ΔCt Values Research Articles

Correlation Between Family Dysfunction and Nonsuicidal Self-Injury in a Sample of Chinese Adolescents: The Mediating Effect of Alexithymia and circRNA_103636.

Adolescent group may be prone to a variety of behavioral disorders, one of which is nonsuicidal self-injury (NSSI), NSSI intervention is limited for its unknown mechanism, so this study aimed to explore the factors associated with and pathological mechanism underlying NSSI from the perspective of family dysfunction, alexithymia, circRNA_103636 in a sample of Chinese adolescents. A total of 200 MDD adolescents with NSSI and 200healthy controls were enrolled via a convenient sampling method in clinical settings. The Family APGAR Index (APGAR), Toronto Alexithymia Scale (TAS-20), and Adolescent Nonsuicidal Self-injury Assessment Questionnaire (ANSSIAQ) were used for mental assessment of the study group and control group participants. Real-time quantitative reverse transcription PCR (qRT-PCR) was utilized to detect circRNA_103636 expression in peripheral blood mononuclear cells (PBMCs). There were significant between-group differences of 134 patients (67%) in the study group and 42 patients in the control group (21%) with moderate or severe family dysfunction (P<0.01). The APGAR score was lower, and the difficulty identifying feeling (DIF), difficulty describing feeling (DDF) and externally oriented thinking (EOT) scores of the TAS-20 and ΔCt value of circRNA_103636 were greater in the study group than in the control group. NSSI behavior and NSSI function were negatively correlated with the APGAR score and positively correlated with DIF, DDF, and the EOT of TAS-20 and the ΔCt value of circRNA_103636. Multiple regression analysis confirmed that EOT, circRNA_103636 expression, and APGAR were predictors of ANSSIAQ, which could explain 40.5% of the variance. Similarly, the alexithymia and circRNA_103636 expression mediated the correlation between family dysfunction and NSSI in the study group, and these mediating effects accounted for 27.25% and 23.33%, respectively, of the total effect. Taken together, family dysfunction, alexithymia, and circRNA_103636 expression have predictive effects on NSSI and alexithymia, circRNA_103636 expressions are mediators between family dysfunction and NSSI in Chinese adolescent. Here, we established a new model for NSSI in which exposure to family dysfunction could induce pathological process by modulating personality traits and epigenetic regulators.

Combining single nucleotide polymorphisms and gene expression of LEP, FABP4, STAT5A and DGAT1 genes for improvement of body weight in Holstein calves

The objective of this study was to investigate the possible association of single nucleotide polymorphisms and gene expression of leptin (LEP), fatty acid binding protein (FABP4), signal transducer and activator of transcription 5A (STAT5A) and diacylglycerol acyltransferase (DGAT1) genes with body weight in Holstein calves. For the RNA extraction process, blood samples from 100 Holstein bull calves were taken. According to farm records, the acquired bull calves were weaned at a body weight of roughly 90 kg, with birth weights ranging from 31 to 38 kg and weaning ages ranging from 60 to 100 days. To account for the non-genetic causes, the dairy calves' body weight was changed to their 205-day body weight. Nucleotide sequence changes in the form of SNPs were found in the LEP, FABP4, STAT5A, and DGAT1 genes in the investigated Holstein calves. The identified SNPs and corrected 205-day body weight showed a significant correlation (P &lt; 0.05). According to ΔCT values, a significant relationship between the mRNA levels of the LEP, FABP4, STAT5A, and DGAT1 genes and body weight was also found (P &lt; 0.05). The effectiveness of the LEP, FABP4, STAT5A, and DGAT1 genes as proxies for growth qualities in Holstein dairy calves is highlighted by this study. Therefore, it was possible to select an animal with a high rate of growth.

8414 Utilization of Cell Free DNA Integrity Index to Differentiate Benign from Malignant Thyroid Nodules

Abstract Disclosure: S. Tarafdar: None. S. Dutta: None. P. Mukhopadhyay: None. N.P. Bhattacharya: None. S. Ghosh: None. Introduction: Recent advances in molecular genetics have evaluated a potential role for plasma cell-free DNA (cfDNA) integrity index as a non-invasive cancer marker. Data on cfDNA integrity in differentiated thyroid cancer is sparse and needs to be investigated further. Aim of the study: The purpose of this study is to investigate the hypothesis that longer DNA strands in plasma may serve as a marker for thyroid cancer. Methods: Patients presenting with thyroid nodules underwent ultrasonography with Thyroid Image Reporting and Data Systems (TIRADS) scoring and FNAC (Bethesda classification).Patients with Bethesda 3, 4, 5, and 6 had surgery and confirmation by histopathology. Patients with Bethesda 2 presenting with compressive symptoms and cosmetic concerns underwent surgery; the remaining patients were considered benign based on clinical follow-up, USG, and FNAC characteristics. Blood sample collected prior to FNAC was used to extract and quantify cfDNA. To assess the integrity index 180/67, 306/67, and 476/67 for the APP gene, we used a quantitative real-time PCR (qPCR) method based on the quantification of four amplicons of varying lengths (67, 180, 306, and 476 bp, respectively). Because culture cells had extremely intact genomic DNA, the genomic DNA obtained from the MDA-T32 papillary thyroid cancer cell line was utilised as a reference to estimate the relative DNA strand integrity in plasma DNA. To determine the (ΔCt) value for 67,180, 306, and 476 bp, the Ct value of the sample was subtracted from that of the MDA-T32 control. Results: A total of 161 subjects were recruited, of these 92 were benign and 69 had differentiated thyroid cancer(confirmed by histopathology). Using the 180/67 bp integrity index benign thyroid nodule patients had significant lower value (median 0.39, 95% CI: 0.33-0.46) compared to patients with differentiated thyroid cancer (median 0.62, 95% CI: 0.56-0.68). A cfDNA integrity index of 180/67 bp could significantly differentiate between benign and malignant thyroid nodules (P&amp;lt;0.001). However, there was no significant difference between malignant and benign thyroid nodules based on the cell free DNA integrity indexes of 306/67 and 476/67. Conclusions: The cfDNA integrity score of 180/67 exhibits potential as a circulating marker for the detection of thyroid nodules and as a means of monitoring cfDNA fragmentation in patients with thyroid cancer. However, further confirmation through prospective trials is necessary. Presentation: 6/3/2024

8414 Utilization Of Cell Free DNA Integrity Index To Differentiate Benign From Malignant Thyroid Nodules

Abstract Disclosure: S. Tarafdar: None. S. Dutta: None. P. Mukhopadhyay: None. N.P. Bhattacharya: None. S. Ghosh: None. Introduction: Recent advances in molecular genetics have evaluated a potential role for plasma cell-free DNA (cfDNA) integrity index as a non-invasive cancer marker. Data on cfDNA integrity in differentiated thyroid cancer is sparse and needs to be investigated further. Aim of the study: The purpose of this study is to investigate the hypothesis that longer DNA strands in plasma may serve as a marker for thyroid cancer. Methods: Patients presenting with thyroid nodules underwent ultrasonography with Thyroid Image Reporting and Data Systems (TIRADS) scoring and FNAC (Bethesda classification).Patients with Bethesda 3, 4, 5, and 6 had surgery and confirmation by histopathology. Patients with Bethesda 2 presenting with compressive symptoms and cosmetic concerns underwent surgery; the remaining patients were considered benign based on clinical follow-up, USG, and FNAC characteristics. Blood sample collected prior to FNAC was used to extract and quantify cfDNA. To assess the integrity index 180/67, 306/67, and 476/67 for the APP gene, we used a quantitative real-time PCR (qPCR) method based on the quantification of four amplicons of varying lengths (67, 180, 306, and 476 bp, respectively). Because culture cells had extremely intact genomic DNA, the genomic DNA obtained from the MDA-T32 papillary thyroid cancer cell line was utilised as a reference to estimate the relative DNA strand integrity in plasma DNA. To determine the (ΔCt) value for 67,180, 306, and 476 bp, the Ct value of the sample was subtracted from that of the MDA-T32 control. Results: A total of 161 subjects were recruited, of these 92 were benign and 69 had differentiated thyroid cancer(confirmed by histopathology). Using the 180/67 bp integrity index benign thyroid nodule patients had significant lower value (median 0.39, 95% CI: 0.33-0.46) compared to patients with differentiated thyroid cancer (median 0.62, 95% CI: 0.56-0.68). A cfDNA integrity index of 180/67 bp could significantly differentiate between benign and malignant thyroid nodules (P&amp;lt;0.001). However, there was no significant difference between malignant and benign thyroid nodules based on the cell free DNA integrity indexes of 306/67 and 476/67. Conclusions: The cfDNA integrity score of 180/67 exhibits potential as a circulating marker for the detection of thyroid nodules and as a means of monitoring cfDNA fragmentation in patients with thyroid cancer. However, further confirmation through prospective trials is necessary. Presentation: 6/3/2024

Targeting ocular tissues with intravenously administered aptamers selected by in vivo SELEX

Ocular diseases cause a significant economic burden and decrease in quality of life worldwide. Drugs and carrier molecules that penetrate ocular tissues after intravenous administration are needed for more efficient and patient-friendly treatment of ocular diseases. Here, ocular barrier penetrating aptamers were selected through the utilization of in vivo SELEX and intravenous injection in rats. Three aptamers – Apt1, Apt2, and Apt5 – were chosen based on their specific accumulation in vascularized ocular tissues and further characterized for their in vivo biodistribution using RT-qPCR. A statistically significant difference between ΔCt values of ocular and control tissues with Apt2 (P < 0.0001) and Apt5 (P < 0.0001) was observed. Interestingly, Apt1 was the most abundant aptamer in the sequencing pool, but did not show a statistically significant difference in in vivo biodistribution between ocular and control tissues. Overall, this study established a functional in vivo SELEX method for discovering ocular tissue targeting aptamers.

B-217 Effective Diagnostic Platform and Method to Differentiate the Infectious From Non-Infectious Viruses With Nucleic Acid Detection Workflow

Abstract Background While qPCR has demonstrated high sensitivity, specificity, and ability to deliver reliable quantitative data in clinical samples, such results do not differentiate between infectious and non-infectious viral particles. In view of the principle of the qPCR method itself, the amplification detects the nucleic acid (DNA/RNA) of the virus, not the virus itself. In many cases, it is still possible to detect DNA/RNA fragments from dead viruses in recovered patients, while they are not infectious anymore. This will lead to prolonged quarantine period or treatment for the patients, which could cause great psychological pressure to the patients and increased healthcare burden. Therefore, an effective detection method that can discriminate viable and inviable pathogens is of great significance. Methods SARS-CoV2 viral RNA, SARS-CoV-2 viruses, or clinical samples with SARS-CoV2 infection were diluted in Delta MTM (Molecular Transport Media) to the respective testing concentrations. The samples were incubated with intercalating dye (PMAxx 100nM) with or without Triton X-100 for 10 min. The mixture was then exposed to the photo-activation device for 5-15 min (@ temperature below 37° C). The samples were then subjected to the standard nucleic acid extraction &amp; purification steps with Qiagen viral RNA extraction kit. The extracted RNA was subjected to amplification with SARS-CoV2 Resolute 2.0 &amp; Fortunate 2.1 assays on BioRAD CFX96. Results The viability PCR (vPCR) efficiency was almost 100% for all SARS-CoV2 RNA input tested suggesting that without viral membrane protection, PMAxx dye effectively bound to the naked RNA after photoactivation process, and the bound RNA was not able to be amplified. The vPCR efficiency was further enhanced with addition of surfactant Triton X-100 suggesting the improved permeability of dye through the viral envelope facilitated the PMAxx binding to the nucleic acid. COVID19 positive clinical samples with viral load ≈ Ct 30 treated with or without PMAxx and photo-activation procedures showed distinctive ΔCt value ranging from 9.27±0.18 to 15.01±0.14. The Ct after dye treatment were all non-numeric (non-detectable) if cut-off Ct sets @ 42.0, indicating the complete inhibition after vPCR pretreatment. Since the clinical samples were undergone heat-inactivation pre-treatment, the result shown here indicating that all samples were non-infectious, which was consistent with the samples tested. Conclusions We developed an effective viability PCR detection method and proprietary vPCR device, which was specifically designed for effective dye photo-activation. In addition, the method takes less than 15 min and could be integrated into the existing standard nucleic acid extraction and amplification workflow. Comparing to the standard PCR test result, this method could provide the information not only on the positivity of the tested pathogen, but also its viability, which could be very essential information for public surveillance and diagnostic decisions for healthcare providers.

Comparative analysis of the safety and effectiveness of Nirmatrelvir-Ritonavir and Azvudine in older patients with COVID-19: a retrospective study from a tertiary hospital in China.

Introduction: Numerous studies have explored the treatment outcomes of Nirmatrelvir-Ritonavir and Azvudine in older patients with COVID-19. However, direct comparisons between these two drugs are still relatively limited. This study aims to compare the safety and effectiveness of these two drugs in Chinese older patients with early infection to provide strategies for clinical treatment. Methods: Older COVID-19 patients (age ≥65) hospitalized during the winter 2022 epidemic in China were included and divided into Nirmatrelvir-Ritonavir and Azvudine. Demographics, medication information, laboratory parameters, and treatment outcomes were collected. All-cause 28-day mortality, delta cycle threshold (ΔCt), nucleic acid negative conversion time, and incidence of adverse events were defined as outcomes. Propensity score matching (PSM), Kaplan-Meier, Cox proportional hazards model, subgroup analysis, and nomograms were selected to evaluate the outcomes. Results: A total of 1,508 older COVID-19 patients were screened. Based on the inclusion and exclusion criteria, 1,075 patients were eligible for the study. After PSM, the final number of older COVID-19 patients included in the study was 375, and there were no significant differences in demographic characteristics between the two groups (p > 0.05). Compared to the Azvudine group, the Nirmatrelvir-Ritonavir group showed a higher incidence of multiple adverse events (12.8% vs 5.2%, p = 0.009). The incidence of adverse events related to abnormal renal function was higher in the Nirmatrelvir-Ritonavir group compared to the Azvudine group (13.6% vs 7.2%, p = 0.045). There were no significant differences between the two groups in terms of all-cause 28-day mortality (HR = 1.020, 95% CI: 0.542 - 1.921, p = 0.951), whereas there were significant differences in nucleic acid negative conversion time (HR = 1.659, 95% CI: 1.166 - 2.360, p = 0.005) and ΔCt values (HR = 1.442, 95% CI: 1.084 - 1.918, p = 0.012). Conclusion: Azvudine and Nirmatrelvir-Ritonavir have comparable effectiveness in reducing mortality risk. Azvudine may perform better in nucleic acid negative conversion time and virus clearance and shows slightly better safety in older patients. Further studies with a larger sample size were needed to validate the result.

O-253 Altered telomerase activity in ovarian follicular cells in patients with polycystic ovary syndrome

Abstract Study question Is telomerase machinery altered in cumulus and granulosa cells of patients with Polycystic ovary syndrome (PCOS) undergoing assisted reproductive treatment (ART)? Summary answer Patients with PCOS presented an altered expression of telomerase inhibitor factors TRF1 and TRF2 and shorter telomeres in cumulus cells (CCs) and granulosa cells (GCs) What is known already PCOS affects 6%-20% of fertile women; one of the most common features is represented by abnormal folliculogenesis, showing a higher number of follicles at all developmental stages. In PCOS, increased levels of reactive oxygen species (ROS) have been related to abnormal follicular development due to oxidative stress-induced DNA damage. Telomeres, present at the ends of the chromosomes, are essential for maintaining their stability during somatic cell proliferation. When guanine-rich telomeric repeats are exposed to oxidative stress including ROS, they undergo oxidative DNA damage that shortens telomeres in non-dividing cells like follicular cells, thereby potentially altering folliculogenesis. Study design, size, duration A prospective experimental study was conducted at IVIRMA Rome and University Unicamillus from September 2022 to January 2024. CCs and GCs were collected from the follicular fluid of patients undergoing in vitro fertilization (IVF). Twenty-eight patients with PCOS and. Thirty-one normal responders were included in the pilot study. Participants/materials, setting, methods Women with PCOS (n = 28), diagnosed according to the Rotterdam criteria, were included in the study. The healthy control group (CONT) included normal responders (n = 31) undergoing IVF. CCs and GCs were collected on the day of oocyte retrieval and subsequently used for immunofluorescence spot counting of TRF1 and TRF2. In addition, telomere length was measured using a specific kit. qRT-PCRs for TRF1, TRF2 and apoptotic marker BAX genes were performed and expressed as 2^-ΔCT values. Main results and the role of chance Immunofluorescence for TRF1 in the PCOS group presented a higher spot number in CCs compared to the CONT (9.90±0.23 vs 7.96±0.33, p = 0.0009) while TRF2 spots significantly decreased (8.85 ±0.24 vs 11.23±0.28; p = 0.0007). An increase of TRF2 gene expression was observed in CCs of the PCOS group when compared to CONT (0.0052±0.0006 vs 0.0030±0.0003; p = 0.062) no differences were found in the expression of TRF1 (0.0343±0.006 vs 0.026±0.047p=0.68; Bax was significantly increased (0.0139±0.001 vs 0.0102±0,0007 p = 0.007) and telomeres have a shortening trend (0.36±0.36 vs 1±0 p = 0.74). In GC a lower number of TRF1 spots in PCOS vs CONT was observed while TRF2 had just an increasing trend (11.16± 0.586 vs 11.70 ± 1.99, p = 0.61; 16.79 ± 3,85 vs 11.34 ± 0.588; p = 0.68). In GCs of PCOS group vs CONT there was an increase in TRF2 gene expression (0,007 ± 0,001 vs 0.003 ± 0.0004; p = 0.03) and no differences were found in TRF1 (0.032± 0.003 vs 0.003± 0.012 p = 0.25). Ultimately Bax was increased in GCs of PCOS samples vs CONT (0.018 ±0.002 vs 0.011 ±0.0005 p = 0.0006) and the telomeres were significantly shorter (0.0246± 0.0349 vs 1± 0 p &amp;lt; 0.0001). Limitations, reasons for caution A limited number of samples were analyzed in the study and different PCOS sub-phenotypes were not considered. The analysis of CCs could be uninformative due to inter-individual variability in the PCOS sub-phenotypes within the same study group. Wider implications of the findings These findings demonstrate that PCOS patients present a higher expression of telomerase-inhibiting factors TRF1 and TRF2, undergo apoptosis and have significantly shorter telomeres supporting the theory that abnormal folliculogenesis in PCOS affects telomere length, therefore shedding new light on this medical condition and possibly explaining its clinical and biochemical features. Trial registration number not applicable

Calibration curve for radiation dose estimation using FDXR gene expression biodosimetry – premises and pitfalls

Purpose Radiation-induced alterations in gene expression show great promise for dose reconstruction and for severity prediction of acute health effects. Among several genes explored as potential biomarkers, FDXR is widely used due to high upregulation in white blood cells following radiation exposure. Nonetheless, the absence of a standardized protocols for gene expression-based biodosimetry is a notable gap that warrants attention to enhance the accuracy, reproducibility and reliability. The objective of this study was to evaluate the sensitivity of transcriptional biodosimetry to differences in protocols used by different laboratories and establish guidelines for the calculation of calibration curve using FDXR expression data. Material and Methods Two sets of irradiated blood samples generated during RENEB exercise were used. The first included samples irradiated with known doses including: 0, 0.25, 0.5, 1, 2, 3 and 4 Gy. The second set consisted of three ‘blind’ samples irradiated with 1.8 Gy, 0.4 Gy and a sham-irradiated sample. After irradiation, samples were incubated at 37 °C over 24 h and sent to participating laboratories, where RNA isolation and FDXR expression analysis by qPCR were performed using sets of primers/probes and reference genes specific for each laboratory. Calibration curves based on FDXR expression data were generated using non-linear and linear regression and used for dose estimation of ‘blind’ samples. Results Dose estimates for sham-irradiated sample (0.020–0.024 Gy) and sample irradiated with 0.4 Gy (0.369–0.381 Gy) showed remarkable consistency across all laboratories, closely approximating the true doses regardless variation in primers/probes and reference genes used. For sample irradiated with 1.8 Gy the dose estimates were less precise (1.198–2.011 Gy) but remained within an acceptable margin for triage within the context of high dose range. Conclusion Methodological differences in reference genes and primers/probes used for FDXR expression measurement do not have a significant impact on the dose estimates generated, provided that all reference genes performed as expected and the primers/probes target a similar set of transcript variants. The preferred method for constructing a calibration curve based on FDXR expression data involves employing linear regression to establish a function that describes the relationship between the logarithm of absorbed dose and FDXR ΔCt values. However, one should be careful with using non-irradiated sample data as these cannot be accurately represented on a logarithmic scale. A standard curve generated using this approach can give reliable dose estimations in a dose range from 50 mGy to 4 Gy at least.

Employing semi-quantitative methods to classify breast cancer markers (ER, PR, HER2, Ki67) expression, using the RT-qPCR based APIS Breast Cancer Subtyping Kit.

e13091 Background: Accurate determination of breast cancer (BC) biomarker status is crucial for guiding patient management decisions. Histopathologists commonly use semi-quantitative scoring systems to convert subjective observations of immunohistochemistry (IHC) marker expression into quantitative data, allowing a more detailed categorisation of marker status. The APIS Breast Cancer Subtyping Kit (BCSK) is an in vitro diagnostic test, detecting the relative expression of seven mRNA target genes ( ESR1, PGR, ERBB2, MKI67, CCNA2, PCNA, and KIF23) from invasive BC tissue. The APIS BCSK reports a positive/negative result for each biomarker alongside a molecular classification. Here, we demonstrate that by implementing additional RNA expression cut-off values, it is possible to generate a semi-quantitative result by further stratifying target expression into negative, low, medium, and high status using the APIS BCSK. Methods: 368 formalin-fixed paraffin-embedded (FFPE) samples of BC cases were used, obtained by core needle biopsy or resection. IHC scores were correlated with copy numbers as determined by an independently validated digital PCR (dPCR) assay and were used to set copy number cut-off values corresponding to IHC classification. The generated dPCR cut-off values were used to evaluate the corresponding ∆Ct values (RNA expression results derived from the APIS BCSK). For each target, three ΔCt cut-offs were established, facilitating classification into negative, low, medium, and high expression. Results: The semi-quantitative approach successfully classified target expression levels ( ESR1, PGR, ERBB2, MKI67). Overlap in ΔCt values was observed in central IHC categories for all targets, likely due to tumor heterogeneity and differing measurement methods for IHC and RT-qPCR. Despite these discrepancies, the derived cut-off values define a semi-quantitative scale for each target (Table). Conclusions: This study has established a methodology that utilises target copy number values and IHC protein quantification to establish ΔCt cut-off points. For each target we have defined four distinct groups as negative, low, medium, and high expression. This approach provides a semi-quantitative scale for evaluating each target with the APIS BCSK and serves as a valuable tool for classifying and understanding target expression levels. [Table: see text]

Discussion on the studies of position-specific carbon isotopes of propane by Li et al. (2018), Zhang et al. (2022) and Shuai et al. (2023)

In a series of recent publications in Organic Geochemistry and elsewhere, investigators from State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry and their collaborators have reported position-specific δ13C of propane obtained from natural reservoirs and pyrolysis experiments. Based solely on the very negative ΔC-T values (δ13Ccenter – δ13Cterminal), ranging from −7 to −10 ‰, they advocated for abiogenic origins of the propanes from sedimentary settings (shale and coal deposits). However, they never demonstrated accuracy of the data and it is likely that correct ΔC-T values for these propanes are close to zero ‰. Therefore, their discussion and interpretation in terms of geochemical processes, including a putative abiogenic origin, are not substantiated.

ADGRA3: In vivo hepatic localization, functional importance, and in vitro activation

G protein-coupled receptors (GPCRs), which encode for more than 3% of all human genes, are the largest protein family in the human genome. However, most GPCRs remain under-characterized with little known about their expression, agonists, and physiological function. To identify understudied receptors in the liver, the largest metabolic organ in the body, we performed a TaqMan array screen of more than 300 GPCRs in both male and female C57BL6 murine livers. Among the most highly expressed orphan receptors is ADGRA3 (GPR125), an adhesion GPCR (aGPCR), whose expression is comparable to that of the glucagon receptor, with ΔCt values of 17.2 and 16.0 respectively. aGPCRs possess a unique N-terminal domain that includes a GPCR auto proteolysis-inducing (GAIN) domain, which contains a GPS cleavage site that allows for the cleavage and non-covalent attachment of the N-Terminal (NTF) and C-Terminal Fragments. It is theorized that the displacement of the NTF exposes a tethered ligand, which ultimately results in ADGRA3 activation. ADGRA3 has an unconventional GPS site and it was unclear if this receptor follows a similar activation sequence. To test this, we synthesized peptides corresponding to the β-strand immediately following the predicted GPS domain and tested for activation using the PRESTO-TANGO assay, a luminescence readout to indicate receptor activation. We confirmed that ADGRA3 is activated by a tethered ligand, observing maximal activation with the complete β-strand and 2 additional C-Terminal amino acids. We then further employed the PRESTO-TANGO assay to deorphanize ADGRA3 by identifying non-self activators in a large, unbiased chemical screen (&gt;500 chemicals). This resulted in the identification of more than 25 unique ligands whose selective binding results in ADGRA3 activation. To date, our studies show that ADGRA3 is best activated by C16H20N2O3. By using RNA scope, we localized Adgra3 transcripts to the cuboidal endothelial cells in the lining of the bile duct and hepatocytes. This localization was confirmed through antibody staining, which revealed that ADGRA3 localizes to the basolateral membrane of cholangiocytes. To investigate physiological function of hepatic ADGRA3, we are characterizing the C57BL6-ADGRA3-GreenLantern mouse (GL-KO), where insertion of a GreenLantern tag within ADGRA3 is predicted to disrupt functionality. To visualize the impacts of ADGRA3 disruption, we employed functional Magnetic Resonance Imaging (MRI) to track the uptake of gadoxetate disodium (EOVIST). Injection of exogenous EOVIST is selectively taken up by the liver through organic anion transporting polypeptides (OATP) and is further cleared into the gallbladder. In wildtype ADGRA3 mice, EOVIST was rapidly taken up by the liver and was found within the common bile duct entering the gallbladder by 60 minutes. In the GL-KO, this clearance was delayed with minimal accumulation in the common bile duct. Given the localization of ADGRA3 within the cholangiocytes, this raises the possibility that ADGRA3 may play an essential role in hepatic development and biliary repair mechanisms. NIH/NCATS. 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.

MOLECULAR DETECTION PROTOCOL OF SARS-COV-2 THROUGH SELF-COLLECTED SALIVA SPECIMENS VERSUS NASOPHARYNGEAL SWABS.

Several reports have shown that saliva specimen is an excellent alternative biofluid sample for SARS-CoV-2 detection. We conducted this study, in order to assess the sensitivity and specificity of using saliva self-collected by adult and pediatric patients, as a biological sample for RT-PCR diagnosis. The present study was carried out to assess the sensitivity and specificity of using saliva self-collected from adult and pediatric patients, as a biological sample for RT-qPCR diagnosis. In this study, 50 symptomatic patients and 40 asymptomatic subjects (adult and pediatric) were enrolled between September 2020 and November 2020 at the Department of Infectious Diseases, Bejaia University Hospital (CHU), and tested simultaneously for the sensitivity and specificity of the SARS-CoV-2 viral genome by RT-PCR on both nasopharyngeal swabs NP swab and saliva samples. Our RT-qPCR results revealed that saliva samples showed the highest sensitivity (95% CI [27.67, 29.82]) followed by a nasopharyngeal swab for symptomatic (95% CI [29.64, 31.49]) as well as for asymptomatic adult patients. Moreover, the saliva of symptomatic and asymptomatic patients was monitored, and the presence of viral RNA was detected in >95% of the asymptomatic patients as well as the symptomatic patients. Surprisingly, the Ct values of ORF1ab and N genes are highly lower in nasopharyngeal swabs compared to saliva. Indeed, the mean difference note that for the ORF1ab gene and N gene, the mean of difference in ΔCt value were respectively 3.683 and 3.578. Together, including symptomatic and asymptomatic subjects, the overall agreement between the saliva sample and the nasopharyngeal is about 84%. The sensitivity of saliva samples remains acceptable; it may still be a viable option in locations where laboratory facilities are lacking for diagnostic purposes in the early phase of the disease.

Assessment of miRNA-10b Expression Levels as a Potential Precursor to Metastasis in Localized and Locally Advanced/Metastatic Breast Cancer among Iraqi Patients.

Breast cancer (BC) stands as the most prevalent form of carcinoma among women, ranking as the second leading cause of cancer-related mortality in the female population. The objective of this study is to assess the expression of miR-10b and determine its diagnostic and prognostic significance in breast cancer patients across various disease stages. The investigation was carried out in Baghdad at the Oncology Teaching Hospital within Baghdad Medical City and the Oncology Unit at Al-Yarmouk Teaching Hospital. A total of 150 samples were included and divided into two groups: the blood group consisting of 90 samples (including control subjects, localized BC patients, and those with metastatic and locally advanced BC) and the tissue group comprising 60 samples (representing both benign and malignant BC cases). The study spanned from March 2022 to January 2023, with patients' ages ranging from 24 to 75 years. The primary focus of this investigation was to identify the gene expression of miRNA-10b in all sample types. This was achieved by measuring gene expression levels and normalizing them to the level of a housekeeping gene (U6), and quantification was carried out considering the ΔCt value and the fold change method (2-ΔΔCt). The results revealed an upregulated fold expression of miRNA-10b, particularly in locally advanced and metastatic BC, where the expression was significantly higher compared to the other groups, with a fold expression of 1.770 ± 0.1070. In localized breast cancer, the fold expression was 1.624 ± 0.064, and in malignant tissue, it measured 1.546 ± 0.06754, all relative to apparently healthy control subjects. In summary, our research provides compelling evidence supporting the classification of miRNA-10b as an oncogenic factor in BC. The central involvement of miRNA-10b in the tumorigenic processes of BC highlights its reference for developing novel targeted therapeutic interventions and detection biomarkers for BC treatment. Notably, elevated expression of miRNA-10b was observed in BC tissues, correlating with an unfavorable distant metastasis-free survival outcome.

The Relationship of Grade, Stage and Tobacco Usage in Head and Neck Squamous Cell Carcinoma With p53, PIK3CA and MicroRNA Profiles.

Head and neck squamous cell carcinoma (HNSCC) has multiple epigenetic modifications including post-transcriptional regulation by microRNAs (miRNAs) as well as alterations in molecular pathways due to mutations. Examining these miRNAs and location-specific molecular alterations is essential to understanding the intricacies of HNSCC and directing focused diagnoses and treatments. To investigate tobacco-related changes in the expression of miRNAs and proteins with clinicopathological parameters of HNSCC and disease-modifying personal habits like tobacco and alcohol use. The study concentrated on oropharyngeal cancers using immunohistochemistry and reverse transcription-polymerase chain reaction. Expression of microRNAs mir15a, mir20b, mir21, mir31, mir33b, mir146a, mir155, mir218, mir363 and mir497 and immunohistochemical expression of P53 and PIK3CA were correlated with grade, stage and personal habits like tobacco and alcohol intake. mir21 and mir15a are under-expressed in higher grades with a trend towards statistical significance (P-value of 0.094 and 0.056 by one-way analysis of variance (ANOVA) on ΔCT values). mir155 and mir146a are overexpressed in stage IV tumours while mir 31 is under-expressed in stage IV tumours but statistical significance was not reached.mir497 showed overexpression in tobacco users, but these results were limited by many tumours not showing any amplification for the miRNA and statistical significance was not reached. There was no statistically significant association found between immunohistochemical expression of p53 and PIK3CA with grade, stage or personal habits. Through the deciphering of complex miRNA patterns and their relationships with clinicopathology, this study attempted to increase our understanding of HNSCC. Some candidate miRNAs showing probable association with grade, stage and personal habits were identified, but larger studies are needed to confirm or refute the importance of these miRNAs.

Human transcriptome array analysis and diffusion tensor imaging in attention-deficit/hyperactivity disorder

The mRNA markers identified using microarray assay and diffusion tensor magnetic resonance imaging (DTI) were applied to elucidate the pathophysiology of attention-deficit hyperactivity disorder (ADHD). First, we obtained total RNA from leukocytes from three children with ADHD and three healthy controls for analysis with microarray assays. Subsequently, we applied real-time quantitative polymerase chain reaction (qRT‒PCR) assays to validate the differential expression of 7 genes (COX7B, CYCS, TFAM, UTP14A, ZNF280C, IFT57 and NDUFB5) between 130 ADHD patients and 70 controls, and we built an ADHD prediction model based on the ΔCt values of aforementioned seven genes (AUROC = 0.98). Finally, in a validation group (28 patients with ADHD and 27 healthy controls), mRNA expression of the above seven genes also significantly differentiated ADHD patients from controls (AUROC value = 0.91). The DTI analysis showed increased fractional anisotropy (FA) of the forceps minor, superior corona radiata, posterior corona radiata and anterior corona radiata in ADHD patients. Moreover, the FA of the right superior corona radiata tract was positively correlated with ΔCt levels of the COX7B gene and the IFT57 gene. The results shed a new light on a genetic profile of ADHD that may help in deciphering the white matter microstructural features in disease pathogenesis.

Detection of the DNA methylation of seven genes contribute to the early diagnosis of lung cancer

BackgroundLow-dose Computed Tomography (CT) is used for the detection of pulmonary nodules, but the ambiguous risk evaluation causes overdiagnosis. Here, we explored the significance of the DNA methylation of 7 genes including TAC1, CDO1, HOXA9, ZFP42, SOX17, RASSF1A and SHOX2 in the blood cfDNA samples in distinguishing lung cancer from benign nodules and healthy individuals.MethodA total of 149 lung cancer patients [72 mass and 77 ground-glass nodules (GGNs)], 5 benign and 48 healthy individuals were tested and analyzed in this study. The lasso-logistic regression model was built for distinguishing cancer and control/healthy individuals or IA lung cancer and non-IA lung cancer cases.ResultsThe positive rates of methylation of 7 genes were higher in the cancer group as compared with the healthy group. We constructed a model using age, sex and the ΔCt value of 7 gene methylation to distinguish lung cancer from benign and healthy individuals. The sensitivity, specificity and AUC (area under the curve) were 86.7%, 81.4% and 0.891, respectively. Also, we assessed the significance of 7 gene methylation together with patients’ age and sex in distinguishing of GGNs type from the mass type. The sensitivity, specificity and AUC were 77.1%, 65.8% and 0.753, respectively. Furthermore, the methylation positive rates of CDO1 and SHOX2 were different between I-IV stages of lung cancer. Specifically, the positive rate of CDO1 methylation was higher in the non-IA group as compared with the IA group.ConclusionCollectively, this study reveals that the methylation of 7 genes has a big significance in the diagnosis of lung cancer with high sensitivity and specificity. Also, the 7 genes present with certain significance in distinguishing the GGN type lung cancer, as well as different stages.

Investigation of Mirna-223-3p Expression in Non-small Cell Lung Cancers.

Lung cancer remains a principal cause of cancer-related mortality worldwide. Non-small cell lung cancer (NSCLC), representing a significant 80-85% of lung cancer diagnoses, often presents at an advanced stage, with many patients demonstrating local growth or metastasis at the time of detection. Consequently, there exists a pressing need for augmented research into the molecular and genetic underpinnings of this malignancy to facilitate the development of innovative therapeutic and preventative strategies. MicroRNA's (miRNAs) are non-coding RNA molecules, consisting of 20-25 nucleotides. Their involvement in epigenetic processes holds a pivotal role in the elucidation of molecular mechanisms. This study examined the potential of miRNA-223-3p as a biomarker in the diagnosis of patients with NSCLC. The expression analysis of miRNA-223-3p was performed in serum samples obtained from 18 patients diagnosed with NSCLC and a control group comprising 15 healthy volunteers. The miRNA-223-3p ΔCT values in the serum samples taken from the patient group exhibited statistically significant elevation compared to those of the control group (p=0.043). The present study corroborates previous literature indicating elevated levels of miRNA-223-3p in NSCLC cases and thereby suggesting its potential role as a biomarker in NSCLC cases.

Intramolecular carbon isotopic rollover in propane from natural gas reservoirs of the Santanghu Basin: Insights into chemical structure of kerogen

An intramolecular isotopic study was conducted on natural gases from the Santanghu Basin in China. Propane samples spanned a wide range of ΔC-T (δ13Ccentral-δ13Cterminal) values from –8.4 to 8.5 ‰, δ13Ccentral and δ13Cterminal values varied from –37.3 to –25.8 ‰ and –35.7 to –28.9 ‰, respectively. During thermal maturation, the position-specific carbon isotopic composition of propane shifted from δ13Ccentral < δ13Cterminal to δ13Ccentral > δ13Cterminal, and the δ13Cterminal decreased by 6.8 ‰ within a Ro range of 0.46–0.50 %. The intramolecular carbon isotopic rollover and a decrease in δ13Cterminal with a slight increase in maturation could be explained by kinetic effects in thermal cracking of kerogen with a low proportion of branched chains. As isomeric groups were rapidly exhausted, propane generation pathway rapidly changed and primary kinetic isotope effect completely shifted from central carbon position to terminal carbon positions in propane. These changes increased carbon isotope fractionation at terminal carbon positions, thereby reducing δ13Cterminal values and shifting δ13Ccentral < δ13Cterminal to δ13Ccentral > δ13Cterminal within a small maturity range. This work demonstrated that position-specific isotope distribution of propane can be applied to elucidate the chemical structure of kerogen.

MicroRNAs in Umbilical Cord Blood and Development in Full-Term Newborns: A Prospective Study.

Exploring the epigenetic regulations, such as microRNA, in newborns holds significant promise for enhancing our ability to address and potentially prevent early-life developmental delays. Hence, this research seeks to investigate if the expression of miRNA in the umbilical cord blood of infants can forecast their developmental outcomes as they grow older. We enrolled 143 full-term newborns, delivered either via cesarean section (CS) or through natural spontaneous delivery (NSD). We then analyzed the profiles of specific miRNAs (miR-486-5p, miR-126-5p, miR-140-3p, miR-151a-3p, miR-142-5p, and miR-30e-5p) in the umbilical cord blood of these infants. Subsequently, we performed follow-up assessments using Bayley-III scores when the cohort reached 1 year of age. Furthermore, we conducted pathway-enrichment analyses on the target genes associated with these examined miRNAs. When comparing newborns delivered via cesarean section (CS) to those born via natural spontaneous delivery (NSD), we observed notable differences. Specifically, newborns through NSD displayed significantly higher ΔCt values for miR-486-5p, alongside lower ΔCt values for miR-126-5p and miR-151a-3p in their cord blood. At 1 year of age, cognitive development was significantly linked to the ΔCt values of miR-140-3p and miR-142-5p, while language development showed a significant association with the ΔCt values of miR-140-3p. Moreover, our pathway enrichment analyses revealed that the target genes of these miRNAs were consistently involved in the pathways related to neurons, such as axon guidance and the neurotrophin signaling pathway. In summary, this study represents a pioneering effort in elucidating the potential connections between miRNA levels in cord blood and the health indicators and neurodevelopment of newborns at 1 year of age. Our findings underscore the significance of miRNA levels at birth in influencing mechanisms related to neurodevelopment.