailability has been linked to better fracture healing, their levels during healing have not been studied. We hypothesized that zinc and magnesium levels transiently decrease at the fracture site compared to intact bone. This study is the first to explore temporal trace element levels during the initial stages of fracture healing. The findings will reveal when each element changes its concentration at the fracture site, which is associated with specific functions necessary for appropriate healing. Understanding the connection between trace element concentration and repair processes could lead to more effective treatment or supplementation during fracture healing. Methods: Following induction of a femur fracture in male BB Wistar rats, injured and intact contralateral femurs were harvested at 1- (n=3), 3- (n=4), and 7-days (n=3) after fracture. Fracture segments 10mm in length were resected 5mm from the center of the callus in either direction. Contralateral non-fractured femur segments were resected from the same region within the femoral diaphysis. Samples were analyzed via inductively coupled mass spectrometry using an Agilent ICP-MS 7900 (Agilent Technologies, Santa Clara, CA). Data was converted from ppb to percent weight zinc and magnesium per weight of bone (w/w%). The difference between zinc and magnesium in contralateral intact bone vs. fractured bone was calculated and averaged (∆) for each group. The average % change were also determined. An independent sample two-tailed t-test was performed comparing average w/w% of zinc and magnesium between fracture and contralateral samples. A p-value of < 0.05 was considered significant. Results: After 1-day post-fracture, there was a -29.09 average % change in zinc levels (∆ 6.90x10^-03 ± 5.05x10^-03) (p = 0.033) and a -25.76 average % change in magnesium levels (∆ 1.21x10^-01 ± 7.36x10^-02 w/w%) (p = 0.241) between fractured and non- fractured femurs. After 3-days post-fracture, there was a -22.51 average % change in zinc levels (∆ 5.45x10^-03 ± 3.95x10^-03 w/w%) (p = 0.022) and a -33.07 average % change in magnesium levels (∆ 1.84x10^-01 ± 1.05x10^-01 w/w%) (p = 0.025). After 7- days post-fracture, there was a -9.40 average % change in zinc levels (∆ 1.90x10^-03 ± 3.98x10^-03) (p = 0.377) and a -17.88 average % change in magnesium levels (∆ 9.04x10^-02 ± 1.24x10^-01 w/w%) (p = 0.159) between fractured and non-fractured femurs. Conclusion: Previous research has established the significance of trace elements, namely zinc and magnesium, in the process of fracture healing. Zinc has been shown to improve fracture healing, while magnesium has been found to play a role regulating inflammation during early fracture healing. The findings of this study corroborate our hypothesis that levels of these elements transiently decline following fracture. By evaluating these changes in trace element levels, we can gain a better understanding of their function in the fracture healing process. These observations may offer insight into potential therapeutic approaches utilizing zinc and magnesium for fracture healing in the future.

Evaluation of Glycolysis and Mitochondrial Function in Endothelial Cells Using the Seahorse Analyzer.

Endothelial bioenergetics have emerged as a key regulator of endothelial barrier function. Glycolytic parameters have been linked to barrier enhancement, and interruption with mitochondrial complexes was shown to disrupt endothelial barrier. Therefore, a new technology that has been introduced to assess bioenergetics and metabolism has also made it possible to determine roles of specific energy production pathways in endothelial health. The Seahorse extracellular flux analysis by Agilent technologies is a state of the art tool that has been more frequently used to evaluate bioenergetics of endothelial cells. This chapter includes details about different assays that can be used to study endothelial cells using the Seahorse analyzer and how interpretation of the results can provide novel insight about endothelial metabolism.

A-033 Development and Validation of a Liquid Chromatography Tandem Mass Spectrometry Bioanalytical Method for Prostaglandin D2 in Serum or Urine

Abstract Background Prostaglandin D2 (PGD2) is a major prostaglandin produced by the brain and by mast cells, and plays a role in vasodilation (“niacin flush”) and in the development of allergic diseases such as asthma and alopecia. We sought to develop a bioanalytical method for multiple specimen types for PGD2 for clinical use. Methods An analytical method was developed using a TX-4 HPLC system (Thermo-Fisher) with Agilent® 1200 pumps (Agilent Technologies, Inc.) and a Sciex® 5500 (Danaher) triple quadrupole mass spectrometer. Calibration curve based on purified PGD2 (Cayman) was prepared in depleted serum (Golden West Biologicals). Sample preparation consisted of an initial isotope dilution using a deuterated heavy isotope internal standard (PGD2-2H4) followed by solid phase extraction, evaporation and reconstitution using a second heavy isotope (PGD2-2H9). Double-isotope dilution allows discrimination between extraction loss and ion suppression as causes of decreased ion current. A reversed phase C18 analytical column was used with an acidified water/acetonitrile/methanol solvent gradient to achieve chromatographic separation of physiological PGD2 from related isobaric metabolites include Prostaglandin E2. Negative mode electrospray ionization (ESI) was used for detection in Multiple Reaction Monitoring (MRM) mode. Results Analytical range was 1–1000 pg/mL (up to 5000 pg/mL with dilution). Inter-assay precision ranged from 8.8% to 10.2% and inter-assay accuracy ranged from 95.7% to 103.3%. Reference intervals for adults was developed using residual serum and urine specimens. No interference was observed in the presence of hemolysis, icteric or lipemia. No difference in result was observed when using collection tubes containing EDTA, Heparin or gel barriers. Conclusion A bioanalytical method for PGD2 has been fully validated for clinical use.

B-186 Investigating LC Column Selectivity for 19 Steroids in a Clinical Research Setting

Abstract Background Steroids are a class of structurally related endogenous compounds. Several have the same chemical formula (and hence the same m/z) and must be chromatographically separated for accurate identification and quantitation by LC-MS/MS. Here, six bonded silica solid core or “core-shell” LC columns (see Table 1) were evaluated to determine selectivity and optimum separation conditions for a 19 analyte steroid panel for clinical research. The optimum column for the separation of estrone and estradiol was also established. Methods Analytical reference standards (Cerilliant®, Round Rock, TX) were used to prepare diluted samples for injection. Six different core-shell 2.6 µm, 50 × 2.1 mm LC columns (Kinetex™, Phenomenex, Torrance, CA) were evaluated (see Table 1). Detection employed a 7500 triple quadrupole LC-MS/MS equipped with an ESI source used in positive and negative mode (SCIEX®, Framingham, MA) coupled to an Agilent® 1290 liquid chromatograph (Agilent Technologies, Santa Clara, CA). Results The traditional C18 column worked best for the complete steroid clinical research panel. Estrogens had the best separation on the biphenyl phase, which demonstrated the highest level of selectivity for estrone and estradiol. The phenyl-hexyl column did not sufficiently separate all steroid isomers in the clinical research panel, however it was sufficient to separate estrone and estradiol. Conclusion The proposed HPLC conditions and the core-shell 2.6 μm 50 × 2.1 mm C18 column provided the best separation for 19 steroids with a fast 8 minute run time. For research focused on estrogens, the core-shell biphenyl column yielded the best separation.

B-187 Evaluation of Clinical Utility of LC-MS/MS Method for Thyroglobulin Measurement in Comparison with Immunoradiometric Assay and Chemiluminescence Microparticle Immunoassay

Abstract Background Thyroglobulin (Tg) is the follow-up index of choice for patients with differentiated thyroid cancer (DTC), especially who were undergone total thyroidectomy. However, due to the commonly used immunoassay’s inherent susceptibility to interference combined with the high prevalence of thyroglobulin antibody (TgAb) in DTC patients, the need for more reliable measurement for Tg independent from TgAb has been brought up. In this study, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for Tg measurement and compared its clinical usefulness and analytical performance with immunoradiometric assay (IRMA) and chemiluminescence microparticle immunoassay (CMIA). Methods We developed trypsin digestion-peptide immunoaffinity LC-MS/MS methods for Tg measurement. Analysis was performed using an LC-MS/MS system consisting of Agilent 1290 LC (Agilent Technologies, Santa Clara, CA) and Qtrap 5500+ MS/MS system(ABSciex, Framingham, MA). Tryptic Tg specific peptide (FSPDDSAGASALLR) and cleavable internal standard peptide (LSFYWTTTGDPFFDSHSDSLL*RSGPYMPQ, L*: 13C6, 15N) were quantitated against 5-point linear calibration curve with Access Thyroglobulin Calibrators (Beckman Coulter, Pasadena, CA). Three levels of serum control were prepared (3.95, 12.5, and 39.4 ng/mL). Lower limit of quantitation (LLoQ), linearity, precision, and carryover were validated for the assay. For method comparison, serum from patients with DTC or Hashimoto thyroiditis was collected. Tg was also measured with IRMA using BRAHMS TG Plus (Thermo Fisher Scientific, Waltham, MA), and CMIA using Architect Thyroglobulin (Abbott laboratories, Illinois, CA). TgAb was measured with IRMA using BRAMS Anti-Tg (Thermo Fisher Scientific) and the cut-off for TgAb was 60 IU/mL. Also, interference of TgAb was evaluated by comparing the Tg recovery of five sets of TgAb negative samples spiked with diluents and high TgAb samples. Results Within-run and between-run imprecisions were CV 7.8%–13.0% and CV 8.9%–12.7%, respectively. LLoQ was 0.5 ng/mL. The assay was linear through 0.5–650 ng/mL. No significant carryover was observed within the analytical measurement range. Out of the total 193 samples, the number of samples above LLoQ in both LC-MS/MS, and IRMA or CMIA were 73 samples (45 TgAb-positive and 28 TgAb-negative samples) and 69 samples (42 TgAb-positive and 27 TgAb-negative samples), respectively. The result well correlated between LC-MS/MS and IRMA (R2 = 0.99), and LC-MS/MS and CMIA (R2 = 0.98) in overall samples. When analyzed according to TgAb status, LC-MS/MS and IRMA (R2 = 0.84), and LC-MS/MS and CMIA (R2 = 0.93) correlated rather poorly in TgAb-positive samples, while TgAb-negative samples were consistent (R2 = 0.99 for IRMA, R2 = 0.97 for CMIA). We found one case with systemic metastatic thyroid cancer that showed significantly high Tg levels in IRMA and LC-MS/MS (above the upper limit of quantitation), whereas below LLoQ in CMIA. The interference of TgAb was negligible in LC-MS/MS, while significant interference was observed in CMIA, with a mean recovery rate of 57.6% (4.3%–82.5%). In one sample, when measured with CMIA, thyroglobulin level was 3.29 ng/mL when mixed with diluent, but below LLoQ (0.14 ng/mL) when spiked with TgAb-positive samples (TgAb 1700 IU/mL). Conclusion The LC-MS/MS method for Tg measurement was less prone to antibody interference than immunoassays. Measurement of Tg using LC-MS/MS would be useful for reliable diagnosis and proper management in TgAb-positive patients.

Features of populations of hemp varieties with different composition of cannabinoid compounds

Aim. To study the biological features of populations of hemp varieties with the absence of narcotic activity – tetrahydrocannabinol (THC) not higher than 0.08 %, but with the presence of non-psychoactive components of cannabinoids: cannabidiol (CBD) and cannabichromene (CBC); cannabigerol (CBG); CBG and CBD. To identify breeding prospects for increasing the content of cannabinoids that have therapeutic properties. Methods. Samples of elite plants were analyzed for cannabinoid content by gas-liquid chromatography (GC Agilent Technologies HP 6890, Series Hewlett Packard). Results. The limits of variation of the population variability of the sign of CBD content of the Medana variety are from 0.0253 to 3.3469 %, 75 % of the plants of which contain CBD in the range of 1.0-2.2 %. Studies of the Vik 2020 hybrid population confirmed a high correlation between CBD and THC and the absence of a relationship between CBD and CBG, THC and CBG. More than 11 % of plants in the population contain CBG from 3.5849 to 4.6207 %. There are no correlations between CBD and CBG, CBD and THC in these plants. The presence of such plants indicates the possibility of further increasing the content of СBG to 8 % and above, without exceeding the THC limit in our country (0.08 %). Conclusions. Conducted research has proven that with the current permissible level of THC, the content of CBD can be increased only to 2.5-3.0 % (Medana variety) through purposeful selection. The hybrid Vik 2020 is especially valuable, because in its population unique plants with the presence of several cannabinoid compounds with a THC content of no more than 0.08 % were found. The obtained valuable breeding material of the Medana variety and the Vik 2020 hybrid is a donor for its inclusion in hybridization and further multi-vector selection in the direction of increasing of the content of CBD, CBC and CBG with the absence of THC.