Negative Control Samples Research Articles

DNA methylation analysis of the SDC2, SEPT9 and VIM genes in fecal DNA for colorectal cancer diagnosis.

Colorectal cancer is one of the most common cancers worldwide. DNA methylation sites may serve as a new gene signature for colorectal cancer diagnosis. The search for representative DNA methylation sites is urgently needed. This study aimed to systematically identify a methylation gene panel for colorectal cancer diagnosis via tissue and fecal samples. A total of 181 fecal and 50 tumor tissue samples were collected. They were obtained from 83 colorectal cancer patients and 98 healthy subjects. These samples were evaluated for DNA methylation of 9 target genes via quantitative bisulfite next-generation sequencing. We employed the rank-sum test to screen the colorectal cancer-specific methylation sites in the tissue and fecal cohorts. A data model was subsequently constructed and validated via the dedicated validation dataset. Compared with the fecal and negative control samples, the colorectal cancer tissue samples presented significantly higher methylation rates for all the selected gene sites. The methylation rates of the tissue and preoperative fecal samples showed the same high and low rates at the same sites. After screening, a panel of 29 loci in the SDC2, SEPT9, and VIM genes proved to be reliable biomarkers for colorectal cancer diagnosis in fecal samples. Logistic regression models were then constructed and validated using this panel. The sensitivity of the model was 91.43% (95% CI = [89.69, 93.17]), the specificity was 100% (95% CI = [100,100]), and the AUC value is 99.31% (95% CI = [99,99.62]). The diagnostic accuracy of the model for stage I and stage II colorectal cancer was 100% (11/11) and 91.3% (21/23), respectively. Overall, this study confirms that the gene locus panel and the model can be used to diagnose colorectal cancer effectively through feces. Our study identified a set of key methylation sites for colorectal cancer diagnosis from fecal samples, highlighting the importance of using tissue and fecal samples to accurately assess DNA methylation levels to screen for methylation sites, and developing an effective diagnostic model for colorectal cancer.

Development and validation of a droplet digital PCR assay for Nipah virus quantitation

BackgroundNipah virus (NiV) is a zoonotic pathogen that poses a significant threat because of its wide host range, multiple transmission modes, high transmissibility, and high mortality rates, affecting both human health and animal husbandry. In this study, we developed a one-step reverse transcription droplet digital PCR (RT-ddPCR) assay that targets the N gene of NiV.ResultsOur RT-ddPCR assay exhibited remarkable sensitivity, with a lower limit of detection of 6.91 copies/reaction. Importantly, it displayed no cross-reactivity with the other 13 common viruses and consistently delivered reliable results with a coefficient of variation below 10% across different concentrations. To validate the effectiveness of our RT-ddPCR assay, we detected 75 NiV armored RNA virus samples, mimicking real-world conditions, and negative control samples, and the RT-ddPCR results perfectly matched the simulated results. Furthermore, compared with a standard quantitative real-time PCR (qPCR) assay, our RT-ddPCR assay demonstrated greater stability when handling complex matrices with low viral loads.ConclusionsThese findings show that our NiV RT-ddPCR assay is exceptionally sensitive and provides a robust tool for quantitatively detecting NiV, particularly in stimulated field samples with low viral loads or complex matrices. This advancement has significant implications for early NiV monitoring, safeguarding human health and safety, and advancing animal husbandry practices.

Development of point-of-need colourimetric, isothermal diagnostic assays for specific detection of Bacillus subtilis using shikimate dehydrogenase gene.

The largest obstacle in the promotion of biopesticides is the existence of counterfeit products available in the market. Identification and quantification of antagonistic organisms in biopesticide products are the key to the reduction of spurious microbial pesticides. In this study, we have developed a simple, sensitive, isothermal-based colourimetric assay for specific detection of Bacillus subtilis from the biopesticide formulations and soil samples. A region specific to B. subtilis which codes for shikimate dehydrogenase was identified through in silico analysis. We employed conventional PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and qPCR for specific detection of B. subtilis in soil samples and biopesticide formulations. Specificity tests showed that the PCR primers amplified an amplicon of 521bp in four strains of B. subtilis only, and no amplification was found in negative control samples. Similarly, the LAMP assay showed sky blue colour in all four strains of B. subtilis and violet colour in negative control samples. Whereas in the RPA assay, upon the addition of SYBR Green dye, a bright green colour was seen in B. subtilis strains, while a brick-red colour was observed in negative control samples by visualizing under a UV transilluminator. The qPCR assay showed specific amplifications with a Ct value of 12 for B. subtilis strains and no amplification in negative control samples. In the sensitivity test, PCR could amplify DNA of B. subtilis up to 500pg/µL. DNA concentration as low as 10pg/µL was enough to show the colour change in the LAMP as well as the RPA assays, whereas the qPCR assay showed sensitivity till 100pg/µL. All four diagnostic assays developed in the study have been validated in soil samples and B. subtilis-based biopesticides. Compared to conventional PCR, the qPCR assay has the advantage of quantification and visualizing the result in real-time, whereas LAMP and RPA assays have the benefits of being colourimetric and less time-consuming. The other advantages are that the results can be visualized with the naked eye, and these assays do not require a costly thermal cycler and gel documentation system. Hence, LAMP and RPA assays are highly suitable for developing point-of-need diagnostic kits and, in turn, help regulators assess the quality of biopesticides in the market.

Discordant performance of mpox serological assays

BackgroundSince July 23, 2022, global mpox cases reached 92,546, with over 31,000 in the United States. Asymptomatic carriage is a critical mechanism influencing the global dissemination of mpox. Seroprevalence studies are crucial for determining the epidemic's true burden, but uncertainties persist in serologic assay performance and how smallpox vaccination may influence assay interpretation. ObjectivesOur study aimed to assess the performance of several diagnostic assays among mpox-positive, vaccinated, and pre-outbreak negative control samples. This investigation sought to enhance our understanding and management of future mpox outbreaks. Study designSerum samples from 10 mpox-positive, five vaccinated uninfected, and 137 pre-outbreak controls were obtained for serological testing. The mpox-positive samples were obtained around 100 days post symptom onset, and vaccinated patients were sampled approximately 90 days post-vaccination. Multiple diagnostic assays were employed, including four commercial ELISAs (Abbexa, RayBioTech, FineTest, ProteoGenix) and a multiplex assay (MesoScale Diagnostics (MSD)) measuring five mpox and five smallpox antigens. ResultsThree commercial ELISA kits had low specificity (<50 %). The Proteogenix ELISA targeting the E8L antigen had a 94 % sensitivity and 87 % specificity. The E8L antigen on the MSD assay exhibited the greatest distinction between exposure groups, with 98 % sensitivity and 93 % specificity. ConclusionsNone of the assays could distinguish between mpox-positive and vaccinated samples. The MSD assay targeting the MPXV E8L antigen demonstrated the greatest differentiation between mpox-positive and pre-outbreak negative samples. Our findings underscore the imperative to identify sensitive and specific assays to monitor population-level mpox exposure and infection.

KHSRP has oncogenic functions and regulates the expression and alternative splicing of DNA repair genes in breast cancer MDA-MB-231 cells

Breast cancer has become the most common type of cancers worldwide. Its high prevalence and malignant features are associated with various environmental factors and molecules. The KH-type splicing regulatory protein (KHSRP) participates in the development of breast cancer, while the underlying mechanisms are largely unknown. In this study, we silenced KHSRP expression in MDA-MB-231 cells by small interfering RNA (siKHSRP), and then assessed its effects on cellular features. Finally, we performed whole transcriptome sequencing (RNA-seq) experiments to explore the downstream targets of KHSRP, and validated their changed pattern using quantitative polymerase chain reaction. We found KHSRP showed higher expression level and was associated with worse prognosis in breast cancer patients. In siKHSRP samples, the proliferation, invasion, and migration abilities were significantly repressed compared with negative control (NC) samples, while the apoptosis level was increased. By investigating the RNA-seq data, we found KHSRP globally regulates the expression and alternative splicing profiles of MDA-MB-231 cells by identifying 1632 differentially expressed genes (DEGs) and 1630 HKSRP-regulated AS events (RASEs). Functional enriched analysis of DEGs demonstrated that cilium assembly and movement and extracellular matrix organization pathways were specifically enriched in up DEGs, consistent with the repressed migration and invasion abilities in siKHSRP cells. Interestingly, the cell cycle and DNA damage and repair associated pathways were enriched in both down DEGs and RASE genes, suggesting that KHSRP may modulate cell proliferation by regulating genes in these pathways. Finally, we validated the changed expression and AS patterns of genes in cell cycle and DNA damage/repair pathways. Expression levels of BIRC5, CCNA2, CDK1, FEN1, FOXM1, PTTG1, and UHRF1 were downregulated in siKHSRP samples. The AS patterns of PARK7, ERCC1, CENPX, and UBE2A were also dysregulated in siKHSRP samples and confirmed PCR experiments. In summary, our study comprehensively explored the downstream targets and their functions of KHSRP in breast cancer cells, highlighting the molecular mechanisms of KHSRP on the oncogenic features of breast cancer. The identified molecular targets could be served as potential therapeutic targets for breast cancer in future.

Detection of Species DNA in Chicken Meat Processed Food Products Using GH (Growth Hormone) Genes as Molecular Markers

The species DNA detection test in processed chicken meat food products using genetic growth hormone (GH) gene markers is a novel approach for authenticity test regarding the presence of a particular species in a product. This research aimed to test the ability of the GH genetic marker to detect the presence of chicken species in processed chicken meat food products. The method used in this research is a molecular biology method using real-time PCR with the SYBR Green kit. The genetic marker primers used were designed independently from the NCBI website. Based on DNA isolation data, the DNA concentration value is 43.60 ng/µL – 45.20 ng/µL with an average of 44.11. The purity value of isolated DNA is 1.855 – 1.925 with an average of 1.906. Chicken species DNA was detected in all samples using real-time PCR analysis, with a Ct value of 24.50 and a Tm value of 78.10. However, the Ct and Tm values were not detected in the negative control (NTC) and specificity (internal) control samples. In the positive control LOD, the Ct value was detected at 27.20 with a Tm value of 79.40. In the positive control, a Ct value of 21.10 and a Tm value of 78.10 were detected. The results indicate that all samples contained the GH gene, and hence, chicken species DNA was detected in all samples. This suggests that the GH genetic marker can be potentially used for species DNA identification testing.

Detection of avian reovirus (ARV) by ELISA based on recombinant σB, σC and σNS full-length proteins and protein fragments.

Introduction. Avian reovirus (ARV) is associated with arthritis/tenosynovitis and malabsorption syndrome in chickens. The σC and σB proteins, both exposed to the virus capsid, are highly immunogenic and could form the basis for diagnostic devices designed to assess the immunological status of the flock.Gap Statement. Commercial ARV ELISAs cannot distinguish between vaccinated and infected animals and might not detect circulating ARV strains.Aim. We aimed to develop a customized test to detect the circulating field ARV strains as well as distinguish between vaccinated and unvaccinated animals.Methodology. We developed ELISA assays based on recombinant (r) σB, σC and the nonstructural protein σNS and tested them using antisera of vaccinated and unvaccinated chickens as well as negative controls. Fragments of σB and σC proteins were also used to study regions that could be further exploited in diagnostic tests.Results. Vaccinated and unvaccinated birds were positive by commercial ELISA, with no difference in optical density values. In contrast, samples of unvaccinated animals showed lower absorbance in the rσB and rσC ELISA tests and higher absorbance in the rσNS ELISA test than the vaccinated animals. Negative control samples were negative in all tests. Fragmentation of σB and σC proteins showed that some regions can differentiate between vaccinated and unvaccinated animals. For example, σB amino acids 128-179 (σB-F4) and σC amino acids 121-165 (σC-F4) exhibited 85 and 95% positivity among samples of vaccinated animals but only 5% and zero positivity among samples of unvaccinated animals, respectively.Conclusion. These data suggest that unvaccinated birds might have been exposed to field strains of ARV. The reduction in absorbance in the recombinant tests possibly reflects an increased specificity of our test since unvaccinated samples showed less cross-reactivity with the vaccine proteins immobilized on ELISAs. The discrepant results obtained with the protein fragment tests between vaccinated and unvaccinated animals are discussed in light of the diversity between ARV strains.

Diversifying color palette of Prangos ferulacea colorants on wool yarns: dual mordant approach for enhanced color, antioxidant, and UV protection properties

Natural dyes have gained significant attention in recent years due to their environmentally friendly characteristics. This research paper focuses on the dyeing of wool yarns with P. ferulacea aerial part extract in conjunction with biomordants and metal mordants. The study focused on optimizing dyeing variables including pH of 2–8, temperature of 65 °C–95 °C, dye concentration of 25%–125% o.w.f., and dyeing time of 15–120 min using a one-factor-at-a-time (OFT) optimization by determining the color depth (K/S) values on wool yarns. The optimized dyeing conditions were a pH of 4, a temperature of 95 °C, a dye concentration of 75.0% (o.w.f.), and dyeing time of 60 min. The color hues, brightness, color intensity, and fastness of the samples varied significantly depending on the kind and nature of the biomordant used. Furthermore, the introduction of metal ions as pretreatment to biomordanted wool yarns exhibited unique characteristics such as the creation of new color shades, the combined enhancement of dye absorption (K/S), and increased color fastness with the most colorfast hues produced by Cr, Cu, and Fe-biomordant combinations. The antioxidant potential of biomordanted wool yarns was evaluated in terms of the inhibition of peroxidation by 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activity and compared to the un-dyed wool yarns. The two-step mordanting process enhanced the antioxidant activity of dyed wool yarn (up to 99.90%) compared to the unmordanted sample (58.30%) and the negative control sample (5.70%). The ultraviolet protection factor (UPF) values were found in a very good to excellent range, with a rating >40 and maximum protection (UPF = 59.80) provided by Fe-YM mordanting. The findings indicate that this efficient technique enabled us to broaden the spectrum of colors generated by the extract of P. ferulacea aerial parts on wool, while simultaneously guaranteeing outstanding antioxidant and UV protection characteristics.

Fetal cardiac troponin I levels decline toward birth in sheep.

Elevated cardiac troponin I (cTnI), a myocardial damage biomarker, has been reported in cord blood of neonates delivered vaginally or by cesarean section. Although the neonatal peak likely reflects the physiological adjustment to extrauterine life, a better understanding of serial prepartum changes is required to determine physiological causes of fetal cTnI release. We longitudinally sampled eight healthy lambs (20 days before spontaneous birth to 5 days postnatal), and from three fetuses receiving intravenous IGF-1. Samples were collected into heparin, and the plasma was stored at -80°C for later determination of high-sensitivity (hs) cTnI levels (BeckmanCoulter UniCel DxI Access IA; log transformed detection limit = 0.30, quantification limit = 0.78, 99th percentile = 1.78). Positive and negative control samples were drawn from an adult ewe during a terminal experiment (myocardial ischemia) and similarly assessed. hs-cTnI data were log transformed from ng/L. Log(hs-cTnI) was 1.47 ± 0.30 (means ± SD) at 20 days before birth and declined to 1.02 ± 0.65 in fetuses 12 ± 4 h before birth (P < 0.0001, R2 = 0.7869). Birth stimulated a delayed, transient peak in hs-cTnI (P = 0.0058). Newborn (43 ± 19 min postnatal) levels were 1.39 ± 0.40 (P = 0.0650 vs. fetus on day of birth) and 2.14 ± 0.63 the day after birth (P = 0.0331 vs. newborn). The second day after birth, levels declined to 1.65 ± 0.48 (P = 0.0238 vs. day 1). IGF-1 infusion increased hs-cTnI levels 25-50% over baseline (P = 0.0252, R2 = 0.9938). Baseline adult ewe log(hs-cTnI) was below the limit of detection; 3 h following coronary artery ligation, levels were 3.21. In conclusion, we newly report that fetal hs-cTnI levels decline concomitantly with reduced proliferation of cardiomyocytes toward term.NEW & NOTEWORTHY Serial blood samples were collected from catheterized, normally developing fetal and newborn lambs and high-sensitivity cardiac troponin I (hs-cTnI) levels were assessed, providing unprecedented insight into the physiological processes leading to high levels in the perinatal period. Moderately high levels of hs-cTnI found in the normally developing fetus declined toward term. An elevation to high levels peaked the day after birth, after which hs-cTnI declined again. Stimulation of fetal cardiomyocyte proliferation with IGF-1 also elevated hs-cTnI.

PSI-5 Evaluation of EhV (surrogate for African swine fever virus) inactivation in corn- and soybean-based feed ingredients and diets at various times and temperatures during storage

Abstract African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA viruses (NCLDVs) and remains stable in a variety of environments, including animal feed ingredients as shown in previous laboratory experiments and simulations. Emiliania huxleyi virus (EhV) is another member of NCLDV, which has a restricted host range limited to a species of marine algae called Emiliania huxleyi. This algal NCLDV has many similar morphological and physical characteristics to ASFV which makes it a safe and reliable surrogate for studying ASFV survival and inactivation in feed ingredient matrices in-situ under various time and temperature storage conditions. The objective of the present study was to investigate the inactivation of ASFV in various feedstuffs under a wide range of time and temperature conditions during storage using EhV as a surrogate. Corn- and soybean-based feed ingredients including corn, corn fermented protein, distiller dried grains with solubles, extruded soybean meal, high protein distiller’s grains, solvent-extracted soybean meal, soybean hulls, complete feed, and a positive control (no matrix) were inoculated with EhV (1 × 108 viruses/mL). Negative controls were inoculated with medium containing no EhV for each matrix. To assess the effects of different times and temperatures of storage, all samples were stored at temperatures of 4, 24, and 34 °C for 1, 5, 60, and 120 d. For each timepoint and temperature combination, samples were evaluated for virus presence (qPCR) and viability using a previously validated viability qPCR (v-qPCR) method. On average, we observed a 0.2 log reduction in viable viral DNA content over the 120-d period, with a 0.1 log reduction across varying temperatures. No reductions in EhV viability exceeding 1 log were detected for any matrix and the positive control at any time and temperature combination evaluated. No EhV was recovered from the negative control samples. Given that the experimental error was calculated to be within the range of 1 log, we concluded that storing EhV at 4, 24, or 34 °C for up to 120 d did not result in the inactivation of EhV in any of the ingredients, complete feed, or the control with no matrix. These results demonstrate that ASFV-like NCLDVs is extremely resilient and can maintain viability in various feed matrices during long-term storage and suggest that extending storage time alone may not be an effective mitigation practice against African swine fever virus.

Circulating Fetal Troponin I May Reflect Cardiomyocyte Proliferation

INTRODUCTION: Circulating biomarkers such as Troponin I (TnI) that are typically indicative of myocardial damage in adults have been reported to be detectable in cord blood of healthy infants delivered both vaginally and by cesarean section prior to labor. We performed serial sampling of healthy fetal sheep from ~20 days before spontaneous birth to 5 days postnatal in order to describe normal developmental values of circulating TnI. METHODS: Seven fetal sheep were surgically instrumented with indwelling catheters advanced into the ascending aorta and the superior vena cava. Following surgical recovery, daily blood samples were taken first thing in the morning into heparin, and the plasma was frozen for later determination of high-sensitivity (hs) TnI levels (BeckmanCoulter UniCel DxI Access IA; log transformed limit of detection =0.30, limit of quantification =0.78, and 99th%ile =1.78). Positive and negative control samples were drawn from an adult ewe during a terminal experimental ligation of the left anterior descending coronary artery and similarly assessed. hs-TnI data were log transformed from ng/L, visually assessed, and outliers removed by the ROUT method. RESULTS: Log(hs-TnI) was 1.47±0.30 at 20 days before birth and declined to 0.88±0.36 in fetuses 12±4 hour before birth (p&lt;0.0001, R2=0.8444). Birth stimulated a delayed, transient peak in hs-TnI (P=0.0058). In the newborn, 43±19 min after birth, levels were 1.39±0.40 (P=0.0650 vs. fetus on day of birth). The day after birth, log(hs-TnI) increased to 2.14±0.63 (P=0.0331 vs. newborn). The second day after birth, levels declined to 1.65±0.48 (P=0.0238 vs. day 1). Adult ewe log(hs-TnI) was below the limit of detection; three hours following coronary artery ligation, levels were 3.21. CONCLUSIONS: This study confirms that hs-TnI levels are moderate to high in healthy sheep fetuses, and reports for the first time that they decline towards term. Why TnI is elevated in the healthy fetus is unknown. If cardiomyocyte TnI is leaked during cytokinesis, the decline towards birth may reflect the diminishing rate of proliferation in this same period. The postnatal hs-TnI peak, above the clinical threshold for evidence of myocardial ischemia in adult humans, may result from hemodynamic, oxidative, or metabolic stresses associated with the establishment of postnatal physiology. This work was funded by an award from the NHLBI (R01HL142483). 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.

Anti-Inflammatory Cytokine (IL-10) Profiles and Ratio of IL-6/IL-10 in Covid-19 Patients

Background: The number of COVID-19 cases in Indonesia has continually increased since the first cases appeared in March 2020. This disease is due to SARS-CoV-2 virus infection in the respiratory system that induces an immune response. The innate and adaptive immune response triggered the secretion of an excessive pro-inflammatory cytokine-caused cytokine storm that became one of the mechanisms of acute respiratory distress (ARDS). The anti-inflammatory cytokines (IL-10, IL-13, and IL-4) were secreted as the immune response in the ARDS condition. Purposes: This study aims to determine the ratio of the IL-6/IL-10 profile as basic information for the therapeutic approach to prevent ARDS. Methods: This cross-sectional study used stored biological material in plasma form from COVID-19 patients in Jakarta Islamic Hospital – Pondok Kopi and Dr. M. Goenawan Partowidigdo Hospital, Cisarua. The plasmas were from severe (n=20) patients and mild to moderate severity (n=25). The negative control sample was collected from 13 healthy subjects. Assessment of IL-10 levels in plasma using ELISA technique. Results: Our analysis showed that IL-10 has no statistical difference between negative control, mild to moderate, and severe categories (p=0.629). Meanwhile, the ratio IL-6/IL-10 presented statistical differences between mild to moderate and severe categories (p=0.011). The average ratio of IL-6/IL-10 in severe categories is two-fold higher than in mild-moderate categories. Conclusion: We conclude that there is a cytokine storm condition in severe COVID-19 patients with an imbalance ratio of pro-and anti-inflammatory cytokines and could be used as basic information for drug development in cytokine storm conditions to prevent ARDS.

Abstract 1171: Estimating sensitivity of exploratory cellular biomarkers in oncology clinical trials

Abstract Background: In the prolonged and complex process of assessing immunotherapeutic oncology drug candidates, flow cytometry plays an essential role. In oncology studies, biomarker measurement requires robust and reliable flow cytometric assays. A crucial element of these assays is to understand the lower limits of measurable surface biomarkers. Methods: We incorporated into the development and validation of flow cytometry assays the measurement of the lower limit of quantitation (LLOQ). The LLOQ is the lowest number of a reportable parameter in a blood or bone marrow study sample that can be quantitatively determined with acceptable precision and accuracy. Two steps are necessary to determine the LLOQ. First in these assessments a measurable range of lower limits of a particular cell type or receptor is estimated. That is, in validating an assay LLOQ, there are three options to select cells expressing target surface biomarkers of interest: using cultured or bioengineered cells, using samples of known high expression of a biomarker, or using pre-titered positive samples. Then decreasing concentrations of the target cell or receptor may be used to precisely determine the LLOQ of a specific assay reportable parameter. Results: By acquiring at least ten negative control sample results, the limit of the blank (LOB) and limit of detection (LOD) of a biomarker is determined. LOB is calculated as mean +1.645 SD, and LOD as mean +3 SD. The negative control samples either lack the fluorescent monoclonal antibodies against the markers of interest or lack the target cell population. Different reportable parameters, detected by one or more biomarkers have varying LLOQ. The LLOQ for a reportable parameter is a precise measurement of a parameter above or equal to the LOD. We found that typical LLOQ values are from 4 to 145 positive cells, with a frequency of 0.01 to 0.1% among a parent population. For example, in the case of tetramer assays, flow cytometry can reliably detect as few as 25 CD8+ antigen-specific cells among three million lymphocytes. Conclusions: LLOQ determinations for exploratory flow cytometry biomarkers are required for clinical research in cancer immunotherapy and cancer vaccine development. This valuable information allows for interpretation of data at the low end of a measurable range. Citation Format: Chengsen Xue, Christina D. Swenson, Thomas W. Mc Closkey. Estimating sensitivity of exploratory cellular biomarkers in oncology clinical trials [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 1171.

Abstract 239: Establishing a murine intratumoral bacterial model

Abstract Background: Long thought to be sterile spaces, current evidence now suggests that host microbiota can be found within cancerous tissue. Emerging data has illuminated that microbes are integral components of tumor tissue itself, which may correlate with tumor progression, treatment responsiveness, and disease prognosis. A recent Cell article from Fu et al. demonstrated that tumor-resident intracellular microbiota can promote metastatic colonization in breast cancer. Others within this research landscape see therapeutic opportunity - harnessing the tropism of these microbes to deliver TME modifying payloads directly to cancerous lesions. Purpose: Before studying the mechanism underlying their tropism to solid tumors, it is necessary to first establish a murine model that would allow for an oncotropic bacteria to be detected and quantified within tissues of interest. My research entailed the design and execution of pilot experiments developing xenograft melanoma and genetic colorectal models for future research projects. Methods: In all mouse studies, an E. coli Nissle 1917 strain called “NisLux” was used, who’s integration with a luxCDABE cassette allowed it to be selectively identified via luminescence using traditional exposure photography or endpoint luminescence detection using a 96-well plate reader. A xenograft melanoma model was developed using C57BL/6J mice and a B16-F10 murine melanoma line. A genetics model was developed using C57BL/6J-Apcmin mice which spontaneously develop colorectal adenomas as they age. Tumors, spleens, and healthy tissue were harvested and processed following tail vein injections of either NisLux or PBS solution. Harvested tissue was normalized by weight and concentration of resident NisLux was calculated from serially diluted samples plated on LB agar. Results: In both the xenograft and genetics murine models, NisLux exhibited tropism to tumor sites, enriching at a concentration above that of healthy background tissue. NisLux dosing correlated with splenomegaly without changes to the mass of the primary lesion compared to control. Conclusions: NisLux exhibits tropism to tumor sites in our xenograft and genetics murine models. Both models should undergo further procedural and control optimization. Two possible modifications to future melanoma xenograft experiments include the collection of non-cancerous skin negative control samples and a further delayed takedown day for tissue processing. In future C57BL/6J-Apcmin mice experiments, healthy colon sections from NisLux dosed C57BL/6J mice should be used as negative controls. Overall, the results from both models were promising and encourage future exploration of the mechanism underlying NisLux’s tropism to solid tumors. Subsequent experiments could involve comparing the tropism of engineered KO strains to WT NisLux or performing comparative sequencing of isolated tumor resident NisLux to the initial dosing population. Citation Format: Ryan A. Hannon, Dongqing Xu, Fengyi Wan. Establishing a murine intratumoral bacterial 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 239.

Insulin-like Growth Factor II mRNA-binding Protein 3 is a Highly Sensitive Marker for Intravascular Large B-cell Lymphoma: Immunohistochemical Analysis of 152 Pathology Specimens From 88 Patients.

Intravascular large B-cell lymphoma (IVLBCL) is a rare type of aggressive extranodal large B-cell lymphoma characterized by the selective growth of lymphoma cells within the lumina of blood vessels, particularly capillaries. IVLBCL lacks mass formation, and its diagnosis can be challenging. We analyzed the utility of insulin-like growth factor II mRNA-binding protein 3 (IMP3) immunohistochemistry for IVLBCL diagnosis in various organs. Double staining with paired box 5 (PAX5) was performed for validation. Overall, 152 pathological specimens (111 positive and 41 negative for IVLBCL) obtained from 88 patients with a diagnosis of IVLBCL were stained for IMP3 and IMP3/PAX5. As negative controls, 40 pathology specimens from 38 patients with no history of IVLBCL or other B-cell lymphomas were stained for IMP3, which comprised 31 benign pathological specimens from 29 patients in whom malignancy was suspected, 7 cases of appendicitis with intravascular and/or intralymphatic lymphoid proliferations, and 2 cases of intravascular natural killer/T-cell lymphoma. All mononuclear cells with cytoplasmic staining were considered positive for IMP3 expression, but expression restricted to germinal center B cells was excluded from evaluation. All 111 IVLBCL pathological specimens were positive for IMP3 and IMP3/PAX5. In addition, 11 of the 41 specimens originally diagnosed as IVLBCL-negative showed IMP3/PAX5 double-positive cells, raising the suspicion of IVLBCL. However, of the 40 negative control samples, IMP3-positive non-germinal center B cells were detected in only 2 samples ( P = 0.0131) and no intravascular IMP3-positive B cells suspicious for IVLBCL were identified. Altogether, IMP3 immunohistochemistry is a highly sensitive marker of IVLBCL and can be a helpful adjunct for IVLBCL diagnosis.

Effect of Applied AC Voltage on the Performance of Non-Faradaic Impedimetric Biosensors

Electrochemical impedance spectroscopy based non-faradaic biosensors are promising low-cost and rapid detection tool for a variety of biomarkers. Unlike faradaic impedimetric biosensors, non-faradaic impedimetric biosensors are redox-free and do not require the use of three electrodes. Instead, two electrodes are enough to carry out biosensing using non-faradaic biosensors. Commonly used AC perturbation during non-faradaic impedimetric biosensors is 10 mV to maintain the linearity of the electrochemical biosensor. In this work, we investigated the effect of different AC voltages (10, 50, 100, 200, 400, 800 and 1600 mV) on bare gold interdigitated electrodes (Au-IDEs). As the AC voltage increased, the capacitance and phase of bare Au-IDEs increased whereas the impedance decreased. At 800 and 1600 mV, non-linear effects started to appear on the Lissajous plots. Then, the effect of all the AC voltages except 800 and 1600 mV on the performance of fully functionalized non-faradaic LDL-cholesterol sensors were investigated. Increasing the AC voltage resulted in better limit of detection and change in capacitance. However, it also resulted in larger change in capacitance due to testing against negative (control samples). The reported study can be useful for the future development of non-faradaic impedimetric biosensors for the detection of different biomarkers and to achieve different limits of detection as per the application requirements.

Stage by stage E- Ecommerce market database analysis by using machine learning models

In the recent era, advertising strategies are far more sophisticated than those of their predecessors. In marketing, business contacts are essential for online transactions. For that, communication needs to develop a database; this database marketing is also one of the best techniques to enhance the business and analyze the market strategies. Businesses may improve consumer experiences, streamline supply chains, and generate more income by analyzing E-Commerce market datasets using machine learning models. In the ever-changing and fiercely competitive world of e-commerce, the multi-stage strategy guarantees a thorough and efficient use of machine learning. Analyzing the database can help to understand the user's or industry's current requirements. Machine Learning models are developed to support the marketing sector. This machine learning model can efficiently operate or analyze e-commerce in different stages, i.e., systematic setup, status analysis, and model development with the implementation process. Using these models, it is possible to analyze the marketing database and create new marketing strategies for distributing marketing objects, the percentage of marketing channels, and the composition of marketing approaches based on the analysis of the marketing database. It underpins marketing theory, data collection, processing, and positive and negative control samples. It is suggested that e-commerce primarily adopt the database marketing method of the model prediction. This is done by substituting the predicted sample into the model for testing. The issue of unequal marketing item distribution may be resolved by machine learning algorithms on the one hand, and prospective customer loss can be efficiently avoided on the other. Also, a proposal for an application approach that enhances the effectiveness of existing database marketing techniques and supports model prediction is made.

Protective effect of colchicine on albumin glycation and cellular oxidative stress: Insights into diabetic cardiomyopathy.

The present work elucidates the role of colchicine (COL) on albumin glycation and cellular oxidative stress in diabetic cardiomyopathy (DCM). Human serum albumin (HSA) was glycated with methylglyoxalin the presence of COL (2.5, 3.75, and 5 µM), whereas positive and negative control samples were maintained separately. The effects of COL on HSA glycation, structural and functional modifications in glycated HSA were analyzed using different spectroscopical andfluorescence techniques. Increased fructosamine, carbonyl, and pentosidine formation in glycated HSA samples were inhibited in the presence of COL. Structural conformation of HSA and glycated HSA samples was examined by field emission scanning electron microscopy, circular dichroism, Fourier transform infrared, and proton nuclear magnetic resonance analyses, where COL maintained both secondary and tertiary structures of HSA against glycation. Functional marker assays included ABTS•+ radical scavenging and total antioxidant activities, advanced oxidative protein product formation, and turbidimetry, which showed preserved functional properties of glycated HSA in COL-containing samples. Afterward, rat cardiomyoblast (H9c2 cell line) was treated with glycated HSA-COL complex (400 μg/mL) for examining various cellular antioxidants (nitric oxide, catalase, superoxide dismutase, and glutathione) and detoxification enzymes (aldose reductase, glyoxalase I, andII) levels. All three concentrations of COL exhibited effective anti-glycation properties, enhanced cellular antioxidant levels, and detoxification enzyme activities. The report comprehensively analyzes the potential anti-glycation and properties of COL during its initial assessment.

Bactericidal Effect of Triple Antibiotic Paste against Enterococcus faecalis in Dentinal Tubules: An Ex Vivo Study.

The aim of this study was to investigate the bactericidal effect of various concentrations of triple antibiotic paste (TAP) against Enterococcus faecalis (E. faecalis) in dentinal tubules using a bacterial culture assay and confocal laser scanning microscope (CLSM). Ninety human teeth were contaminated with E. faecalis (ATCC 29212) and randomly allocated into 5 groups; the negative control (without TAP), 1 mg/ml, 5 mg/ml, 7.5 mg/ml and 10 mg/ml TAP (n=18). After a 3-week TAP treatment, samples were collected from the root canal space, root dentin at 100-μm and 200-μm depth. The collected samples were subjected to a bacterial culture assay (n=10). Eight roots from each group underwent CLSM analysis to determine the live/dead bacterial cells. The bacterial culture assay results indicated that the negative control samples were all culturable. The number of culture-positive samples decreased after TAP treatment at 1, 5, 7.5 and 10 mg/ml, with 2, 2, 1 and 0 culturable samples, respectively. However, there was no significant difference among the TAP treatments. Surprisingly, the CLSM analysis demonstrated live bacteria in the dentinal tubules in all samples. The negative control had 52.36%+-3.24 live bacteria. TAP treatment at 10 mg/ml had the lowest percentage of live bacterial cells (40.58%+-5.40), followed by 7.5 mg/ml (44.14%+-6.03), 5 mg/ml (46.31%+-5.32) and 1 mg/ml (52.55%+-8.82). The percentage of live cells in the 10 mg/ml, 7.5 mg/ml and 5 mg/ml TAP groups were significantly lower than the 1 mg/ml TAP and negative control groups. TAP treatment significantly decreased the percentage of viable E. faecalis cells in the dentinal tubules and its bactericidal effect was dose-dependent.

A small-scale external quality assessment for PCR detection of group B streptococcus in China

BackgroundGroup B streptococcus (GBS) is considered a leading cause of maternal and infant morbidity and mortality. Molecular diagnosis is a routinely used approach for GBS screening to protect pregnant women and prevent early-onset GBS neonatal disease. The objective of this study was to identify issues and guarantee the dependability of GBS molecular diagnosis by an external quality assessment (EQA) scheme. MethodsThe EQA panel comprised eight samples spiked with 10-fold dilutions of GBS suspension (20–2,000,000 copies/mL), and 2 negative control samples. The panels were coded randomly and distributed to participating laboratories for GBS detection. ResultsIn total, 44 participating laboratories submitted results with eight commercial GBS PCR assays and one in-house assay. Among them, 36 obtained an acceptable or higher performance score, while 8 required improvement. Among the 440 results returned, 62 (14.1 %) were incorrect, including 5 false positives and 57 false negatives. ConclusionsOur small-scale EQA showed that most participating laboratories have reliable diagnostic capacities for GBS PCR detection. Nonetheless, further improvements in the detection performance of some laboratories are required, particularly with low-concentration samples. Our survey also reinforces the use of EQA as an essential tool to evaluate the overall proficiency of clinical laboratories.