Reagent Kit Research Articles

Abstract LB309: Influence of pathological pericytes on PDAC tumor progression and tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in men and women in the U.S., with a 5-year survival rate of 12 %. This poor prognosis is partly due to the aggressive nature of the disease, lack of early detection methods, its complex and dense tumor microenvironment (TME), and lack of effective treatment options. Within the PDAC TME, morphologically aberrant leaky vessels are responsible for hypoxia and impaired immune response, which likely reduces the efficacy of cancer therapies. We and others showed that pericyte coverage significantly correlates with vascular integrity/function and intratumoral hypoxia. Therefore, understanding perivascular heterogeneity and the unique contribution of each phenotype to the TME evolution is a critical step toward personalized treatment options for PDAC. Consequently, we hypothesize that correcting such undesirable phenotype via vascular normalization will alleviate the harsh TME and enhance the treatment efficacy. Our study revealed that tumor-associated pericytes across all PDAC tumor tissues exhibited ectopic αSMA expression up to 10 times higher than normal pericytes. This aberrant pericyte phenotype correlated with vascular leakiness, hypoxia, and vessel leakiness. Our attempt to elucidate the underlying mechanism of pericyte phenotype switching using an in-vitro culture system shows that pancreatic cancer cell-derived extracellular vesicles are a potent inducer of αSMA expression in pericytes present mechanical abnormalities and immune-suppressive features. To further determine the pathological signature of tumor pericytes and identify potential target molecules toward vascular normalization, we performed single-cell RNA sequencing (scRNA-seq). Pericytes were isolated from WT pancreas and KPC tumors at 15-20 weeks to obtain desired viable single-cell population suitable for 10x library preparation and Illumina sequencing.We isolated and barcode at least 5000 cells using the 10X genomics system. Sequencing libraries were prepared using the Chromium Single Cell 3ʹ reagent kit v3. They were sequenced until a sequencing depth of 100,000 reads per cell was achieved, and the data was analyzed using Cell Ranger and various other analytical tools (e.g., Seuret, Conos, ScMerge, singleR). We generated custom scripts from the single-cell data to answer the relevant questions. Sequences were demultiplexed based on barcodes and assigned unique molecular identifiers (UMIs). We visualized clusters using graphing methods, such as t-SNE plots and UMAP clustering. This approach helped identify groups of cells with unique and differential transcriptional landscapes and group them into cellular subtypes based on their gene expression profiles. In conclusion, our study indicates that tumor-associated pericytes undergo phenotype switching under the influence of pancreatic cancer cells, and these aberrant pericytes might contribute to non-optimal vascular integrity and function. Single-cell RNA sequencing helped understand the molecular signature of the pericyte subpopulation, providing a powerful tool to develop novel targeting strategies for vascular normalization. Future work includes exploring the vascular normalization approach by suppressing pericyte phenotype switching to enhance vascular function and chemo and immunotherapeutic efficacy. Citation Format: Vikneshwari Natarajan, Sangdeuk Ha, Jiha Kim. Influence of pathological pericytes on PDAC tumor progression and tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB309.

Abstract LB381: Plasma microbiota as novel biomarkers of early epithelial ovarian cancer detection

Abstract Background: Ovarian cancer accounts for 2% of all female cancers in the United States yet it is the 5th leading cancer death among women. Advanced epithelial ovarian cancer (EOC) is associated with an overall survival rate of 30% but can be cured in up to 90% of cases if diagnosed early when the disease is still limited to the ovaries and fallopian tubes. While there is no effective screening method for EOC, developing novel and minimally invasive clinical tests is paramount to identify women with early disease as well as those who are at increased risk. Plasma-based microbiota signatures specific to EOC could become leading biomarkers of early disease in the field that complement existing clinical tools. Methods: We utilized an established institutional biobank to access 180 biobanked plasma samples from: (1) women with EOC; (2) women with non-EOC solid tumors; (3) women undergoing gynecologic-related procedures and non-EOC surgeries; and (4) healthy controls. Batch 1 included 96 plasma samples (n=24 per group) and Batch 2 included 84 samples (n=19 per group and 8 repeat EOC samples). Microbial profiles of plasma specimens were assessed using an amplicon-based sequencing method that targets the 16S rRNA gene in the bacterial genome to study bacterial phylogeny and taxonomy in diseased and healthy women. A stringent bioinformatic pipeline was applied to filter out suspicious bacterial contaminants at the genus level. Bacterial decontamination procedures in the pipeline included evaluating repeat plasma samples of EOC cases and sampling nucleic acid isolation kit reagents to serve as negative controls (only in Batch 2) . Differential abundances of the plasma microbiota among the groups were assessed using analysis of compositions of microbiomes (ANCOM) as implemented in R software package ANCOM-BC. Results: The 16S rRNA gene sequencing identified, classified, and quantified 564 bacterial genera in the circulation of women diagnosed with EOC, non-EOC solid tumors, benign gynecologic conditions, and healthy controls. Bioinformatic decontamination analysis removed bacteria: (1) with differing bacterial abundances between batches; (2) with higher bacteria in negative controls; (3) with differing bacterial abundances in repeat samples; and (4) known as contaminants in the literature that resulted in the removal of 225 high-risk bacterial contaminants. A total of 339 high quality bacteria remained for evaluation. Because we did not collect negative controls in Batch 1, only Batch 2 plasma samples (n=84; 27 in EOC group; 19 in each other group) were included in the final analysis. Women with EOC, which were primarily of the high-grade serous ovarian carcinoma histologic subtype had significantly distinct differential abundances of genera Brevibacterium (p <0.001), Chloronema (p <0.001), Facklamia (p <0.001), Zymomonas (p <0.001), and Sutterella (p <0.001) when compared to women with non-EOC solid tumors, benign gynecologic conditions, and healthy controls. Conclusions: These findings suggest that plasma EOC-associated bacteria could be exploited in the development of a microbial-based blood test for the diagnosis and early detection of EOC in routine practice settings. Citation Format: Diane E. Mahoney, Prabhakar Chalise, Dong Pei, Rachel Griffard, Trisha Home, Harsh Pathak, Shahid Umar, Andrew K. Godwin. Plasma microbiota as novel biomarkers of early epithelial ovarian cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB381.

Performance Evaluation of Newly-Developed Tumor Markers Assay Kit Based on Flow Fluorescence Assay.

The purpose of this study is to evaluate the performance of recently developed tumor marker clinical kits in China, with the aim of encouraging local medical technology innovation and thus narrowing the research and development gap with foreign kits. The newly established reagent kits were analyzed on the TESMI F3999-Luminex200 flow lattice instrument to verify precision, sensitivity (blank limit), linearity, anti-interference ability, carry-over contamination rate, hook effect, and reference interval verification. Additionally, the newly established reagent kits were compared to other commercially available detection kits (reference reagent kits) to analyze the correlation between the two types of kits. The intra-assay and inter-assay precision had coefficients of variations (CVs) less than 3.50% and 6.91%, respectively. The tumor marker blank limits were lower than the manufacturer's statement. The newly established reagent kits demonstrated excellent linearity (r > 0.99). Rheumatoid factor, triglycerides, bilirubin, and hemoglobin did not have significant interference with the determination of tumor markers. The carry-over contamination rates were all much lower than 3%. At extremely high concentrations of AFP (277,335 ng/mL and 1,031,424 ng/mL), the measured tumor marker values were higher than the upper limit of the linear range and no hook effect occurred. The reference interval was suitable for use in clinical laboratory settings. Correlation analysis indicated a satisfactory relevance and consistency between the newly developed reagent kits and reference reagent kits, with correlation coefficients of r > 0.967 among 654 patients and healthy individuals. The newly developed reagent kits for tumor markers performed well in all evaluated parameters, having the potential for clinical promotion and application.

Validation of a Field-Portable, Handheld Real-Time PCR System for Detecting Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome in Bats.

White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has decimated bat populations across North America. Despite ongoing management programs, WNS continues to expand into new populations, including in US states previously thought to be free from the pathogen and disease. This expansion highlights a growing need for surveillance tools that can be used to enhance existing monitoring programs and support the early detection of P. destructans in new areas. We evaluated the feasibility of using a handheld, field-portable, real-time (quantitative) PCR (qPCR) thermocycler known as the Biomeme two3 and the associated field-based nucleic acid extraction kit and assay reagents for the detection of P. destructans in little brown bats (Myotis lucifugus). Results from the field-based protocol using the Biomeme platform were compared with those from a commonly used laboratory-based qPCR protocol. When using dilutions of known conidia concentrations, the lowest detectable concentration with the laboratory-based approach was 108.8 conidia/mL, compared with 1,087.5 conidia/mL (10 times higher, i.e., one fewer 10× dilution) using the field-based approach. Further comparisons using field samples suggest a high level of concordance between the two protocols, with positive and negative agreements of 98.2% and 100% respectively. The cycle threshold values were marginally higher for most samples using the field-based protocol. These results are an important step in establishing and validating a rapid, field-assessable detection platform for P. destructans, which is urgently needed to improve the surveillance and monitoring capacity for WNS and support on-the-ground management and response efforts.

Circulating microRNA-21-3p in patients with arterial hypertension

Objective — to study changes in the blood plasma levels of microRNA‑21‑3p in patients with arterial hypertension (AH) and their relationship with the characteristics of the course of the disease.
 Materials and methods. Examinations involved 24 patients with hypertension stage II and degree 2—3 (11 men, 13 women) aged from 42 to 60 years. The control group consisted of 12 practically healthy individuals, age and gender matching. The examinations were carried out in the Department of Hypertension and Kidney Diseases of the GI «L. T. Mala National Therapy Institute of NAMS of Ukraine». The degree, stage of hypertension and the presence of risk factors for cardiovascular diseases (CVD), established CVD and kidney disease were assessed in accordance with current recommendations. All patients underwent general clinical laboratory and instrumental examinations. The blood plasma level of circulating microRNA‑21‑3p was determined by quantitative reverse transcriptase‑polymerase chain reaction (qRT‑PCR) using reagent kits for monitoring and analyzing microRNA expression «TaqMan microRNA Assay» (ThermoFisher Scientific, USA). Small nuclear RNA U6 was used as a reference. Relative expression was calculated in relative units (r.u.) using the ΔΔCt method. Statistical analysis of the obtained data was carried out using standard methods with application packages Microsoft Excel and SPSS 19.0.
 Results. A significant decrease in the plasma levels of microRNA‑21‑3p in patients with hypertension was established in comparison with practically healthy individuals. The most pronounced decrease in plasma microRNA‑21‑3p levels was observed in patients with the most severe hypertension (stage 3 AH) and in subjects, in whom antihypertensive therapy did not result in the achieving target levels of «office» blood pressure (BP). Significant negative correlations were found between the plasma microRNA‑21‑3p levels and hemodynamic parameters (BP levels) and the left ventricular myocardial mass index — an indicator of the severity of left ventricular hypertrophy, one of the key lesions of target organs. The obtained results indicate the presence in patients with hypertension of a significant deficiency in the production of microRNA‑21‑3p, which, according to the literature data, has pronounced antihypertensive and cardiovascular‑ and renoprotective properties.
 Conclusions. A significant decrease in the blood plasma levels of microRNA‑21‑3p, the endogenous regulator of gene expression with antihypertensive and organoprotective properties, was established in patients with hypertension in comparison with practically healthy subjects. The revealed decrease in the blood plasma levels of this microRNA was enhanced at severe and uncontrolled hypertension and associated with the degree of hypertensive heart damage. The data obtained indicate the important role of microRNA‑21‑3p in the hypertension pathogenesis and the prospects of finding methods for correcting its production.

Abstract 4635: RNF208: A novel diagnostic and prognostic biomarker for colorectal cancer and key regulator of malignant behavior

Abstract Background: RNF208, a member of the RING finger protein family, has been implicated in ubiquitin-mediated degradation of Vimentin, regulating invasion in breast cancer, and serves as a prognostic factor in uveal melanoma. However, its diagnostic and prognostic value in colorectal cancer (CRC), as well as its role in regulating malignant behavior in CRC, remains unclear. This study aims to reveal the potential of RNF208 as a diagnostic and prognostic marker for CRC and its function in regulating malignant behavior in CRC through bioinformatics analysis and experimental validation. Methods: In this study, based on the ExoRbase2.0 human plasma exosome database (CRC=35; Healthy=118) and TCGA-COAD dataset (CRC=478; Healthy=41), we conducted bioinformatics analysis of the transcriptomic differences in plasma exosomes between CRC patients and normal individuals using ExoRbase2.0. Survival data from the TCGA-COAD dataset were integrated to select key molecules for CRC diagnosis and prognosis. Plasma samples from 39 CRC patients and 16 normal individuals were collected from hospital, and exosomes were isolated and purified using a reagent kit. qPCR was employed to validate the levels of RNF208 in plasma exosomes, confirming the results of bioinformatics analysis. Biological behavior was assessed through CCK-8 proliferation assays, flow cytometry experiments, and Transwell migration assays to investigate the relationship between RNF208 and malignant behavior in the HCT116 colorectal cancer cell line. Results: Bioinformatics analysis revealed a significant correlation between elevated RNF208 expression in plasma exosomes and the incidence of CRC, as well as poor prognosis. The qPCR results from clinical samples further confirmed this correlation, and ROC curves demonstrated high sensitivity and accuracy, suggesting RNF208's potential as a diagnostic and independent prognostic factor for CRC. CCK-8 experiments showed that RNF208 knockdown suppressed proliferation activity in HCT116 cells, flow cytometry indicated that RNF208 knockdown promoted apoptosis in HCT116 cells, and Transwell migration assays demonstrated that RNF208 knockdown inhibited the migration of HCT116 cells. Conclusion: RNF208 serves as a novel diagnostic and prognostic biomarker for colorectal cancer, promoting the proliferation and migration of colorectal cancer cells and contributing to the further deterioration of the disease. Citation Format: Fangyue Guo, Shanfeng Zhang, Pei Li. RNF208: A novel diagnostic and prognostic biomarker for colorectal cancer and key regulator of malignant behavior [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 4635.

Abstract 2405: Harnessing liquid biopsy and genetic barcoding in evaluating diversity within triple-negative breast cancer

Abstract Introduction: While liquid biopsy shows promise as a non-invasive precision medicine tool, its ability to comprehensively represent the complexity of a tumor or only a subset of its clones remains uncertain. This issue is particularly crucial in the context of triple-negative breast cancer (TNBC), a type of cancer known for its significant heterogeneity. The goal of this study is to examine the clonal diversity within Circulating Biomaterials (CBMs): namely circulating tumor cells (CTCs), extracellular vesicles RNA (evRNA), and circulating tumor DNA (ctDNA), employing genetic barcoding and patient-derived TNBC xenografts. Methodology: Cells from TNBC patients' resected tumors were tagged with a unique barcode sequence through vector transduction. These barcoded cells were subsequently introduced into immunodeficient mice to form xenografts. When these xenografts grew to a certain size (15mm in diameter), the mice were humanely euthanized, and the tumor tissues as well as CBMs from their blood and plasma were collected. CTCs were isolated through negative enrichment and DEPArray technology; evRNA was extracted using ultracentrifugation and TRIzol™ Plus RNA Purification method; ctDNA was retrieved using the QIAamp DNA Micro Kit. Tumor tissues were processed for RNA and DNA using RNeasy micro kit and Qiamp DNA micro kit, respectively. RNA sequencing of the tumor tissues was carried out using the Lexogen - CORALL Total RNA-Seq Kit and Illumina NextSeq high output reagents. Results: Analysis of xenografts from four patients showed that ctDNA was the predominant genetic material detected through liquid biopsy. Crucially, clonal analysis using Next Generation Sequencing (NGS) demonstrated that ctDNA provides an accurate reflection of the primary tumor's clonal heterogeneity. The method efficiently identified both main tumor clones and subclones, even those constituting as little as 1% of the tumor, with a 50% detection probability. Transcriptomic analysis of the tumor tissues uncovered a distinct mesenchymal and pro-metastatic signature, more pronounced in tumors with higher ctDNA release. Clonal analysis of CTCs and evRNA is still ongoing. Conclusions: This investigation affirms the effectiveness of ctDNA analysis in liquid biopsy for identifying subclonal diversity in TNBC. Our results suggest that liquid biopsy via ctDNA is capable of managing TNBC's extensive intra-tumoral heterogeneity, including the identification of smaller subclones. Furthermore, a link was identified between the release of ctDNA and a mesenchymal, prometastatic gene expression pattern in the tumors. Citation Format: Davide Ceresa, Barbara Cardinali, Antonio Daga, Roberta Tasso, Andrea Sciutto, Irene Appolloni, Daniela Marubbi, Franca Carli, Piero Fregatti, Paolo Malatesta, Lucia Del Mastro. Harnessing liquid biopsy and genetic barcoding in evaluating diversity within triple-negative breast cancer [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 2405.

Abstract 1149: Spatial transcriptomic analysis of tumor microenvironment in patients with triple negative breast cancer with residual disease after neoadjuvant chemotherapy with immune checkpoint inhibitor

Abstract Recent advance of treatment strategy for triple negative breast cancer (TNBC) including immune checkpoint inhibitor improved survival outcome. Especially, adding pembrolizumab on neoadjuvant chemotherapy (NAC) increased pathologic complete response (pCR) rate at the time of curative surgery. Previous studies suggested that PD-L1 expression in both tumor and immune cells was associated with the response of pembrolizumab in TNBC, but PD-L1 expression state were not associated with pembrolizumab response in NAC setting. In addition, neoadjuvant pembrolizumab increased event-free survival (DFS) rate after NAC even though they had not achieved pCR. However, there were no biomarker for EFS increasement of using pembrolizumab. We designed prospective translational research for evaluating tumor microenvironment (TME) of residual TNBC which had treated with NAC followed by curative surgery. We collected formalin fixed paraffin embedded (FFPE) surgical specimens after NAC with or without pembrolizumab. TNBC samples were used for spatial transcriptomics (ST) analysis, with H&E staining and inspection by an experienced pathologist. Libraries were prepared using Visium Spatial Gene Expression Reagent Kit protocol for FFPE (10x Genomics). Sequencing was conducted on the NovaSeq 6000 System (Illumina). From February 2023 to October 2023, 23 TNBC specimens from 22 patients were collected. Twenty specimens were breast tissues and three were lymph nodes, 18 TNBCs had been treated with NAC with pembrolizumab and four with only NAC, and five had residual cancer burden(RCB) class I, 11 of RCB class II and one of RCB class III. We collected BC and LN samples from BC with class III. Of 23 samples, we performed spatial transcriptomic analysis in 12 samples. In intrinsic subtyping, eight specimens were categorized into normal like, two of basal like and two of luminal A BC subtypes. In TNBC subtyping, mesenchymal stem cell like (MSL) were in six specimens, three of stem cell like (SL), two of luminal androgen receptor (LAR) and one of immunomodulatory (IM). We also performed cell type annotation with unsupervised method and categorized into 11 cell types; three cancer cell types, two macrophages, B and T cells, adipocytes, normal epithelium and perivascular-like and cancer associated fibroblasts. Among these 11 cell types, increasement of immune cells including B cells, T cells and macrophage proportions was associated with pembrolizumab treatment in NAC setting. In our study, we evaluated TME state of TNBC after NAC with or without pembrolizumab. We suggested that MSL or SL TNBC subtypes were associated with residual disease after NAC regardless of pembrolizumab use and pembrolizumab may recruit more immune cells in TME of TNBC. For concreating our suggestion, further analyses would be warranted. Citation Format: Ji-Yeon Kim, Kyunghee Park, Yoon La Choi, Jeong Eon Lee, Byung Joo Chae, Jonghan Yu, Seok Won Kim, Seok Jin Nam, Jin Seok Ahn, Woong-Yang Park, Yeon Hee Park. Spatial transcriptomic analysis of tumor microenvironment in patients with triple negative breast cancer with residual disease after neoadjuvant chemotherapy with immune checkpoint inhibitor [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 1149.

Abstract 491: Aerosol delivery of immunotherapy and hesperetin nanoparticles in murine lung cancer model

Abstract Purpose: Radiation is the most common modality for lung cancer treatment method. Radiationinduces apoptosis, causing cancer cells to be fragmented and expose the cancer-associatedantigens to the tumor microenvironment. These can be recognized by antigen-presenting cells(APC) to induce the antineoplastic effect by activating cytotoxic T cells. Our prior studies showadding immunoadjuvant like AntiCD40 with radiation can further activate the APCs in its anti-tumor (M1) format. Recent studies have shown that flavonoids like Hesperetin, an ACE2receptor agonist also can induce apoptosis in cancer cells whereas ACE2 receptors are abundant in lung cancer cells.This project aims to develop a novel treatment for non-small cell lung cancer (NSCLC) using nanotechnology for inhalation drug delivery Hesperetin, a flavonoid with antioxidant and pro-apoptotic activity; bound to PLGA-coated nanoparticles (Hesperetin Nanoparticles, HNPs) andAntiCD40 aiming for localized targeted dosing to reduce the treatment-related systemic toxicity.We plan to test the efficacy and safety of this aerosol spray. Methods: In-vitro studies were performed in human A549 (ATCC) and murine LLC1 (ATCC)lung cancer cell lines for growth (MTT) and clonogenic survival assays to demonstrate the anti-cancer cell activity of Hesperetin (Sigma). Next, HNP was prepared using NanoFabTx™ nano-formulation reagent kits (Millipore-Sigma). A syngeneic orthotopic murine model of Lungadenoma was generated in wild (+/+) C57/BL6 background mice with similar background LLC1cell lines. We developed syngeneic orthotopic murine lung tumors with luciferase genetransfected LL/2-Luc2 (ATCC) Lewis Lung Cancer cell line in wild-type C57BL/6 mice(Techonic). Lung tumor-bearing mice were treated with HNP, antiCD40, or both with aerosolspray with a control group treated with the solvent (dH2O) only. To assess the drug delivery withaerosol treatment ex-vivo lung tissue was analyzed for Fluorescence tagged antiCD40 andnanoparticle uptakes. A survival assay was performed to analyze the efficacy of aerosoltreatment of HNPs with or without AntiCD40. All cohorts were also analyzed for body score, body weight, and liver and kidney functions. Results: We were able to successfully develop an aerosol drug delivery model to administer bothAnti-CD40 and HNP in a murine lung cancer model. In our analysis of an orthotopic murinelung cancer model, we demonstrate a high intake of the HNP and AntiCD40 by the cancer cellssparing the normal lung tissue. Moreover, the highest percentage of survival rate was observed with the combination aerosol treatment with HNP+AntiCD40 (p<0.001), compared to CD40alone (p<0.01) or HNP (p<0.01) alone. Comments: This treatment model will allow us to make the lung cancer treatment method easilyavailable for the mass population without having hazardous radiation treatment in the lungcancer model. Citation Format: Sayeda Yasmin-Karim, Geraud Richard, Amanda Fam, Alina-Marissa Ogurek, G. Mike Makrigiorgos. Aerosol delivery of immunotherapy and hesperetin nanoparticles in murine lung cancer model [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 491.

Role of ixodid ticks in tick-borne pathogen spread and circulation in the Belarusian Lakeland

Results of ixodid tick analysis for their ecological, epizootological and epidemiological significance for tick-borne pathogen spread across the Belarusian Lakeland are presented. The ticks were collected in publicly accessible areas of the Vitebsk Raion in April – November 2022: 8 routes were tracked, 18 flag-km were passed, 529 tick specimens were collected, including 350 imago ticks and 179 nymph ticks. The ixodid tick genus and species were determined using N. A. Filippova’s ixodid tick determinator. All caught ticks were tested for Borrelia spp., Anaplasma spp. (Ehrlichia spp.), Babesia spp. and Tick-borne encephalitis virus genetic materials with real-time polymerase chain reaction using the reagent kit for nucleic acid extraction from environmental samples in accordance with the manufacturer’s instruc­tions. The specimens were grouped in accordance with the MG 3.1.1027-01 “Collection, recording and preparation for laboratory tests of blood-sucking arthropods being vectors of natural focal infections”; therewith, one specimen includes only one tick. Differences in the numbers of ixodid ticks and the occurrence of genetic markers of tick-borne pathogens in them were found to be associated with ecological characteristics of the examined territories. The following epidemically and epizootically significant ticks contributing to transmissible infection and invasion spread were found in the Belarusian Lakeland: ticks of Ixodes and Dermacentor genera; their frequency index was 70.1 and 29.9%, respectively. Tick-transmitted pathogen prevalence rate in the examined territories of the Vitebsk Raion was as follows: 61.7% for Borrelia spp., 25.8% for Anaplasma spp. (Ehrlichia spp.) and 25% for Babesia spp., mixed infections were found in 10.8% of the ticks. No tick-borne encephalitis virus genetic materials were found in the specimens. Total infection rate for ixodid ticks was 22.7%.

Kandungan Nutrien Nitrat dan Fosfat Pada Air di Kawasan Estuari DAM, Badung, Bali

Nitrate and phosphate nutrients are important nutrients in waters, but in conditions that exceed the limit will cause problems for the aquatic environment. One of the aquatic environments that has the potential to experience increased nitrate and phosphate content is the DAM Estuary located in Badung, Bali. This study aims to determine the current condition of nitrate and phosphate content in water in the DAM Estuary. Water sampling was carried out at 4 different station. Station 1 represents the inlet of the DAM Estuary, Station 2 and 3 represent the middle part of the DAM Estuary, while Station 4 represents the outlet of the DAM Estuary. Nitrate was measured using Hanna HI 713 Reagent, while Phosphate was measured using Salifert Nitrate KIT Reagent. In addition, temperature, pH, TDS and DO measurements were also carried out in water. The results showed that the nitrate and phosphate content in the water in the DAM Estuary was still below the water quality standard for all classes according to Government Regulation no. 82 of 2001 concerning Water Quality Management and Water Pollution Control. Other water quality parameters also show good conditions in accordance with these regulations.

Phylogenetic position and genetic features of HIV-1 in CNS

Background. Due to the wide coverage with antiretroviral therapy, the life expectancy of HIV infected people has significantly increased. Against the background of a decrease in mortality from HIV infection, HIV-associated neurocognitive disorders, which develop even during effective treatment, are of high importance. The overall prevalence of this pathology among HIV-infected people reaches 42.6%.
 The objective of the study was to research the genetic features and phylogenetic position of HIV-1 persisting in the central nervous system.
 Materials and methods. The clinical study group consisted of 38 patients with severe neurocognitive disorders against the background of HIV infection in stage 4B. The viral load of HIV-1 in blood plasma and cerebrospinal fluid (CSF) was measured using the "AmpliSens HIV Monitor-FRT" reagents kit. Sanger sequencing was performed using the AmpliSens HIV-Resist-Seq assay kit on an Applied Biosystems 3500 analyzer. Phylogenetic analysis of the pol gene fragments of HIV-1 strains (the site encoding the viral protease and part of the reverse transcriptase) was carried out using maximum likelihood method with the GTR+G nucleotide substitution model. Comparisons of the tertiary structure of viral proteins were performed according to three-dimensional models of the protease and p51 and p66 reverse transcriptase subunits obtained by homologous reconstruction using the SWISS-MODEL tools.
 Results. The viral load in the sample of patients with severe CNS lesions in blood plasma was 6.27 times higher than in CSF and amounted to 4.67 and 3.87 lg copies/ml respectively by median (p = 0,004).
 Phylogenetic analysis with the use of all available HIV-1 genomes from GenBank, which differed from the studied ones by less than 5% showed close genetic relations of viruses circulating in Chelyabinsk region, apart from strains circulating in Russian Federation, with viruses circulating in neighboring countries, in most abundance — from Ukraine and Kyrgyzstan, slightly less — from Belarus, Tajikistan, Kazakhstan and Armenia and also with strains from certain foreign countries: Poland and Germany. Phylogenetic analysis of 38 HIV-1 genomes revealed significant genetic distances between HIV isolates from blood plasma and CSF in 5 patients, 4 of whom were PWID, which may indicate an event of superinfection.
 The amount of independent amino acid substitutions in protease in isolates from blood plasma ranged from 1 to 3, in isolates from CSF — from 1 to 2. An amount of such substitutions in a fragment of reverse transcriptase in isolates from blood plasma ranged from 1 to 6, while in isolates from CSF, it ranged from 1 to 7. HIV isolates from blood plasma and CSF from 5 patients had differences in the tertiary structure of HIV-1 reverse transcriptase p51 subunit in amino acid positions 16–20 and 210–235. Isolates from 3 other patients differed in the tertiary structure only in amino acid positons 210–235. Isolates from 3 patients differed in the structure of HIV-1 RT p66 subunit in a non-nucleoside reverse transcriptase inhibitor binding pocket (NNRTI) region. Fixed differences in the tertiary structure of p51 subunit required at minimum only 1 amino acid substitution to emerge. Alterations in the tertiary structure of p66 subunit required at least 3 amino acid substitutions.
 Conclusion. Microevolution of HIV-1 proceeds in parallel within the same patient, in different compartments, which is reflected in the accumulation of amino acid substitutions different from another compartment in the conserved pol gene. There is a weak correlation between the viral load level in plasma and in CSF. The genetic heterogeneity of HIV strains from patients of the Chelyabinsk region indicates a high frequency of reintroduction of HIV infection in the region from other countries. Differences in the tertiary structure of HIV-1 reverse transcriptase between blood plasma and CSF isolates are regularly fixed in certain domens, which also confirms the presence of parallel HIV microevolution during virus persistence in tissues separated by the blood-brain barrier which allows a better understanding of the fixation trends of individual amino acid substitutions during HIV-induced damage to central nervous system.

The SOX2/PDIA6 axis mediates aerobic glycolysis to promote stemness in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is an aggressive and rapidly expanding lung cancer. Abnormal upregulation or knockdown of PDIA6 expression can predict poor prognosis in various cancers. This study aimed to investigate the biological function of PDIA6 in NSCLC. SOX2 and PDIA6 expression in NSCLC tissues and regulatory relationship between them were analyzed using bioinformatics. GSEA was performed on the enrichment pathway of PDIA6. qRT-PCR was utilized to examine expression of SOX2 and PDIA6 in NSCLC tissues and cells, and dual-luciferase reporter assay and ChIP experiments were performed to validate their regulatory relationship. CCK-8 experiment was conducted to assess cell viability, western blot was to examine levels of stem cell markers and proteins related to aerobic glycolysis pathway in cells. Cell sphere formation assay was used to evaluate efficiency of cell sphere formation. Reagent kits were used to measure glycolysis levels and glycolysis products. High expression of PDIA6 in NSCLC was linked to aerobic glycolysis. Knockdown of PDIA6 reduced cell viability, expression of stem cell surface markers, and cell sphere formation efficiency in NSCLC. Overexpression of PDIA6 could enhance cell viability and promote aerobic glycolysis, but the addition of 2-DG could reverse this result. Bioinformatics predicted the existence of upstream transcription factor SOX2 for PDIA6, and SOX2 was significantly upregulated in NSCLC, and they had a binding relationship. Further experiments revealed that PDIA6 overexpression restored repressive effect of knocking down SOX2 on aerobic glycolysis and cell stemness. This work revealed that the SOX2/PDIA6 axis mediated aerobic glycolysis to promote NSCLC cell stemness, providing new therapeutic strategies for NSCLC.

A multilevel core-shell structure Fe3O4@SiO2@InGaO@SiO2 magnetic particles for DNA extraction

DNA extraction is greatly significant in biological detection fields, including species identification, pathogen detection, environmental monitoring, biosafety, and food safety. Scientific research and practical application must conduct high-quality trace DNA separation, purification and extraction. Herein, a two-step strategy successfully prepares a multilevel core–shell structure Fe3O4@SiO2@InGaO@SiO2 magnetic particles for trace DNA extraction. The morphologies, structures, elemental compositions, components, and properties of the materials are systematically characterized. Fe3O4@SiO2@InGaO@SiO2 magnetic particles in the chaotropic salt solutions are used to directly target DNA binding due to the existence of Si-O–H bond. The maximum DNA adsorption rate can highly reach 83.1 % after 700 °C annealing in the air for 30 min, far exceeding the adsorption rate of commercial reagent kit, which is attributed to the increased specific surface area. The excellent result is conducive to rapid DNA extraction in practical applications, especially in daily criminal cases, further promoting social security and stability.

Minimally Invasive Method of Measuring Anti-Müllerian Hormone Level in Dried Blood Spots of Indonesian Infertile Population

This study aims to evaluate the certainty of anti-Müllerian hormone (AMH) levels in dried blood spots (DBS) compared to serum AMH. A total of 109 infertile women who planned to undergo their controlled ovarian stimulation cycle were recruited. Serum and DBS samples were collected from each patient for the quantification of AMH levels. Serum AMH was measured with an automated clinical chemistry assay (Roche) while DBS AMH was measured using the Ansh Labs DBS AMH ELISA reagent kit. Mean AMH level in DBS was [Formula: see text][Formula: see text]ng/mL, which ranged from 0.01 to 17.3[Formula: see text]ng/mL, while the mean AMH in serum samples was [Formula: see text][Formula: see text]ng/mL (0.01–10.8[Formula: see text]ng/mL). Comparable accuracy for low AMH quantification was observed between the two methods over basal antral follicle counts (AFC) as a clinical comparator (AUC 86%, cutoff [Formula: see text][Formula: see text]ng/mL, [Formula: see text], [Formula: see text] vs. 85%, cutoff [Formula: see text][Formula: see text]ng/mL, [Formula: see text], [Formula: see text], respectively for DBS and serum sample). Likewise, DBS AMH achieved high concordance for low AMH discrimination in comparison to serum AMH method (AUC 99% vs. 93%, respectively). A strong linear Spearman regression ([Formula: see text], [Formula: see text]) and sufficient agreement (94.5%, mean difference of −1.6, 2.7 to −5.9) was demonstrated among studied groups with mathematical Passing and Bablok regression equation of [Formula: see text]. The confidence interval of intercept (−0.12, 95% CI [−0.29 to −0.01]) and slope (1.80, 95% CI [1.66–1.94]) indicated a constant and proportional difference between the two methods but it was not significant ([Formula: see text]). AMH measurement in DBS could be useful as an alternative approach to screening ovarian reserve through a more convenient remote sample collection.

Diagnostic performance evaluation of urine HIV-1 antibody rapid test kits in a real-life routine care setting in China

ObjectivesTo evaluate the diagnostic performance of urine HIV antibody rapid test kits in screening diverse populations and to analyse subjects’ willingness regarding reagent types, purchase channels, acceptable prices, and self-testing.DesignsDiagnostic...

Динамика выявления РНК вируса SARS-CoV-2 у пациентов и сотрудников ФГБУ «НМИЦ гематологии» Минздрава России в первые 2 года пандемии новой коронавирусной инфекции COVID-19

Background. COVID-19 required fundamental changes in healthcare management, also in medical care for oncological and hematological patients. Visits to healthcare organizations were minimized, 75 % of doctor appointments were converted to telemedicine consultations. The solutions aimed at preventing further spread of COVID-19 included establishing of observational units, distinguishing between patient and employee flows, regular SARS-CoV-2 RNA testing, reducing hospital stays and transferring patients with positive COVID-19 tests to the remodeled hospitals specializing in the novel coronavirus infection, as well as providing only emergency medical treatment and, as far as feasible, converting systemic chemotherapy to per os treatment, etc.
 Aim. To assess SARS-CoV-2 RNA detection dynamics at the National Research Center for Hematology from April 2020 to January 2022 during the implementation of epidemic control measures.
 Materials & Methods. The study was based on SARS-CoV-2 RNA testing of naso- and oropharyngeal samples obtained from patients and employees of the National Research Center for Hematology (hereafter referred to as Center). Besides, bronchoalveolar lavage fluid, lung tissue biopsies, and sputum were examined for SARS-CoV-2 RNA. The study was performed at the Center’s Virusology Department with the use of Sintol reagent kit “ПЦР-РВ-2019-nCov”.
 Results. The study was based on 107,470 tests: 58,141 (54 %) of employees and 45,126 (46 %) of patients; 35,508 (33 %) of men and 71,962 (67 %) of women. In 1318 cases SARS-CoV-2 RNA was detected which accounted for 1.15 % of total test number. In the groups of employees/patients, virus detection rate was 1.42 %/1.09 % (p < 0.001), and in male/female groups it was 1.3 %/1.2 %, respectively (p = 0.154). The rate of infection in the groups of tumor and non-tumor hematological patients, as proved by SARS-CoV-2 RNA testing, was 1.24 % and 0.92 %, respectively (p = 0.147). In employees and patients of the Center, a wave-like virus detection rate was observed. The largest number of infections was registered in April-June 2020 (79 patients and 170 employees), October-December 2020 (126 patients and 190 employees), and January 2022 (59 patients and 203 employees), which corresponded to the first, second, and fifth COVID-19 waves in Russia.
 Conclusion. The analysis of data obtained at the National Research Center for Hematology demonstrated a wave-like SARS-CoV-2 RNA detection rate in employees and patients of the Center, which corresponded to the general trend in Russia. The SARS-CoV-2 RNA detection rate did not depend on sex of subjects under study and was not significantly different in the groups of tumor and non-tumor hematological patients. Although the patients in hematological hospital are more exposed to the risk of severe infectious complications, they showed laboratory markers for COVID-19 less frequently than the Center employees.

Quality control data management with unity real-time in molecular virology

IntroductionQuality control (QC) is one component of an overarching quality management system (QMS) that aims at assuring laboratory quality and patient safety. QC data must be acceptable prior to reporting patients’ results. Traditionally, QC statistics, records, and corrective actions were tracked at the Johns Hopkins Molecular Virology Laboratory using Microsoft Excel. Unity Real-Time (UnityRT), a QMS software (Bio-Rad Laboratories), which captures and analyzes QC data by instrument and control lot per assay, was implemented and its impact on the workflow was evaluated. The clinical utility of real-time QC monitoring using UnityRT is highlighted with a case of subtle QC trending of HIV-1 quantitative control results. MethodsA comprehensive workflow analysis was performed, with a focus on Epstein Barr Virus (EBV) and BKV quantitative viral load testing (Roche cobas 6800). The number of QC steps and time to complete each step were assessed before and after implementing UnityRT. ResultsOur assessment of monthly QC data review revealed a total of 10 steps over 57 min when using Microsoft Excel, versus 6 steps over 11 min when using UnityRT. HIV-1 QC monitoring revealed subtle trending of the low positive control above the mean from November to December 2022, correlating with a change in the reagent kit lot. This associated with a shift in patients’ results from positives below the lower limit of quantification to positives between 20 and 100 copies/mL. ConclusionsUnityRT consolidated QC analyses, monitoring, and tracking corrective actions. UnityRT was associated with significant time savings, which along with the interfaced feature of the QC capture and data analysis, have improved the workflow and reduced the risk of laboratory errors. The HIV-1 case revealed the value of the real-time monitoring of QC.

Features of <i>ESCHERICHIA COLI</i> samples from patients with diarrheal syndrome in the Republic of Guinea

Introduction. Diarrheal diseases are a global public health issue and cause 15% of deaths in children under 5 years old, of which about 80% occur in the regions of Africa and Southeast Asia. According to the Global Enteric Multicentre Study (GEMS) conducted in a number of African countries, one of the leading pathogens of high risk of death in infants and young children is diarrheagenic E. coli (DEC). In recent decades, antimicrobial resistance (AMR) has become globally ubiquitous. The Republic of Guinea urgently needs large-scale studies devoted to assessing DEC distribution and antibiotic resistance. The purpose of the study is to assess the pattern of E. coli infections and to test the susceptibility to antibiotics in strains of diarrheagenic E. coli sampled from individuals residing in the Republic of Guinea.
 Materials and methods. From 2019 to 2022, we studied 724 samples of faeces of patients with acute diarrhea, among them 72 (9.9%) children aged 1–5 years, 128 (17.7%) children aged 6–17 years, and 524 (72.4%) people aged 18 years and older; a method of polymerase chain reaction (PCR) was applied with the use of the AmpliSense® Escherichioses-FL reagent kit to identify the genetic determinants of DEC: EPEC, EHEC, ETEC, EIEC, and EAgEC (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia). Susceptibility to 15 antimicrobial agents was found by the disc-diffusion method using Mueller–Hinton agar (Russia) and Oxoid discs (UK). Results were interpreted according EUCAST criteria, versions 2019–2022 (https://www.eucast.org/ast_of_bacteria/previous_versions_of_documents).
 Results. For the period from 2019 to 2022, the percentage of E. coli infections in the etiological pattern of acute intestinal infections amounted to 51.7%. In the age-related manner, DEC was significantly more common in young children aged 0–5 (96.9%, p 0.05) as compared to school age children aged 6–17 (53.9%) and adults (45.6%). In all years of observation, EAgEC strains prevailed, accounting for 38.4%. Other DEC pathotypes, EPEC, ETEC, EIEC and STEC, accounted for 27.2%, 17.5%, 11.8%, and 5.1%, respectively. DEC strains are susceptible to meropenem, amikacin, and nitrofurantoin. The activity of other antibiotics ranged from 11.3% for ampicillin, 28.3% for trimethoprim-sulfamethoxazole, and 34.0% for tetracycline to 73.6% for cephalosporins, 84.0% for aminoglycosides, and 98.1% for fluorinated quinolones.
 Conclusion. To reduce the burden of diarrheal diseases in the Republic of Guinea, it may be necessary to conduct targeted epidemiological and microbiological studies to identify DEC and monitor the development of antimicrobial resistance of E. coli infection pathogens in the population.

СПЕКТР МИКРОФЛОРЫ ПАРОДОНТАЛЬНОГО КАРМАНА У ПАЦИЕНТОВ С ХРОНИЧЕСКИМ ПАРОДОНТИТОМ СРЕДНЕЙ СТЕПЕНИ ТЯЖЕСТИ

The purpose of the study. To evaluate the quantitative composition of the microflora of the periodontal pocket in patients with chronic generalized periodontitis of moderate severity. Materials and methods. The study group included 39 patients, of which: 41% (16/39) were men, 59% (23/39) were women, with chronic generalized periodontitis of moderate severity at the age of 48.9±2.3 years. To determine the dental status, clinical research methods were used (questioning, examination), determination of the Green-Vermillion Oral Hygiene Index (OHI-S), (PMA) modified by Parma, the Mulleman-Cowell bleeding index, the degree of pathological tooth mobility was determined using the Miller scale (Miller) modified by Flesar, the complex periodontal index (CPI), identified furcation defects according to the classification of Lindhe J. (1983).Quantitative assessment of the periodontal pockets microflora was performed. Reagent kits “RIBO-prep” (Next-Bio LLC, Russia), DENTOSKRIN® (NPF Litech LLC, Russia) were used to isolate RNA/DNA from clinical material and subsequent analysis by polymerase chain reaction. Spectrum of identifiable bacteria: Porphyromonas endodontalis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia, Tannerella forsythia.The results of the study and their discussion. Fusobacterium nucleatum was found in 84.6% (33) of patients in periodontal pockets, 30.8% (12) – Treponema denticola, 28.2% (11) – Prevotella intermedia, 17.9% (7) – Porphyromonas gingivalis and Porhyromonas endodontalis. Aggregatibacter actinomycetemcomitans was isolated only in 5.1% (2) of individuals, Tannerella forsythia – in 2.6% (1). Combinations of 4 microorganisms were detected in 20.5% (8), from 3 - in 12.8% (5), from 2 - 15.4% (6). In 12.8% (5) of the examined patients were not isolated from any of the studied periodontal pathogens. When checking correlations between the level of hygiene and the number of pathogenic microorganisms detected, a moderate direct relationship was established between the value of the OHI-S index and the amount of Fusobacterium nucleatum r=0.36 (p<0.05) and Prevotella intermedia r=0.30 (p<0.05), as well as a moderate direct relationship between the value of the PMA index and the amount of Porphyromonas gingivalis r=0.33 (p<0.05), Porphyromonas endodontalis r=0.31 (p<0.05), Prevotella intermedia r=0.30.Conclusion. In patients with chronic periodontitis of moderate severity, a microbial biofilm is formed on the surface of the periodontium, represented by anaerobic bacteria. In periodontal pockets, the amount of Fusobacterium nucleatum and Prevotella intermedia increases with the deterioration of the hygienic condition of the oral cavty. With the development of inflammation in the gum tissues, periodontal pathogens Porphyromonas gingivalis and Porphyromonas endodontalis are more often detected, having more aggressive invasive and toxic properties in relation to periodontal tissues.