Duplicate Samples Research Articles

Intravenous administration of gold nanoparticles in rats exhibit alterations in sphingomyelins, bile acids, sphingolipids, and cholesterol esters levels

AbstractNanoparticles (NP) have gained significant attention in biomedical research due to their unique properties and potential applications in drug delivery, imaging, and diagnostics. Gold (AuNPs) and silver (AgNPs) NPs are among the important nanoplatforms that received extensive attention recently for various biomedical applications. Understanding the complex interaction of these NP in biological systems is essential to unveil their pharmacological, Pharmacokinetic and toxicological attributes. Metabolomics has proven invaluable in providing detailed insights into NP’s biodistribution, metabolic effects, and potential toxicity. This study aims to investigate the underlying metabolic pathways affected by in vivo exposure to NP using a robust metabolomics approach. In this work, spherical polyethylene glycol (PEG) modified AuNPs (13 nm, diameter) or AgNPs (20 nm, diameter) were synthesized and dosed into rats via intravenous route to study the associated metabolic changes. Rats (n = 14) were divided into three groups: control (n = 2), AuNPs (n = 6) and AgNPs (n = 6), to mimic potential biomedical exposure scenarios. Duplicate serum samples were collected 24 h post-dosing, and comprehensive metabolite profiling was performed using liquid chromatography-mass spectrometry (LC-MS/MS) and flow injection analysis-mass spectrometry (FIA-MS/MS). Metabolite extraction followed the MxP Quant 500 Kit protocol, with chromatographic separation using the Xevo TQS system. Metabolite identification and quantification were conducted with isotopically labelled internal standards and multiple reaction monitoring (MRM), utilizing optimized conditions under mass spectrometry (MS) as provided by Biocrates. Annotation of metabolites was determined by retention times and specific MRMs for each compound. Results indicate that AuNPs treatment significantly impacted several metabolic pathways. Notably, there was an increase in sphingomyelin (SM 34:2) levels (estimate 0.23, p ≤ 0.001), which are critical for cell membrane structure and signalling. Additionally, a decrease in glycochenodeoxycholic acid levels was also triggered by treatment with AuNPs, suggesting modulation of bile acid metabolism with potential effects on lipid homeostasis. Furthermore, treatment with AuNPs caused significant alterations in cholesterol ester levels, essential for lipid storage and transport, indicating disruptions in these mechanisms. These metabolic changes suggest that gold nanoparticles can disrupt fatty acid metabolism, pyrimidine/purine metabolism, and amino acid synthesis. These findings contribute to the growing understanding of nanoparticle toxicity profile and underscore the need for further research to ensure the safe application of nanoparticles in biomedical and other fields.

Rapid Analysis for α-Tocopherol and Its Oxidative Products in the Pisum sativum L. Leaf Using Supercritical Fluid Chromatography-Medium Vacuum Chemical Ionization Tandem Mass Spectrometry.

A method for the rapid determination of α-tocopherol (α-T) and its oxidative products in plant tissue has been developed using supercritical fluid extraction (SFE) coupled with supercritical fluid chromatography (SFC) and medium vacuum chemical ionization (MVCI) with tandem mass spectrometry. The method is designed to study changes in levels for α-T and its oxidative products in plant cells during photosynthesis, aiming to observe the light response curves. α-T oxidation is a non-enzymatic self-defense mechanism in plant cells. Unlike enzyme-involved reactions, it cannot be stopped, so the oxidation continues in crude extracts even after extraction. Therefore, a real-time in-situ method is essential for tracking the light response curves. To optimize the selective reaction monitoring method, the reaction mixture of α-T and singlet oxygen (1O2), generated by rose Bengal under light illumination, was used as the source of oxidative products. The relative abundance changes in α-tocopherylquinone and 8a-hydroperoxy tocopherone in Pisum sativum L. (Pea) leaves under excessive light illumination have been preliminarily analyzed as part of the light response curve study. The method archives a throughput of 10-15 minutes for analyzing duplicate leaf samples. This process includes cutting off the leaf, sectioning it, placing the sample in a frozen SFE vessel, and conducting SFE/SFC analysis. Consequently, the average throughput is approximately 5-7 minutes per sample.

B-004 Method Validation of Lactate in the Plasma of Rats

Abstract Background The objective of this study is to validate Lactate (LAC) measurement in rat plasma using the ADVIA 1800 analyzer. Methods LAC was validated in rat plasma using the ADVIA 1800 analyzer. Validation testing included intra-assay and inter-assay precision, accuracy, linearity of dilution, limit of quantitation (LOQ), limit of detection (LOD), reference interval, carry-over, correlation, recovery, and stability. Samples were collected into tubes containing sodium fluoride. Intra-assay precision was determined by analyzing two biological samples and the two quality control levels of Bio-Rad Lyphochek Assayed Chemistry Control 10 consecutive times. Inter-assay precision was determined by analyzing the two quality control levels in duplicate for six days over a 10-day period. Accuracy was calculated using the data obtained from the inter-assay precision testing. For linearity of dilution, Audit MicroControl General Chemistry Linearity Kit and biological samples were diluted and analyzed in duplicate. The LOQ was determined by diluting the calibrator material to produce a value at the low end of the reportable range. Diluted material was tested six times. The LOD was determined by assaying the appropriate blank 10 times. The reference interval was determined by analyzing 21 plasma samples. Correlation between the two ADVIA 1800 analyzers was determined by assaying 10 samples on both analyzers once. The carry-over of the assay was determined by analyzing the high-level quality control material followed by the low-level quality control material. Recovery was determined by analyzing a pair of test samples in duplicate that have been spiked and diluted. The proportional error between the two test samples was calculated. Stability of plasma samples was tested on wet ice, refrigerated, and frozen (at -70°C) for various durations of storage. Results Measurement of LAC in rat plasma met the acceptance criteria for precision, accuracy, linearity of dilution, limit of quantitation, carry-over, recovery, and stability. For intra-assay precision in specimen and control material, the %CV was within ±20%. The inter-assay precision %CV was within ±20%. The total error observed (TEobs) was within ±20% for accuracy and the obtained mean of the control material was within the manufacturer’s acceptable range. For linearity of dilution, the coefficient of determination was ≥ 0.9000, the slope was within 1.00 ± 0.25, and TEobs for each plasma and linearity kit dilution was ≤20%. The LOQ was set at the lowest concentration for which the TEobs was within ±20%. The LOD was calculated by adding the mean concentration obtained and three times the standard deviation. The reference interval was set to the 2.5th to 97.5th percentile interval using Excel software. The carry-over was acceptable at ≤2%. Correlation between the two ADVIA 1800 analyzers was determined to describe any bias in result interpretation. Recovery was acceptable as the proportional error was within ±20%. Stability at all storage conditions was found to be acceptable with a mean %difference within ±20%. Conclusions LAC in rat plasma met the acceptance criteria for all required validation parameters. The ADVIA 1800 analyzer can be used for preclinical studies to test LAC in rat plasma.

A-038 Interlaboratory Comparison of Parathyroid Hormone Analytical Results Across Clinical Assays from 8 Manufacturers - Steps Towards Standardization

Abstract Background Parathyroid hormone (PTH) is an 84 amino acid peptide produced by the parathyroid gland and is a key biomarker used for the diagnosis and treatment of Chronic Kidney Disease-Mineral and Bone Disorder, as well as hypo- and hyperparathyroidism. PTH clinical assays were previously reported to exhibit significant inter-assay measurement variability, which limits the utility of PTH as a diagnostic biomarker and can result in the misclassification of patients. Thus, there is a need for PTH clinical assay standardization to improve clinical assay measurement agreement. The Centers for Disease Control and Prevention’s Clinical Standardization Program (CDC-CSP) will launch a PTH standardization program, which is in support of and coordinated with the IFCC Committee on Bone Metabolism. In preparation, the CDC-CSP conducted an Inter-Laboratory Comparison Study to evaluate the current status of PTH inter-assay variability and to investigate potential sources of variability. Methods Eight PTH assay manufacturer’s laboratories measured PTH in 42 deidentified, fresh-frozen single donor serum samples in duplicate over 2 independent runs. Sample PTH concentrations ranged from 1.3 - 324 pg/mL, based on initial screening values. Five manufacturers submitted data for 2 different assays, resulting in PTH measurements from 13 assays total. Data were submitted to the CDC CSP for analysis. Results Assay precision was <5% for 10/13 measurement procedures (MP) assessed and 3 MPs exhibited precisions between 5 - 18%. Assay-specific PTH measurements demonstrated good linear correlation compared to the all-assay mean concentration. Interestingly, inter-assay variability increased with increasing PTH concentrations, with individual sample standard deviations increasing from 2.9 - 123, with a mean SD of 24.1 and individual sample CVs increasing from 18.9 - 44.5%, with a mean CV of 24.2%. Conclusions This study provides new insights into PTH inter-assay variability and assay performance for assays commonly used in the clinical setting. The findings of this PTH Interlaboratory Comparison Study establish a baseline for PTH inter-assay variability and will guide future PTH standardization efforts.

First Detection and Characterization of Smacovirus in the Human Vagina in Two Sequential Samples over a Twelve-Day Interval

Background: Smacovirus is a CRESS-DNA virus identified almost exclusively in transient fecal samples from various vertebrate species. Objective: We evaluated human vaginal samples for the presence and maintenance of Smacovirus. Methods: Viral metagenomics analysis was performed on vaginal samples collected from 28 apparently healthy women in New York City, USA. Twenty-one of the women provided duplicate samples over a 12–21-day interval. Results: Phylogenetic analysis identified two samples from the same individual, collected over a twelve-day interval, that were positive for the complete Smacovirus genome. All detected sequence contigs belonged to a single variant of CRESS-DNA. Conclusions: The continuous presence of Smacovirus in the human vagina over a twelve-day period was identified.

Abstract B024: Multiplexed 3D atlas of state transitions and potential prognostic role of poly- ADP ribose glycohydrolase (PARG) in chronic pancreatitis and PDAC

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC), a disease predominantly associated with chronic inflammation, ranks as the third-leading cause of cancer-related mortality. The limited efficacy of current PDAC therapies is, in part, attributed to the challenges posed by late- stage diagnosis, highlighting the urgent need for early detection techniques to improve treatment outcomes. Sustained pancreatic inflammation induces the production of reactive oxygen species, leading to the activation of the DNA damage repair network, including enzymes such as poly-ADP ribose glycohydrolase (PARG). PARG is crucial for degrading poly-ADP ribose (PAR) chains catalyzed by PARP and, thus, regulating key cellular responses to DNA damage. However, the role of PARG in the molecular etiology of pancreatic diseases remains unclear. As a first step, we conducted a single-cell spatial analysis on a multiplex immunofluorescence cyclic dataset focusing on pathways involved in: immune and stromal alterations influencing cell-state transitions; differentiation; and critical cellular signaling pathways. Specifically, we evaluated the expression patterns of DNA damage enzymes (i.e., PARG), to elucidate their potential roles in the transformation from chronic inflammation to malignancy. Method: The human pancreatitis tissue microarray (TMA), consisting of 6 normal, 36 pancreatitis, 36 pancreatic intraepithelial neoplasia (PanINs), and 6 PDAC samples in duplicate, obtained from patients with pancreatitis or pancreatic cancer diagnosed at Oregon Health and Science University. We generated a comprehensive 48-plex image dataset that was processed through unsupervised clustering and manual gating to identify common cell types across different stages of pancreatic disease, including epithelial, stromal, immune, and endothelial. Result and conclusion: We observed moderate to strong cytoplasmic PARG expression in pancreatitis samples compared to normal tissue (p < 0.001), which significantly decreased in PanINs (p < 0.001) and PDAC samples (p < 0.001). Notably, the PARG expression was primarily in CK19-positive epithelial cells. Further evaluation showed that DNA damage markers, such as pRPA and γH2AX, increased as the disease progressed from pancreatitis to PanINs to PDAC. Therefore, there was an inverse correlation between PARG expression and DNA damage marker expression in cells exhibiting DNA damage. Additionally, PARG expression was inversely correlated with proliferating cells, marked by Ki67 and PCNA, at the single-cell level. In conclusion, our single-cell spatial analysis supports the notion that PARG expression correlates with resolution of DNA damage and that its reduced expression associates with disease progression and cellular proliferation. Overall, these findings support early phase clinical trials using PARG inhibitors in the oncologic space, and as a potential target for early detection of PDAC and treatment of other pancreatic diseases. Mechanistically, PARG loss may provide a setting for faulty DNA repair and thus, an accumulation of driver mutations. Citation Format: Vidhi M Shah, Jennifer Eng, Koei Chin, Brett C. Sheppard, Jonathan R. Brody, Rosalie C. Sears. Multiplexed 3D atlas of state transitions and potential prognostic role of poly- ADP ribose glycohydrolase (PARG) in chronic pancreatitis and PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B024.

Towards similar alignment and unique uniformity in collaborative filtering

Representation learning, with its desired properties, including Alignment and Uniformity, has recently emerged as an effective approach in collaborative filtering (CF) for recommender systems. Alignment measures the distance between positive pairs, and uniformity describes the distribution of all samples on the unit hypersphere. Despite the effectiveness of existing research, the challenges of insufficient alignment and uniformity bias persist in studies on the desired properties of recommender systems: (1) Sparse interaction information leads to an insufficient alignment representation. (2) Calculating the uniformity loss based on duplicate samples introduces bias, which affects the overall representation uniformity.Building on aforementioned challenges, we propose a Similar Alignment and Unique Uniformity model called SUAU. SUAU mitigates insufficient alignment by introducing additional item-similar item pairs for model training and employing a uniqueness strategy to prevent uniformity bias. More specifically, we propose a similar items generation method that identifies the most similar items based on the interaction behavior of the original items’ related users to form item-similar item pairs. The experimental results obtained on three highly sparse datasets demonstrate that the propose model achieves superior alignment and uniformity compared with state-of-the-art models, notably surpassing the best CF methods in terms of recommendation performance. We have open-sourced the source code on a publicly accessible website: https://github.com/ZzYUuuu/SUAU.

The group of hypermethylated long noncoding RNA genes is associated with different types of metastasis in ovarian cancer

Background: Ovarian tumors are characterized by asymptomatic progression until their late stages, when at the time of diagnosis the patient already has extensive metastatic disease. In addition to lymphogenous and hematogenous metastasis in ovarian cancer, there are peritoneal dissemination and metastasis to the greater omentum with ascites; moreover, peritoneal carcinomatosis is the predominant route of metastasizing of ovarian cancer. Epigenetic factors, such as gene methylation, regulatory microRNAs and long non-coding RNAs (lncRNAs), contribute to progression of this cancer. Our previous bioinformatic and experimental studies have identified 13 genes of lncRNAs (GAS5, HOTAIR, LINC00472, LINC00886, MAFG-DT, PLUT/PDX1-AS1, SNHG1, SNHG6, SNHG12, SNHG17, TINCR, TP53TG1, TUG1) hypermethylated in the ovarian neoplasms. Aim: To evaluate the clinical significance of methylation levels of 13 lncRNA genes (GAS5, HOTAIR, LINC00472, LINC00886, MAFG-DT, PLUT/PDX1-AS1, SNHG1, SNHG6, SNHG12, SNHG17, TINCR, TP53TG1, TUG1) associated with various types of ovarian cancer metastasis, including lymphogenous, peritoneal, omental, and distant metastases. Methods: The methylation levels of lncRNA genes GAS5, HOTAIR, LINC00472, LINC00886, MAFG-DT, PLUT/PDX1-AS1, SNHG1, SNHG6, SNHG12, SNHG17, TINCR, TP53TG1, TUG1 were analyzed by quantitative real-time methylation-specific polymerase chain reaction. We tested 122 duplicate samples of ovarian neoplasms, including 104 malignancies and 18 borderline tumors, as well as 45 peritoneal macro metastases, collected in the N.N. Blokhin National Medical Research Center of Oncology in 2020 to 2023. The study included 21 samples of primary tumor from patients with lymphogenous metastases, 45 samples from patients with peritoneal dissemination, 61 from those with omental metastases, 49 from patients with ascites, and 9 with distant metastases. Results: The tumor samples from the patients with lymphatic nodes metastases showed a significant increase in the methylation level of two lncRNA genes: SNHG6 (p = 0.044) and SNHG12 (p = 0.006). Peritoneal dissemination was associated with hypermethylation of four lncRNA genes: GAS5, HOTAIR, LINC00472 (p 0.05), and most significantly TINCR (p = 0.001). GAS5, HOTAIR, LINC00886 (p 0.05) and most significantly LINC00472 (p 0.001) hypermethylation was typical for omental metastasis, and that of LINC00472 and LINC00886, with ascites (p 0.05). Peritoneal macroscopic metastases demonstrated increased methylation of MAFG-DT (p 0.001) and TP53TG1 (p 0.001) and desmethylation of LINC00886 (p = 0.003) and SNHG12 (p = 0.002), compared to their primary tumors. Conclusion: We performed the analysis of clinical significance of 13 hypermethylated lncRNA genes in ovarian cancer and were the first to show that 10 genes (GAS5, HOTAIR, LINC00472, LINC00886, MAFG-DT, SNHG6, SNHG12, TINCR, TP53TG1, TUG1) were associated with various types of ovarian tumor metastasis. Also, we were able to determine certain panels of lncRNA, which, if demonstrate abnormal methylation, were specific for lymphogenous and peritoneal metastasis of ovarian tumors.

Genomic and Spatial Analysis on the Recent Transmission of Mycobacterium tuberculosis in Eastern China: A 10-Year Retrospective Population-Based Study.

Understanding the mode of Mycobacterium tuberculosis (M. tuberculosis) transmission is crucial for disease prevention and control. Compared to traditional genotyping methods, whole genome sequencing (WGS) provides higher resolution and comprehensive genetic information, enabling the tracing of infection sources and determining of transmission routes to resolve extensive tuberculosis (TB) outbreaks. We conducted a ten-year study on the transmission of M. tuberculosis in a population in eastern China. We selected Lianyungang, an eastern city in China, as the study site. Patients diagnosed with active pulmonary TB from 2011 to 2020 were enrolled as the study subjects. We isolated and sequenced 2252 M. tuberculosis. Strains with pairwise genetic distances of less than 12 single nucleotide polymorphisms were defined as genomic clusters and which were considered recent transmissions. Kernel density estimation and K-function analysis were applied to explore the spatial distribution of recently transmitted strains. After excluding non-tuberculous mycobacteria and duplicated samples, 2114 strains were included in the final analysis. These strains comprised lineage 2 (1593, 75.35%) and 4 (521, 24.65%). There were 672 clustered strains, with a recent transmission rate of 31.79%. The logistic regression model showed that the risk of recent transmission was high in students [adjusted odds ratio (aOR): 2.68, 95% confidence interval (CI): 1.63-4.49, P<0.001] and people infected with L2.2.1 strains (aOR: 1.59, 95% CI: 1.20-2.12). Higher spatial aggregation of TB transmission has been concentrated in Haizhou, Donghai, and Guanyun for the past 10 years. Three outbreaks affecting 46 patients were spatially spaced, with 11 to 23 persons each. Different groups exhibited varying geographic distances between the initial and later cases. There are areas with a high risk of transmission for M. tuberculosis in the research site, and the risk varies among different populations. Accurate prevention strategies targeted at specific regions and key populations can help curb the prevalence of TB.

Sequence analysis of isolated strains of herpes zoster virus among patients with shingles.

Herpes zoster, or shingles, is caused by the varicella-zoster virus (VZV), which initially presents as chickenpox in children. VZV is a global health concern, especially in winter and spring, affecting 10-20% of adults over 50 and posing a 30% risk for the general population. This study used PCR to detect VZV, confirming results with duplicated DNA samples and identifying 234 bp fragments by targeting the gpB gene. This study examined 50 herpes zoster cases from October 2020 to April 2021, involving 30 males and 20 females aged 10 to 90, diagnosed by dermatologists. Data were collected via a questionnaire. PCR detected VZV by amplifying the gpB and MCP genes from skin lesion samples. Six positive 234-bp PCR products were sequenced at Macrogen Inc. in Seoul, South Korea. Six DNA samples with 234 bp amplicons were sequenced, showing 99-100% similarity to human alpha herpesvirus sequences in the gpB gene. NCBI BLAST matched these sequences to a reference (GenBank acc. MT370830.1), assigning accession numbers LC642111, LC642112, and LC642113. Eight nucleic acid substitutions caused amino acid changes in the gpB protein: isoleucine to threonine, serine to isoleucine, and threonine to Proline. These variants were deposited in NCBI GenBank as gpB3 samples. The study found high sequence similarity to known VZV sequences, identifying six nucleic acid variations and eight SNPs. Notable amino acid changes in the gpB protein were deposited in NCBI GenBank as the gpB3 sample.

Improved estimates of expanded measurement uncertainty.

Measurement uncertainty (MU) is often estimated (sometimes initially as repeatability) using a limited number of single measurement values (n ≪ 30), or of duplicate measurements on duplicated samples, in order to include the contribution from sampling. In cases such as this where the uncertainty estimate is based on limited data, the common use of a coverage factor of 2.0, based on an assumed normal distribution, to give an approximate 95% confidence interval can result in an underestimate of the expanded MU. Where n is much lower than 30 (e.g., n = 8), this can lead to a serious underestimate. More accurate estimation of the coverage factor, for both the classical and robust ANOVA (e.g., in new software RANOVA v4.0) gives more reliable estimates of the expanded MU by calculating coverage factors based on the t-distribution. A case study for nitrate in lettuce shows that the more accurate coverage factor (of ∼2.3) substantially increases the expanded MU estimates by 13-14%.

Field validation as a means for continual monitoring of approved test kit’s fitness for purpose in the commercial market

Testing accuracy of a chemical contaminant requires use of a testing platform that conforms to validation criteria outlined in quality literature and standards. This study explores the application of commercial field data measured by qualified analysts using a United States Department of Agriculture – Federal Grain Inspection Service approved kit for measuring fumonisin in maize to augment method validation procedures. Analysts from seven grain testing facilities were qualified in official USDA sampling, sample preparation, and testing methodology using the Charm LF-FUMQ-WETS5. A duplicate sample was tested in the Office of the Texas State Chemist (OTSC) laboratory using UPLC-MS-MS. Data were subject to four statistical techniques using continuous and categorical methodology. This approach enabled researchers to explore if a single test or multiple comparisons were best suited to assess a field kit’s fitness for purpose across facility, toxin level, and year. The study concluded that a paired t-test and correlation analysis provided a quick and meaningful evaluation of kit performance. The correct placement of samples within the correct bin (violative versus non-violative) aligns well with market forces and regulatory compliance. The results of this study also provide a useful tool to assess all field kits’ performance at the beginning of the harvest season and subsequent years. The combination of statistical techniques presented in this research is an important tool in assessing mycotoxin field test kits fitness for purpose and represents a key step in a continuous improvement-quality systems process meant to protect the feed and food supply.

Adjustment of lactate dehydrogenase concentration results according to the haemolysis index following in vitro haemolysis

In the presence of haemolysis, the interpretation of the Lactate dehydrogenase (LDH) activity result is a major operational challenge for medical laboratories: if the origin is intravascular, then the measurement will reflect the clinical reality, but in extravascular haemolysis, the laboratory will be confronted with an artefactual increase leading to false-positive high results. The aim of our study was to evaluate the adjustment of LDH concentration results according to the haemolysis index (HI). After designed a mathematical model to correct the LDH measured as a function of the haemolysis index using a Cobas 8000 analyser (Roche diagnostics, Mannheim, Germany), LDH measurement of seventy-four duplicate samples were tested before and after exposure to extravascular haemolysis process. After in vitro haemolysis process, a significant increase haemolysis index (Man-Whitney U-Test p < 0.0001) were observed. Before process the HI median was 4 [2.0 − 6.75] and after HI median was 18 [10 − 35.75]. Without correction, LDH results showed a significant increase (p < 0.001) after haemolysis process and substantial analytical discrepancies (31/74) were observed according to TEa of CLIA. After correction, data showed no significant difference (p = 0.497) and the mathematical algorithm allowed to reduce the analytical discrepancies (2/74). If haemolysis was present in vitro, the mathematical algorithm increased the accuracy of the LDH results. However, the lack of discrimination between in vivo and in vitro haemolysis requires caution and the results should be reported only as a commentary to inform the clinician.

Precision Nutrition – Evaluating Dietary Protein and Amino Acid Intake among Children Using Duplicate Dietary Samples

Introduction: Precision nutrition, at the intersection of science and health, offers tailored dietary intervention, particularly vital for vulnerable populations like children. Focusing on protein and essential amino acids (AA), crucial for health, personalized approaches are keys for addressing deficiencies, and moving beyond generalized methods. While research on the dietary duplication technique has explored various nutrients, but there is a gap in understanding its use for assessing protein and amino acid intake, especially in children. Aim: The study aims to use the dietary duplication technique to assess the precise dietary intake of protein and AA in children, focusing on determining their exact portions and analyzing duplicate samples for content. Methods: A cross-sectional study in Chennai involved 12 children aged 4–9 years. Duplicate food portions for a day were collected from mothers in low-density polythene bags. Samples were analyzed for protein and amino acid content in a National Accreditation Board for Testing and Calibration Laboratories-accredited laboratory. Results: Girls exhibited deficient levels of histidine, lysine, phenylalanine, threonine, and tryptophan, while isoleucine met the Recommended Dietary Allowances threshold. A t-test showed significant differences in threonine levels among 4–6-year-old children, with no notable variations in 7–9-year-old children. Conclusion: This study emphasizes the importance of precise nutrition, particularly protein and essential AA, crucial for children’s health. Addressing undernutrition is crucial, and highlighting the urgency of this concern.

Bioprospecting of Indigenous Microbial Species for Bioethanol Production from the Fruits of Some Selected Exotic Ornamental Plants

The study determined the ability of some selected indigenous microbial isolates, namely Bacillus subtilis, Zymomonas mobilis and Saccharomyces cerevisiae, in the production of bioethanol from the fruits of some exotic plants (i.e. Ixora coccinea, Duranta repens and Syzygium guineense). B. subtilis was isolated from soil, while Z. mobilis and S. cerevisiae were isolated from palm wine using cultural methods. The organisms were identified based on their colony characteristics and morphological features, which were observed by microscopic examination of the stained smears of the isolates. The identity of the organisms was confirmed through the molecular study of the 16S rRNA and 18S rRNA regions of the microbial genomes using Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis (PCR - DGGE). The fruit samples were collected, washed, blended, filtered using a clean piece of cloth and pretreated using ultrasonication and autoclave. Concentrations of reducing sugars in the fruit filtrates were determined using DNSA-UV-visible spectrophotometry. Seven (7) conical flasks (500ml capacity) were arranged for each fruit sample in duplicate, and 250ml of the fruit filtrates were dispensed in the flasks accordingly. The fermenting organisms were inoculated in the flasks containing the fruit filtrates singly and in combination. Flasks were incubated in shaking incubator for seven days at 35oC. After incubation, the concentrations of bioethanol produced were determined using the UV-visible spectrophotometry. Highest reducing sugars concentrations of 43.7% and 43.6% were found in I. coccinea and S. guineense, respectively, while 36.4% was recorded for D. repens. Highest bioethanol yield of 27.5m/L was found in I. coccinea fermented by S. cerevisiae and Z. mobilis, while the lowest yield of 17.25m/L was found D. repens. The yield in bioethanol production was higher in samples containing a combination of fermenting organisms compared to ones with individual cells. It was recommended that the fruits of these exotic plants should be considered for commercial production of bioethanol.

Vincristine (VCR) pharmacokinetics (PK) in infants dosed using a body surface area (BSA) banded dosing table and in older children dosed based on BSA: A pediatric early phase clinical trial network study (PEPN22P1).

10015 Background: The Children’s Oncology Group developed and implemented a unified method of dosing anticancer drugs in infants and small children with a BSA &lt; 0.6 m2. The dosing tables use BSA-based dose banding and account for deliverable doses/volumes of the drugs. We studied the PK of VCR (1.5 mg/m2 dose level) in infants and children to determine whether this new infant dosing method achieves uniform drug exposures across the pediatric age span. Methods: We designed a sampling strategy to limit the blood volume required by focusing on the elimination (β) phase of the VCR concentration-time curve, which accounts for &gt; 80% of the total area-under the curve (AUC). Duplicate samples to detect contamination were drawn at 3 post-infusion time points (2, 6-8 and 18-24 hr) through a central venous catheter (CVC) after a 90 min flush. VCR concentrations were measured at the Children’s Hospital of Philadelphia using a validated high-pressure liquid chromatography/tandem mass spectrometry method with a lower limit of quantitation (LLQ) of 0.25 ng/mL. A one-compartment model was fit to the concentration-time data using population PK methods (Phoenix NLME). The AUCβ was derived from the dose and clearance for each patient. Patients were accrued in 4 age groups (≤6 mo, goal n = 20); &gt; 6 mo to ≤12 mo, goal n = 10; &gt; 12 mo to ≤36 mo, goal n = 10; and &gt; 36 mo to ≤12 yr, goal n = 10) to focus on infants and ensure representation across the pediatric age range. Results: 52 eligible patients have been enrolled and 39 are evaluable for PK analysis, including 9 ≤6 mo, 8 &gt; 6 mo and ≤12 mo, 12 &gt; 12 mo and ≤36 mo, and 10 &gt; 36 mo and ≤12 y,. The median (range) age is 13.5 mo (1.15-112 mo). VCR was quantifiable in all samples, and there was no evidence of contamination in the duplicate samples drawn through the CVC. The mean ± SD AUCβ in the 39 evaluable patients was 54.7 ± 19.8 ng⋅h/mL. As shown in the Table, the AUCβ is similar in patients with a BSA &lt; 0.6 m2 dosed according to the BSA-banded infant dosing table and patients with a BSA ≥0.6 m2 dosed by multiplying their BSA times 1.5 mg/m2 (p = 0.53, Mann Whitney test). Conclusions: The VCR BSA-banded infant dosing table appears to provide uniform VCR exposure for infants and young children compared to conventional BSA-based dosing in older children. The study design and sampling strategy facilitated rapid accrual of a young population and generated more VCR PK data in infants within a year than has been published since the drug was approved in 1963. This study demonstrates the feasibility of systematically studying other anticancer drugs in infants and other special populations to ensure they are receiving safe and effective doses. VCR AUCβ by dosing method. Clinical trial information: NCT05359237 . [Table: see text]

Metagenomic analysis of sewage for surveillance of bacterial pathogens: A release experiment to determine sensitivity.

Accurate monitoring of gastro-enteric and other diseases in large populations poses a challenge for public health management. Sewage represents a larger population, is freely obtainable and non-subject to ethical approval. Metagenomic sequencing offers simultaneous, multiple-target analysis. However, no study has demonstrated the sensitivity of metagenomics for detecting bacteria in sewage. In this study, we spot-released 1013 colony-forming units (CFU) of Staphyloccus hyicus (non-pathogenetic strain 842J-88). The strain was flushed down a toilet into the sewer in the catchment area of a public wastewater treatment plant (WWTP), serving a population of 36,000 people. Raw sewage was continuously sampled at the WWTP's inlet over 30- and 60-minute intervals for a total period of seven hours. The experiment was conducted twice with one week in-between release days and under comparable weather conditions. For the metagenomics analyses, the pure single isolate of S. hyicus was sequenced, assembled and added to a large database of bacterial reference sequences. All sewage samples were analyzed by shotgun metagenome sequencing and mapped against the reference database. S. hyicus was identified in duplicate samples at both of two release days and these sequence fragment counts served as a proxy to estimate the minimum number of sick people or sensitivity required in order to observe at least one sick person at 95% probability. We found the sensitivity to be in the range 41-140 and 16-36 sick people at release days 1 and 2, respectively. The WWTP normally serves 36,000 people giving a normalized sensitivity in the range of one in 257 to 2,250 persons.

Abstract PO5-14-03: Analytical Validation of the StemPrintER Assay in Patients with Breast Cancer

Abstract Discussion: Breast cancer is the most common malignancy diagnosed amongst women, with more the two million new cases identified globally each year. Thus, considerable resources have been devoted to a better understanding of the disease and how to optimize outcomes for patients. Genetics and genomics have become part of routine care with the latter used to inform treatment planning for patients with ER+ breast cancer (ER+ BC). A multitude of assays may be used to determine the need for adjuvant chemotherapy but none of the current tests on the market are guideline-recommended for making decisions related to neoadjuvant chemotherapy, surgery, or radiation. The 24-gene StemPrintER assay was developed to measure the “stemness” of tumors, or how much a tumor behaves like stem cells, which could have important and novel implications for breast cancer treatment planning. The assay has been validated for predicting prognosis using formalin-fixed paraffin-embedded (FFPE) tissue blocks from several clinical cohorts, including TransATAC. The goal of current and future research will be to determine whether the assay can determine the ideal surgical intervention for ER+BC patients. Early in silico work demonstrated a trend toward this predictive capacity for the assay and suggests the assay may be able to identify the benefit of mastectomy versus quadrantectomy. Here, we report on the ability to set up and run the assay in our lab using synthetic oligonucleotides and archival tissue blocks. Methods: Synthetic oligonucleotides and archival FFPE breast cancer tissue were used to set up and analytically validate the assay’s performance in two phases. The initial phase was intended to confirm the individual assay design performance by assessing linearity and precision and the ability to amplify RNA from FFPE, which confirmed compatibility with standard extraction methods. The second phase was intended to determine whether these methods could be implemented to reliably extract and amplify RNA from FFPE &amp;gt;10 years old (range: 10-14 years) in order to generate a StemPrintER score. The assay was run according to standard operating procedures for StemPrintER. Analyses were conducted Python 3.9.12. Results: Two assay runs with duplicate samples for each run using a 7-point dilution series demonstrated linearity (coefficient of correlation, r2) results ranging from 0.978 to 0.999 for each of the 24 genes in the assay. Amplification efficiency was between 100-101% for each gene. Using 14 FFPE samples from patients with triple-negative breast cancer, we demonstrated a consistent extraction yield of RNA from tumor blocks which produced amplification of each of the targets. Analysis of intra- and inter-run precision resulted in coefficients of variation (CV) ranging from 0.2-1.07% and inter-run CV’s ranging from 0.66%-5.56%. Utilizing FFPE &amp;gt;10 years old from ER+BC patients demonstrated that the lab could extract RNA from 100% of samples and amplify the material to obtain binary StemPrintER results. Conclusion: These data demonstrate that StemPrintER can be run reliably on FFPE tissue up to 14 years old. In addition, our laboratory has demonstrated that assay results are highly linear and reproducible. Additional studies are planned to build on preliminary surgical prediction data and determine whether the assay can meet the unmet need of identifying the ideal surgical intervention in patients with early-stage ER+/HER2- breast cancer. Citation Format: Catie Cronister, Brock Schweitzer, Hannah Gilmore, Tyler Nielsen. Analytical Validation of the StemPrintER Assay in Patients with Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-14-03.

Confirmed bacteriological diagnosis and cure of non-severe Gram-positive clinical mastitis cases enrolled in a randomized clinical trial based on results of on-farm culture

Our objectives were to describe the confirmed bacteriological diagnosis of mastitis pathogens cultured from cases of non-severe clinical mastitis (CM) identified as Gram-positive (GP) using on-farm culture and to compare differences in bacteriological cure (BC) based on antimicrobial (AM) therapy. Cows that developed non-severe CM in a single quarter were enrolled in a randomized clinical trial based on microbial growth on GP selective agars. Cows were randomly assigned to receive a once-daily intramammary treatment: 3d hetacillin (n = 69), 3d ceftiofur (n = 69), 8d ceftiofur (n = 70) or to a non-treated group (n = 32). Etiologies were confirmed with MALDI-TOF using frozen duplicate milk samples. The crude proportion of BC was determined using milk samples collected at 14, 21 and 28 d after enrollment for cases caused by Streptococci, non-aureus staphylococci (NAS), Strep-like organisms (consisting of Enterococcus and Lactococcus.; SLO) and Staph. aureus. Multivariable models were used to determine the effect of AM treatment on BC cure at d 21 for a subset of cases that had complete data for all covariates (n = 88). Growth on GP agar resulted in enrollment of 240 cases with confirmed etiologies distributed as Strep. spp (21.7%; n = 52), Lact. spp (19.2%; n = 46), NAS (16.3%; n = 39), Staph aureus (6.3%; n = 16), Entero spp (5.0%; n = 12) and others (10%; n = 24). Thirty-five (14.5%) duplicate milk samples collected from enrolled cows had no significant growth in the lab while 16 (6.7%) were contaminated. Among sampling dates, combined crude BC for cases caused by target pathogens ranged from 68 to 71%. Crude BC ranged from 58 to 73% for the small group of non-treated cases (n = 12) and 69–71% for the combined group of cases that received IMM AM (n = 113). Among all groups and all follow-up dates, crude BC was least for cases caused by Staph. aureus (ranging from 33 to 43%), followed by cases caused by SLO (58–67%), NAS (79–80%) and Strep. spp. (81–91%). The proportion of BC at 21d (LSM ± SE) did not vary between all cases that received AM (0.77 ± 0.06) and cases in the non-treated group (0.73 ± 0.16). The odds of BC at 21d was about 5x greater for cases that occurred in primiparous as compared with multiparous cows and decreased 1.3-fold for each 1 log unit increase in SCC before the case. While this study does not have sufficient power to detect small differences in BC among pathogen groups or treatments, our results demonstrate that CM is caused by a wide diversity of GP bacteria with varying ranges of BC. These results provide justification for inclusion of negative control groups in future studies and confirm that important characteristics such as parity and history of subclinical mastitis influence the probability of BC.

Repeatability of Platelet Aggregation Characteristics Using Impedance Aggregometry With and Without Aspirin Intervention

Nearly 40% of adults over 50-years-old regularly take aspirin for primary or secondary prevention of cardiovascular disease and/or adverse cardiovascular events. The effcacy of aspirin therapy is commonly assessed with light transmission aggregometry. However, this method requires removal of large platelets as well as the mechanical disturbance of platelets remaining to be tested in the sample. Impedance aggregometry presents a solution to this problem, but the repeatability of impedance aggregometry analysis remains to be tested. We hypothesized repeated impedance aggregometry analysis would show decreased platelet aggregation activity following aspirin intervention compared with control visits. Venous blood samples were obtained from ten participants on three occasions each, separated by at least two weeks. One sample followed seven days of aspirin intervention (81 mg once daily; ASA) and the other two with no intervention (control). Platelet aggregation was induced using 1 μg/ml collagen (Col1), 5 μg/ml collagen (Col5), and 0.5 mM arachidonic acid (AA) via impedance aggregometry to determine area under the aggregation curve (AUC, au), time to aggregation initiation following addition of reagent (Lag, s), the rate of aggregation (Slope), and the maximal aggregation (Amp, Ohms). These characteristics were statistically analyzed for an impact of aggregometer channel (intra-test repeatability), visit (inter-test repeatability), intervention (ASA or control), and incubation (samples were incubated with either saline or Terutroban solution to block thromboxane receptors). Results were analyzed by multiple paired t-tests and represented with lowest relevant p-value if insignificant or highest relevant p-value if significant. Impedance aggregometry revealed no difference in any aggregation characteristics (AUC, Lag, Slope, or Amp) for duplicate samples (all p ≥ 0.10) or visits (all p ≥ 0.06). ASA decreased AUC, Slope, and Amp while increasing or completely inhibiting Lag response to the addition of all reagents (all p ≤ 0.01). Terutroban incubation had no effect on aggregation characteristics following the ASA intervention (all p ≥ 0.09), but attenuated the AUC, Slope, and Amp (all p ≤ 0.01), and entirely prevented or substantially increased the Lag response to AA (p ≤ 0.01) without impacting the responses to Col1 or Col5 (all p ≥ 0.24). These findings suggest the impedance aggregometry approach is a repeatable method for platelet aggregometry analysis for the characteristics of AUC, Lag, Slope, and Amp. NIH R01 Grant 1R01AG067471-01 NIH F31 Grant 1F31HL170616-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.