Analysis Of Samples Research Articles

Quantification and Functional Studies of Neutrophilic Cells Identifies Distinct Papilloma Phenotypes.

To characterize the distribution of immune cell subsets within laryngeal papillomas and to study the function of potentially immunosuppressive neutrophilic and regulatory T cells (Tregs). Fresh clinical papilloma specimens were collected at the time of surgery and studied with multiparameter flow cytometry. Papilloma infiltrating neutrophilic cells and Tregs were sorted and studied functionally with ex vivo T cell suppression assays. Flow cytometric analysis of fresh laryngeal papillomas samples from 18 adult patients with recurrent respiratory papillomatosis revealed patterns in immune constituency between patients. Clearly divergent phenotypes based primarily on the degree of neutrophilic and T cell infiltration were identified. Relative neutrophilic cell enrichment and T cell depletion were observed in 50% of samples and neutrophilic cell depletion and T cell enrichment were observed in the others. Greater papilloma neutrophilic cell enrichment was positively associated with the number of clinically indicated interventions required in the 12 months prior to sample collection, linking papilloma neutrophil inflammation to disease severity. Functional assays revealed the ability of both papilloma infiltrating neutrophilic and Tregs to suppress T cell function at roughly equal magnitudes, but substantially increased infiltration of neutrophilic cells compared to Tregs across samples. Neutrophilic cells are an important contributor to immunosuppression within the respiratory papilloma microenvironment. Given these data and the association between greater neutrophilic cell infiltration and lack of clinical response to therapeutic vaccination, additional study of strategies aimed at limiting neutrophilic cell infiltration or function within papillomas is warranted. 4 Laryngoscope, 134:3238-3244, 2024.

Screening for emerging chemical risks in the food chain (SCREENER)

Abstract In the European Union, all chemical substances that are manufactured, imported or used in tonnages equal or higher than one tonne per year must be registered under the REACH regulation. This regulatory framework requires the collection and dissemination of detailed information on the properties, uses, and potential health and environmental impacts of chemicals. However, some of these chemicals may pose emerging risks in the food chain, necessitating the development of approaches to identify and assess such risks. The SCREENER project addressed this critical issue by analysing food items for the presence of chemicals that may pose unrecognised hazards. Building on the findings from the previous REACH 1 and REACH 2 projects, which identified a suspect list of 212 chemicals, qualitative multi‐residue high‐resolution mass spectrometry (HRMS) methods have been developed. Diverse food items such as wheat flour, kale, carrots, potatoes, peas, strawberries, oranges, meat, hen's eggs, and trout were pooled from 3 samples each, while cow's milk, and other fish types (salmon and herring) were pooled from 4 samples each to form a single sample. This pooling procedure resulted in 194 analytical samples, which were screened for the presence of the chemicals in the suspect list. Additionally, non‐target analysis (NTA) was performed on the same samples, focusing on detecting halogenated compounds. The aim was to detect substances potentially introduced into the food chain unintentionally through industrial and anthropogenic activities, which were previously unrecognised in the food chain. In the final stage of the project, 15 chemicals were further prioritized for identification and quantitative analysis. Quantitative methods were developed and then applied to the same 194 samples, allowing for the identification and quantification of those chemicals. Subsequently, hazard characterization, exposure assessment, and risk characterisation were conducted. A preliminary characterization of the potential risk posed by the chemicals found in the samples during quantitative confirmatory analysis indicated no risk to human health in all but three cases where additional analysis on occurrence and detailed evaluation of the hazard can be considered as a possible follow‐up action in the future.

THC Content on a Dry Weight Basis: Implications for Hemp Legality.

The 2018 Farm Bill defines hemp as Cannabis sativa L. with a tetrahydrocannabinol (THC) concentration not more than 0.3% on a dry weight basis where THC is implied to be total THC (THCTotal) including both acid (Δ9-THCA) and neutral (Δ9-THC) forms. Effect of temperature and mass were studied to determine the quickest time to prepare fresh hemp suitable for grinding without affecting THCTotal. Proficiency testing program data were also analyzed to compare THCTotal contents on a dry versus wet weight basis. Fresh hemp of 150 and 400 g were freeze-dried or heat-treated at 50 °C, 60 °C, and 70 °C for various lengths of time up to 72 hours. Ground hemp was analyzed for moisture content via AOAC 934.01 and THCTotal via liquid chromatography/mass spectrometer (LC/MS) or gas chromatography with flame ionization detection (GC/FID). A data set analyzed from a proficiency testing program included moisture and THCTotal from 20 to 67 laboratories on 12 analytical samples circulated from 2020 through 2022. The quickest drying time of 24 h occurred at 70 °C with 150 g. These conditions did not affect THCTotal content on a dry weight basis. Twelve proficiency testing program analytical samples ranged from 5.8 to 11.4% moisture and 0.144 to 0.399% THCTotal on a wet weight basis. An increase in reported THCTotal on a dry weight basis compared to a wet weight basis ranged from 0.002 to 0.027% with a significant difference occurring in only one analytical sample. Suitable condition for drying hemp without altering THCTotal content was 24 h at 70 °C. Determining residual moisture in oven-dried ground hemp to calculate THCTotal content on a dry weight basis provides no benefit given minimal difference between contents on dry versus wet weight bases, interlaboratory variability, and terpene volatilization.

Determination of 3-acetyl-11-keto-β-boswellic acid in analytical and biological samples using streamlined and robust RP-HPLC method.

AKBA (3-acetyl-11-keto-β-boswellic acid) is a phytoconstituent derived from Boswellia serrata extract and utilized in the management of rheumatoid arthritis. Drug delivery approaches showed interest in delivering AKBA with advanced nanotechnology. There is a need for a simple, sensitive, and robust HPLC method that can determine AKBA in complex nanoformulation and in vitro and ex vivo samples. In the proposed work, the RP-HPLC method was developed using a mobile phase comprising a mixture of acetonitrile : milli Q (90 : 10) at detection λmax 250 nm. The method exhibited a linearity of 250 to 20 000 ng mL-1 with a high correlation coefficient of 1. The limit of detection and limit of quantification for the analytes were found to be 41.32 ng mL-1 and 125.21 ng mL-1, respectively. In the accuracy study, the % recovery of AKBA was found to be 98% to 102%, and the precision study showed less than 2% relative standard deviation. The developed method was found to be robust under chromatographic conditions with changes in pH and mobile phase mixture ratio. The method was also explored for forced degradation study, and the results showed the successful separation of degradation products from the AKBA. Further, the RP-HPLC method was applied for the quantification of AKBA in topical nanoformulations and different matrices, such as skin matrices and adhesive tapes. The method was able to measure entrapment efficiency (93.13 ± 1.94%), drug loading (25.83 ± 0.54%), drug assay in a gel matrix (96.99 ± 3.89%), drug amount in stratum corneum (7.90 ± 0.62 μg cm-2), and drug amount in viable skin layers (33.94 ± 0.21 μg cm-2) with high-speed reproducibility. The developed method can be utilized for the routine analysis of AKBA in conventional and complex formulations in academia and industry.

Salivary and serum haptoglobin, adenosine deaminase, and immunoglobulin G in growing pigs

BackgroundIdentification of animals in need of medical treatment is important in porcine health management, where analytical samples applicable at farm level could be utilized. Several biomarkers are measurable in saliva, which is less stressful to collect than blood. Saliva sampling is easy to learn and repeatable, making it suitable for monitoring purposes. Previous research suggests that porcine health biomarkers are dependent on production stage and gender, and that combining biomarkers improves diagnostic sensitivity. However, proper monitoring of biomarkers during the complete production cycle has not been studied. We aimed to describe the dynamics of salivary and serum haptoglobin (Hp), adenosine deaminase (ADA), and immunoglobulin G (IgG) in four production stages (suckling, early growing, late growing, finishing), on commercial Finnish pig farms using a total of 117 piglets. The relationship between gender and biomarker dynamics was investigated, as well as the relationships between these biomarkers in saliva and serum.ResultsThe highest salivary concentrations of Hp, ADA and IgG were measured in suckling piglets. The differences between production stages were generally larger in saliva than for the corresponding serum biomarkers. All correlation coefficients between salivary biomarkers were positive in each production stage and the strength of the correlation varied from 0.245 to 0.762. No similar trend was observed regarding correlation coefficients either between serum biomarkers or between salivary and serum biomarkers. Gender was associated with some biomarker concentrations.ConclusionsThe biomarker dynamics supported previous findings that collection of analytical samples should be conducted in age-matched populations. Positive and even strong relationships between salivary biomarkers indicate the potential to use especially saliva for health monitoring. Our results also suggest the importance of considering gender effects when assessing some salivary or serum biomarkers.

Legionella detection from clinical and environmental samples

Several methods are used for diagnosis of legionellosis: culture, urinary antigen detection, serologic tests, PCR etc., characterised by different level of specificity, sensitivity and rapidity. Microbiological diagnosis is an essential tool to for the prompt adoption of an adequate antimicrobial therapy and to understand the real diffusion of legionellosis. Likewise, the environmental samples analysis allows us to know the distribution of the Legionella in the environment and to detect the origin of the infection during an outbreak by comparing clinical and environmental strains.

Evaluation of conditions to minimize mercury losses from leaching test eluates

A series of experiments are described that provide recommendations of modifications to leaching test specifications to minimize losses of mercury from eluates during leaching tests and management of analytical samples. EPA methods of the Leaching Environmental Assessment Framework (LEAF) were used as a reference point for what steps are most likely to see losses, including filtration, storage of unpreserved mercury contaminated material, and materials of construction for vessels. The potential sorption of mercury to borosilicate glass and PTFE lab materials was tested to determine what materials are suitable to replace HDPE and polypropylene. The experimental findings indicate that bottles constructed of PTFE and Type I (borosilicate) glass are recommended as extraction and containment vessels for mercury-containing liquids. Type I syringe filtration of aqueous mercury samples at pH 2 through 9 through 0.45 µm PTFE filters housed in polypropylene is acceptable; however, sorption losses were observed to occur at pH > 9. Storage of unpreserved liquids containing mercury over extended leaching test intervals did not lead to significant losses of mercury when coupled with minimized headspace. All mercury-containing eluates should include preservation with Optima HNO3 to a 1 % concentration and gold (III) standard to a concentration of 200 µg/L, followed by refrigeration at <6 °C prior to analysis.

Evaluation of the Performance of Wastewater Treatment Plant at Steel Mill Karachi, Pakistan

The purpose of this study is to evaluate how well Steel Mill Karachi's wastewater treatment system works. Wastewater treatment facilities employ the activated sludge technique. Several influent and effluent locations allowed for the collection of influent and effluent samples. A number of physicochemical parameters, such as total dissolved solids (TDS), biochemical oxygen demand (BOD5), total suspended solids (TSS), chemical oxygen demand (COD), electric conductivity (EC), pH, and other metals, such as potassium (K), iron (Fe), sulphate (SO4-2), calcium (Ca), sodium (Na), zinc (Zn), and magnesium (Mg), were measured in order to evaluate the effectiveness of wastewater treatment plants. In a lab environment, the sample's analysis findings were compared to Sindh Environmental Quality Standards (SEQS) for Pakistan. EC, BOD5, COD, SO4-2, Ca, Mg, Na, K, TDS, TSS, EC, BOD5, COD, SO4-2, Ca, Mg, Na, K, Fe, and Zn were removed to different degrees: 42.93%, 60.33%, 43.63%, 51.53%, 40.5%, 35.56%, 26.33%, 22.12%, 34.09%, 20.75%, 55.87%, 22.12%, and 76.10%. Only two metrics, the COD and Zn concentrations, were over the SEQS limits, keeping other effluent results parameters within the limits. Additionally, it is advised that proper maintenance be carried out to increase the effectiveness of wastewater treatment plants.

Combining TIGIT blockage with MDSC inhibition hinders breast cancer bone metastasis by activating anti-tumor immunity.

Bone is the most common site of breast cancer metastasis. Bone metastasis are incurable and are associated with severe morbidity. Utilizing an immunocompetent mouse model of spontaneous breast cancer bone metastasis, we profiled the immune transcriptome of bone metastatic lesions and peripheral bone marrow at distinct metastatic stages, revealing dynamic changes during the metastatic process. We show that crosstalk between granulocytes and T cells is central to shaping an immunosuppressive microenvironment. Specifically, we identified the PD-1 and TIGIT signaling axes and the pro-inflammatory cytokine IL1b as central players in the interactions between granulocytes and T cells. Targeting these pathways in vivo resulted in attenuated bone metastasis and improved survival, by reactivating anti-tumor immunity. Analysis of patient samples revealed that TIGIT and IL1b are prominent in human bone metastasis. Our findings suggest that co-targeting immunosuppressive granulocytes and dysfunctional T cells may be a promising novel therapeutic strategy to inhibit bone metastasis.

Measuring Linguistic Growth in Sentence-Level Writing Curriculum-Based Measures: Exploring Complementary Scoring Methods.

Picture-word writing curriculum-based measures (PW CBM-Ws) are technically sound, formative measures of descriptive, sentence-level writing but cannot estimate underlying linguistic skills. The purpose of this exploratory alternative scoring investigation was to apply metrics from language sample analysis (LSA) to PW CBM-Ws as a complementary measure of underlying language skills in beginning writers' sentence-level writing. LSA metrics were applied to 104 typically developing first through third graders' PW CBM-W samples across fall and spring semesters. Factorial analyses of variance with post hoc Bonferroni pairwise comparisons were applied after obtaining alternate-form reliability and criterion-related validity estimates. Analyses revealed reliable discrimination between grades and significant growth between fall and spring semesters for three LSA metrics: mean length of T-unit in words, mean length of T-unit in morphemes, and number of different words. While mean length of T-unit in words and morphemes demonstrated evidence of discrimination and growth in first grade only, number of different words showed evidence of reliable discrimination and growth in first and third grades. Mean length of T-unit in words, mean length of T-unit in morphemes, and number of different words showed evidence of adequate criterion-related validity, discrimination among grades, and sensitivity to growth when calculated using PW CBM-W samples to gauge underlying linguistic skills in first- and third-grade students. Implications and future directions for research are discussed. https://doi.org/10.23641/asha.25050290.

The effect of potassium levels on electrocardiographic data in calves with neonatal diarrhea

Objective: The objective of the current study was to assess the potassium levels and electrocardiographic (ECG) data in a cohort of 40 calves (age &lt; 30 days) diagnosed with newborn diarrhea, originating from dairy farms located in Ankara, Turkey.&#x0D; Materials and Methods: This study was conducted on a dairy farm located in Ankara, involving a population of 500 dairy animals. The calves were divided into two groups: one group with normal potassium levels (n=24) and another group with hyperkalemia (n=16) due to neonatal diarrhea. After the initial clinical examination was conducted on the farm, electrocardiographic investigations were carried out utilizing an ECG equipment. The biochemical analysis of blood samples that were collected in red and dry collecting tubes was carried out with the assistance of an automated analyzing machine &#x0D; Results: The findings indicate that there were no statistically significant differences observed across all ECG variables between the two groups.&#x0D; Conclusion: Acidemia and metabolic acidosis are commonly observed in calves suffering from hyperkalemic diarrhea, as documented in previous investigations, often accompanied by heart rhythm problems. In contrast, the current investigation revealed that neonatal calves with diarrhea and hyperkalemia had just a deepening of the T wave, without any additional abnormalities. Nevertheless, it is important to note that acidemia and metabolic acidosis were not of a severe nature. The implementation of Holter monitoring is indicated for calves experiencing hyperkalemia in conjunction with diarrhea.

A Cone Beam CT Bronchoscopy Study of the Ultrathin Cryoprobe for Biopsy of Peripheral Lung Lesions.

Compared with the standard cryoprobe, the novel ultrathin 1.1mm cryoprobe (UTCP) has improved ergonomics, shape memory, and flexibility. The performance of UTCP has demonstrated promising results in several small trials. In this single-center, retrospective review, we examine 200 (N=200) consecutive patients referred for cone beam CT bronchoscopic biopsy of peripheral lung lesions. We utilized an extended multimodality approach, including transbronchial needle aspirate, brush, traditional forces biopsies, UTCP biopsies, and BAL. We analyzed tool in lesion, tool touch lesion, center strike rates, and diagnostic yield. We assessed for molecular adequacy and analyzed safety. A total of 222 lesions were biopsied. We achieved a tool in lesion or tool touch lesion confirmation for all biopsy attempts (100%) and a center strike rate of 68%. AQuIRE diagnostic yield was 90%, with 60% malignant, 30% benign lung nodules, and 10% nondiagnostic. UTCP was diagnostic in 3.6 % of peripheral lung lesions biopsies when all other modalities were nondiagnostic; thus, raising our overall diagnostic yield from 86.4% to 90.1%. Our analysis demonstrates superior adequacy for molecular analysis for histologic samples (TBBX or UTCP) versus cytologic samples (FNA) ( P <0.001). Three patients (1.5%) had a pneumothorax, and 1 patient (0.5%) had moderate bleeding. UTCP was diagnostic in 3.6% of peripheral lung lesions when all other modalities were nondiagnostic. In the setting of CBCT guidance, UTCP has a similar safety profile to standard biopsy tools. Future trials are warranted to assess UTCP and its impact on peripheral lung lesion biopsies.

Characteristic Features of Kolludere Valley (Bitlis-Hizan) Honey

This study was carried out to reveal the characteristic features of honey produced in the Kolludere Valley, which is located within the borders of the Hizan district of Bitlis province. This area is isolated as there are no seasonal migratory beekeepers. In 2022, a flora study was carried out in the area and 133 plant taxa belonging to 19 families from which bees receive nectar or pollen were identified. 23 of these taxa are endemic. Content analysis of honey samples taken from the study area was carried out. Proline value, which is an important parameter of honey quality, was determined as 809.41 mg kg-1, diastase number 28.9, HMF 2.9 mg kg-1, and sucrose 0.2 g/100g. All other parameters (humidity, acidity, pH, fructose+glucose, fructose/glucose, saccharose, maltose, electrical conductivity) were also met standart according to the values of the Food Codex Honey Communiqué and European Union Standards. In addition, pollen analysis of honey samples was made and the data were compared with the flora. In honey samples, pollen is generally minor or rare, and pollen of a dominant taxon was found in only one sample. For this reason, most of the honey produced in the Kolludere Valley was evaluated as flower honey.

Rostrupomyces, a new genus to accommodate Xerocomussisongkhramensis, and a new Hemileccinum species (Xerocomoideae, Boletaceae) from Thailand.

A new genus, Rostrupomyces is established to accommodate Xerocomussisongkhramensis based on multiple protein-coding genes (atp6, cox3, tef1, and rpb2) analyses of a wide taxon sampling of Boletaceae. In our phylogeny, the new genus was sister to Rubinosporus in subfamily Xerocomoideae, phylogenetically distant from Xerocomus, which was highly supported as sister to Phylloporus in the same subfamily Xerocomoideae. Rostrupomyces is different from other genera in Boletaceae by the following combination of characters: rugulose to subrugulose pileus surface, white pores when young becoming pale yellow in age, subscabrous stipe surface scattered with granulose squamules, white basal mycelium, unchanging color in any parts, yellowish brown spore print, and broadly ellipsoid to ellipsoid, smooth basidiospores. In addition, Hemileccinuminferius, also from subfamily Xerocomoideae, is newly described. Detailed descriptions and illustrations of the new genus and new species are presented.

Incorporating machine learning and PPI networks to identify mitochondrial fission-related immune markers in abdominal aortic aneurysms

PurposeThe aim of this study is to investigate abdominal aortic aneurysm (AAA), a disease characterised by inflammation and progressive vasodilatation, for novel gene-targeted therapeutic loci. MethodsTo do this, we used weighted co-expression network analysis (WGCNA) and differential gene analysis on samples from the GEO database. Additionally, we carried out enrichment analysis and determined that the blue module was of interest. Additionally, we performed an investigation of immune infiltration and discovered genes linked to immune evasion and mitochondrial fission. In order to screen for feature genes, we used two PPI network gene selection methods and five machine learning methods. This allowed us to identify the most featrue genes (MFGs). The expression of the MFGs in various cell subgroups was then evaluated by analysis of single cell samples from AAA. Additionally, we looked at the expression levels of the MFGs as well as the levels of inflammatory immune-related markers in cellular and animal models of AAA. Finally, we predicted potential drugs that could be targeted for the treatment of AAA. ResultsOur research identified 1249 up-regulated differential genes and 3653 down-regulated differential genes. Through WGCNA, we also discovered 44 genes in the blue module. By taking the point where several strategies for gene selection overlap, the MFG (ITGAL and SELL) was produced. We discovered through single cell research that the MFG were specifically expressed in T regulatory cells, NK cells, B lineage, and lymphocytes. In both animal and cellular models of AAA, the MFGs' mRNA levels rose. ConclusionWe searched for the AAA novel targeted gene (ITGAL and SELL), which most likely function through lymphocytes of the B lineage, NK cells, T regulatory cells, and B lineage. This analysis gave AAA a brand-new goal to treat or prevent the disease.

Exploring the potential of SrTi0.7Fe0.3O3 perovskite/Chitosan nanosheets for the development of a label-free electrochemical sensing assay for determination of naproxen in human plasma samples

Naproxen is a nonsteroidal anti-inflammatory drug used to treat nonrheumatic inflammation, migraine, and gout. Therefore, the determination of naproxen in pharmaceutical and biological samples is of particular importance. In the present work, SrTi0.7Fe0.3O3 perovskite/Chitosan nanosheets were used to modify the surface of a glassy carbon electrode (GCE) for highly sensitive determination of naproxen. To ensure the successful synthesis of the perovskite nanosheets, morphological studies including scanning electron microscopy (SEM), Energy-dispersive X-ray (EDX), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS) were carried out. The electrochemical investigations of naproxen on the modified surface of GCE were investigated and the limit of detection (LOD) and limit of quantification (LOQ) were acquired 0.50 and 1.67 μM, respectively. Additionally, the linear range (LR) of 1.99–130.84 μM was obtained for the oxidation of naproxen. The obtained results have been proved that the mentioned method is simple, sensitive, and specific with a short analysis time. The dominant analytical features of the designed sensor are possessing a low detection limit, excellent stability, repeatability, and high selectivity in the presence of naproxen. For investigation of the applicability of the designed assay in real sample analysis, human plasma samples have been examined and a recovery index was acquired 95%.

Correlation of Geotechnical and Mineralogical Properties of Lithomargic Clays in Uttara Kannada Region of South India

The present study explores the intricate relationship between the geotechnical and mineralogical properties of lithomargic clays in the Uttara Kannada region of south India. Lithomargic clays, characterized by their unique composition of clay minerals and calcareous content, play a crucial role in the geotechnical behavior of soils. The study aims to provide a comprehensive understanding of the interplay between the mineralogical composition and engineering characteristics of lithomargic clays, shedding light on their suitability for various construction and infrastructure projects. The research methodology involves a systematic analysis of lithomargic clay samples collected from different locations in the Uttara Kannada region. Geotechnical investigations, including particle size distribution, Atterberg limits, unconfined compressive strength (UCS), California bearing ratio (CBR) and triaxial tests, are conducted to assess the engineering properties of the clays. Concurrently, mineralogical analyses, such as X-ray diffraction (XRD) and scanning electron microscopy (SEM), are employed to identify and quantify the clay mineral constituents within the samples. The findings of this study reveal correlations between specific mineralogical features and geotechnical behaviors of lithomargic clays. Understanding these relationships is crucial for predicting the response of these clays to different engineering applications, including slope stability, foundation design and embankment construction. The research contributes valuable insights to the scientific and engineering communities, aiding in the informed utilization of lithomargic clays in geotechnical projects in the Uttara Kannada region and beyond. The outcomes of this investigation, such as the correlation of geotechnical properties with the variation in minerals in various sample locations, enhance our understanding of the complex nature of lithomargic clays, providing a foundation for more sustainable and effective engineering practices in the geologically diverse landscapes of south India.

A Novel Validated GC-MS/MS Method for the Estimation of N-Nitroso Dimethyl Amine and N-Nitroso Diethyl Amine in Zidovudine.

A novel method has been developed for the estimation of N-Nitroso dimethyl amine impurities (NDMA) and N-Nitroso diethyl amine (NDEA) in Zidovudine by using Gas chromatograph Triple Quadrupole Mass with Liquid autosampler (GC-MS/MS) and the method is validated as per International Conference on Harmonization recommendations. Sample analysis was executed for Zidovudine by developed method. Both NDMA and NDEA were detected in below quantitation limit for the Zidovudine batches. Efficient chromatographic separation was achieved on a DB-WAX 30m length × 0.25mm internal diameter, 0.5-μm film thickness, Triple quad-8040 GC-MS/MS. Quantification was carried out at Triple quad electron ionization source was at a column flow of 1.5mL/min at a column oven temperature 50°C. The precision was in the range of 0.9-2.5% for NDMA and 0.8-2.3% for NDEA, and regression analysis shows as r value (correlation coefficient) of is >0.99. This method is capable to detect the NDMA and NDEA impurities in Zidovudine at a level of 0.006ppm for limit of detection and 0.018ppm for limit of quantitation with respect to test concentration of 66.66mg/mL.

Abstract 4039: Enhanced anti-cancer efficacy of the AhR Inhibitor DA-4505 in combination with anti-PD-1 treatment: Attenuation of lung metastasis and elimination of tumor

Abstract Background: The Aryl hydrocarbon receptor (AhR) is one of the most predominant regulators of cancer metabolism. AhR plays a crucial role in inhibiting the activation of immune cells and promoting the growth of tumor cells. Here, we propose that a best-in-class AhR inhibitor, DA-4505, improves anti-tumor efficacy in combination with anti-PD-1. Methods: To explore the anti-tumor effects, DA-4505 was administered at a daily dose of 10 mg/kg either alone or in combination with anti-PD-1 (10 mg/kg) in a syngeneic mouse model. Tumor volume, survival rates, and metastasis were measured, and immune profiles were evaluated using mIHC, flow cytometry, and scRNAseq. Results: The synergistic anti-tumor effects of the DA-4505 and aPD-1 combination therapy were observed across four mouse tumor models (CT26, 4T1, LLC, TC1). The combination group demonstrated a significant improvement in anti-tumor efficacy compared to the monotherapy groups. Despite the moderate response of aPD-1 in the CT26 colon tumor model, the combination group displayed heightened anti-tumor effects through increase in CD8 T cell activity (P&amp;lt;0.05). In the LLC and TC1 lung cancer models, the combination therapy demonstrated improved anti-tumor effects and survival rates (P&amp;lt;0.001). Notably, the LLC model exhibited a significant increase in M1-type macrophages, while the TC1 model showed a significant augmentation in functional markers of CD3+ T cells within the combination group (P&amp;lt;0.05). In both models, there was a significant reduction in the ratio of immunosuppressive M-MDSC and PMN-MDSC in the combination group. Immune depletion assays elucidated that DA-4505 mediated anti-tumor effects by increase in CD8+ T cells, NK cells, and macrophages in the CT26 model. In contrast, the LLC model exhibited the contribution of macrophages and CD4+ T cells to anti-tumor effects in the combination group (P&amp;lt;0.05). To assess the impact on metastasis inhibition, an orthotopic model of 4T1 mouse tumor was established, revealing a significantly increased anti-tumor effects and survival rate in the combination group, accompanied by a reduced lung metastasis ratio. H&amp;E staining confirmed diminished migration of tumor tissue to adjacent areas. scRNAseq analysis of CT26 tumor samples indicated consistent changes at the gene expression level, reflecting a decrease in metastasis-related MMP and VEGF genes in the combination group and an increasing trend in cytotoxicity-related genes following DA-4505 treatment. Conclusion: The AhR inhibitor, DA-4505, has demonstrated enhanced anti-tumor efficacy when administered in combination with anti-PD-1. Furthermore, it not only promoted tumor elimination but also demonstrated significant efficacy in inhibiting metastasis. This study highlights the potential of combining AhR inhibitors with immunotherapies for clinical treatment. Citation Format: Dong Kwon Kim, Sujeong Baek, Seung Min Yang, Young Taek Kim, Joon Yeon Hwang, Seul Lee, Seong-san Kang, Mi hyun Kim, Heekyung Han, Kwangmin Na, Chai Young Lee, Yu Jin Han, Taedong Han, Hyounmie Doh, Jongho Cho, Dajeong Kim, Daewon Cha, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Mi Ran Yun, Jii Bum Lee, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. Enhanced anti-cancer efficacy of the AhR Inhibitor DA-4505 in combination with anti-PD-1 treatment: Attenuation of lung metastasis and elimination of tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4039.

Abstract 4920: A nanopore sequencing approach characterizes cell-free DNA methylation-fragmentomics profiles indicative of breast cancer in a large cohort

Abstract The detection of breast cancer from cell-free DNA (cfDNA) offers a new approach with the potential to benefit individual screening and early detection. However, identifying breast cancer from cfDNA remains a major challenge when one uses conventional short-read sequencing. Methylation and DNA fragmentation characteristics provide highly informative biomarkers for detecting and monitoring cancer. To characterize these genomic features from cfDNA, we developed an approach using single molecule Nanopore sequencing. Single cfDNA molecules are sequenced intact with the methylation and fragment size directly extrapolated from Nanopore data. Moreover, our approach requires small amounts of cfDNA to discern specific genomic features from individual molecules. Thus, the use of Nanopore sequencing provides both methylation and fragment size as directly measured from native cfDNA without the use of any chemical processing or other molecular manipulation. We studied an extended breast cancer cohort with in total 1080 of samples, consisting of 440 untreated breast cancer patients and 640 healthy controls. Nanopore sequencing analysis of these cfDNA samples identified a range of 1 to 4 million CpG sites from 1 ml of plasma. Leveraging the cohort, we developed an ensemble classifier for breast cancer detection based on cfDNA methylation and fragmentation. The classifier leveraged a leave-one-out cross-validation (LOOCV) to generate predicted probability. We created nucleosome-specific methylation scores as the input features to the classifier, which scores quantified the similarity of each sample's methylation profile to a training set on a selected set of CpG sites associated with cancers. Furthermore, we also identified DNA fragmentation features, revealing distinct mono-nucleosome and di-nucleosome patterns that distinguish cancers from healthy controls. Aggregating the DNA nucleosome-specific scores and fragmentation features, we built an ensemble LOOCV random forest model. We applied the model to the breast cancer cohort and achieved a detection specificity of 74%, a sensitivity of 71%, and an overall accuracy of approximately 73%. The area under the receiver operating characteristic curve (AUROC) reached 80%. We validated the classifier with two external independent cohorts. Validation cohort 1 demonstrated as a specificity of 85%, a sensitivity of 80%, and overall accuracy of 83% with AUROC of 90%. Validation cohort 2 without controls demonstrated that the classifier achieved a sensitivity of 94%. Both validation sets confirm that our model can achieve excellent performance for early breast cancer detection. In summary, our findings demonstrate that nanopore sequencing provides cfDNA methylation and fragment profiling that enables breast cancer detection. This approach holds the potential to improve cancer diagnosis and treatment outcomes. Citation Format: Xiangqi Bai, Billy T. Lau, Alison Almada, Sue Grimes, Tianqi Chen, Hojoon Lee, Hanlee P. Ji. A nanopore sequencing approach characterizes cell-free DNA methylation-fragmentomics profiles indicative of breast cancer in a large cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4920.