qPCR Results Research Articles

Abstract 7443: Investigating the impact of ferroptosis in a high-fat and high-oxidative stress hepatic steatosis environment

Abstract An iron-dependent mechanism of cell death, ferroptosis, occurs with intracellular iron accumulation and is associated with lipid peroxidation. Previous studies have shown that lipid peroxidation-induced ROS leads to DNA damage and promotes the progression of steatosis to advanced HCC. Therefore, we aim to study ferroptosis in a cell model with hepatocytes co-cultured with ACM (adipocyte culture medium) and low dose H2O2 for a high-fat/high ROS environment in vitro; an animal model with high-fat content and accompanying low dose carbon tetrachloride in vivo to observe the possibility of hepatocyte steatosis due to ferroptosis. Animal model: Mice were fed a Western diet and provided with high fructose/glucose-containing sugar drinking water (WFSD) for 7 months while receiving a monthly extra-low dose of CCl4. Tissue staining: Liver sections were performed to observe histological abnormalities in each group. Cell model: Pre-adipocytes, 3T3L1 were differentiated by adding IBMX, Dexamethasone, and Insulin (10 μg/ml). After differentiation, ACM was collected. The XTT assay was used to examine the relationship between cell death and ferroptosis by a ferroptosis inducer (Erastin) in high-fat and high-ROS environments.qPCR: To detect the expression of iron transport-related genes.Western Blot: To ascertain protein levels associated with ferroptosis. Mitochondrial assay: Mitochondrial membrane potential (MMP), mitochondrial dynamics-related proteins, and fluorescence mitochondrial tracking and staining. Ferritin, GPX4, IRP2, and ferroportin were upregulated when ACM co-culture with the hepatocytic AML12 cell line, and it also increased the sensitivity of ROS-related cell cytotoxicity, which was related to ferroptosis by the inducer, Erastin, in the high-fat/high-ROS cell environment. However, these results can be suppressed by UAMC3203, a ferroptosis inhibitor. As for the animal model with high-fat and carbon tetrachloride accompanied experiments, the mice had higher weight, Alanine Aminotransferase (ALT) levels, and developed tumors. Their liver sections showed lipid accumulation, histological fibrosis, and iron accumulation in both the WD and WD+CCl4 groups, which corresponds to the IRP1 and IRP2 upregulated in both groups by qPCR result. Our research findings demonstrate that ferroptosis correlates with high-fat and high-ROS-induced hepatocellular damage. The related mitochondria dynamics changes in liver tissue and high-fat and/or high ROS provide evidence for ferroptosis-related hepatic steatosis. We have established the effect of ferroptosis on hepatic steatosis in vivo and in vitro in nonalcoholic steatosis hepatitis (NASH). These results may enable the development of precise health prevention strategies by inhibiting ferroptosis to prevent the occurrence of liver diseases including hepatic steatosis and HCC. Citation Format: Chi-Sheng (Wayne) Chen, Shu-Chi Wang. Investigating the impact of ferroptosis in a high-fat and high-oxidative stress hepatic steatosis environment [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 7443.

Abstract 6587: Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray

Abstract MYC deregulation is a key driver and feature of many human cancers. Yet, despite its importance in cancer progression, MYC is considered “undruggable” by small molecules due to its disordered conformation. Therefore, interfering with its interactions with protein partners to modulate MYC activity appears viable. FBXW7 is an E3 ligase, which serves as a constituent of the SCF (Skp1-Cul1-F box) ubiquitin ligase complex. It mediates the degradation of both c-Myc and Cyclin E through ubiquitin-dependent proteolysis. FBXW7 mutations are found in various human tumor types, establishing its role as a general human tumor suppressor. Specifically, FBXW7 down-regulation increases MYC level, and its expression correlates to lethal outcomes of breast epithelial cells, poor prognosis in glioblastoma, and T cell acute lymphoblastic leukemia remission. Therefore, our project aims to discover potent small molecule glues that would enhance the interaction between FBXW7 and MYC, leading to MYC degradation through proteolysis and eventually suppression of various cancers. Taking advantage of our high throughput screening technology, Small Molecule Microarrays (SMMs), we successfully identified 29 compounds that potently bind to both c-Myc and FBXW7 after screening our 65,000 ‘drug-like’ small molecule library. We then performed secondary assays using a mCherry-GFP MYC reporter cell line to identify our lead compound, KI-FBX-001, which decreases MYC’s expression:transcription ratio by over 50% compared to control. Additionally, this down-regulation can be rescued by protease inhibitor MG132, indicating that the effect is achieved via proteolysis. In vitro dose-response and time-course treatment revealed an optimal drugging concentration at 15uM in K562 cells, with inhibition reaching its peak at the 4-hour time point at over 60%. Using the Qiagen MYC reporter assay, the in vitro IC50 of KI-FBX-001 is found at 7.42uM. Beyond the protein level, the qPCR result also reveals a decrease in MYC mRNA level at over 70%. Surface Plasmon Resonance (SPR) is used to confirm direct binding between FBXW7 and KI-FBX-001, which showed promising binding with kd=0.17µM. Proximity Ligation Assay further confirms KI-FBX-001’s effect in enhancing FBXW7:MYC interaction in the cell. In summary, our findings proved that KI-FBX-001 is a promising molecular glue in the FBXW7:MYC interaction, and can effectively modulate the MYC level for future therapeutic purposes. Citation Format: Shenghao Guo, Morgan Stilgenbauer, Bocheng Wu, Yatin Mankan, Angela N. Koehler. Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray [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 6587.

Discovery of potential components characteristic by conjugated enone from the branches and leaves of Croton lauioides with anti-neuroinflammatory activity via regulating the NF-κB pathway

In this study, the chemical composition and pharmacological activity of Croton lauioides were investigated for the first time. The bioactive and HPLC-UV guided isolation led to the discovery of twenty-three conjugated enone-type components (1–23), including nine previously unknown sesquiterpenoid derivatives (1–4, 9–10, 12–14). Notably, compounds 1 and 12 are epoxides containing an endoperoxide bridge (1) or a unique dioxaspiro core (12), respectively. Compounds 2–7 are non-benzenoid aromatics featuring a tropone function, while 9–11 possess a rare rearranged scaffold with tropone shift into benzene. Extensive characterization was performed using NMR spectra, HRESIMS data, and electronic circular dichroism (ECD) calculations. Furthermore, we evaluated the bioactivities of all isolated compounds against neuroinflammation in LPS-stimulated BV-2 microglial cells. Remarkably, most sesquiterpenoid derivatives exhibited significant NO inhibit activities, and compound 5 showed the most potent effect with an IC50 value of 0.14 ± 0.04 μM. Structure-activity relationship (SAR) analysis revealed that sesquiterpenoids modified with endocyclic enone conjugation may serve as a key pharmacophore for NO inhibition, particularly involving aromatic tropone moiety. The qPCR and Western blot results demonstrated that 5 exerted an inhibitory effect on the mRNA levels of iNOS, TNF-α and COX-2 in a time-dependent manner, as well as suppressed the protein expression of iNOS, TNF-α, COX-2. In mechanism, 5 could prevented activation of NF-κB pathway by suppressing phosphorylation of p65 and IκB-α. These findings revealed C. lauioides might be a promising resource for drug candidate development targeting neuroinflammation.

Ultrasensitive, label-free, electrochemical detection of miRNA-206 in human plasma: A potential biomarker associated with Alzheimer’s disease

An impedance-based biosensor for the ultrasensitive, selective, and label-free detection of a blood miRNA associated to Alzheimer disease (AD), miRNA-206, was developed. The principle was grounded in the changes in the charge transfer resistance (RCT) as an effect of intramolecular forces between miRNAs and ferro/ferricyanide in a well-structured transducer platform. A compact well-ordered mixed monolayer made of co-immobilized miRNA capture to 6-mercapto-1-hexanol (MCH) in a 1:4 M ratio (at 37 °C), uplifted the performance of the sensor through effectively assisting the orientation of the oligonucleotides. In this work, the remarkable response of the sensor was generated through new insights into the use of different moieties of miRNA capture to MCH, aiming to control interfacial constants, surface densities, and hybridization efficiency.A very low limit of detection, 0.15 aM, is achieved and the sensor has a wide linear dynamic range (from 1 aM to 1 μM), high selectivity to mismatches, low non-specific binding of proteins (BSA) and good stability (<10 % change in response after 14 days storage). Importantly, the sensor successfully measured miRNA-206 concentrations in real plasma samples (>95 % recovery), correlating directly with qPCR results. Nanomolar concentrations of miRNA-206 were found in the plasma of confirmed AD patients, while healthy controls, had a concentration of pM or lower. The biosensor's ability to quantitatively detect miRNA-206 in plasma without target amplification, e.g., using PCR, is significant, opening the possibility of developing a point-of-care diagnostic device for AD screening, contributing to clinical trials and patient care.

Distinct cellular microenvironment with cytotypic effects regulates orderly regeneration of vascular tissues

Regeneration of the architecturally complex blood vascular system requires precise temporal and spatial control of cell behaviours. Additional components must be integrated into the structure to achieve clinical success for in situ tissue engineering. Consequently, this study proposed a universal method for including any substrate type in vascular cell extracellular matrices (VCEM) via regulating selective adhesion to promote vascular tissue regeneration. The results uncovered that the VCEM worked as cell adhesion substrates, exhibited cell type specificity, and functioned as an address signal for recognition by vascular cells, which resulted in matching with the determined cells. The qPCR and immunofluorescence results revealed that a cell type-specific VCEM could be designed to promote or inhibit cell adhesion, consistenting with the expression patterns of eyes absent 3 (Eya3). In addition, a 3D vascular graft combined with VCEM which could recapitulate the vascular cell-like microenvironment was fabricated. The vascular graft revealed a prospective role for cellular microenvironment in the establishment of vascular cell distribution and tissue architecture, and potentiated the orderly regeneration and functional recovery of vascular tissues in vivo. The findings demonstrate that differential adhesion between cell types due to the cellular microenvironment is sufficient to drive the complex assembly of engineered blood vessel functional units, and underlies hierarchical organization during vascular regeneration.

Analysis of diagnostic test outcomes in a large loiasis cohort from an endemic region: Serological tests are often false negative in hyper-microfilaremic infections.

The parasitic disease loiasis is associated with significant morbidity and mortality. Individuals with hyper-microfilaremia (greater than 20,000 microfilariae per mL of blood) may suffer from serious treatment-related or spontaneous adverse events. Diagnosing loiasis remains complex and primarily relies on direct parasite detection. In this study, we analyzed the performance of various diagnostic tests and the influence of parasitological and clinical factors on test outcomes in samples from individuals living in an endemic region. Data and samples were collected from rural Gabon. Loiasis was defined as either detectable microfilaremia, or a positive history of eyeworm as assessed by the RAPLOA questionnaire. Diagnostic testing included a quantitative PCR (qPCR) for detection of Loa loa DNA in blood samples, an in-house crude L. loa antigen IgG ELISA, and a rapid test for antibodies against the Ll-SXP-1 antigen (RDT). Sensitivity and specificity were determined for each test and factors potentially influencing outcomes were evaluated in an exploratory analysis. ELISA, RDT and qPCR results were available for 99.8%, 78.5%, and 100% of the 1,232 participants, respectively. The ELISA and RDT had only modest diagnostic accuracy. qPCR was specific for L. loa microfilaremia and Cycle threshold values correlated with microfilarial density. Anti-L. loa IgG levels were highest in occult loiasis, and antibody levels correlated inversely with L. loa microfilarial density as did RDT line intensities. Only 84.6% and 16.7% of hyper-microfilaremic individuals tested positive by ELISA (11/13) and RDT (2/12), respectively. None of the tests demonstrated high sensitivity and specificity for loiasis. Indirect diagnostic assays were characterized by low specificity. Additionally, hyper-microfilaremic individuals often tested negative by RDT and ELISA, indicating that these tests are not suitable for individual case management in endemic populations.

Inhibition of SIRT1 in the nucleus accumbens attenuates heroin addiction-related behavior by decreasing D1 neuronal autophagy.

This study aimed to investigate the effects of SIRT1 modulation on heroin addiction-like behavior and its possible biological mechanisms. Wild-type C57BL/6J and Sirt1loxp/loxp D1-Cre mice were used in this experiment, and Sirt1 loxp/loxp D1-Cre(-) mice were used as a control for conditional knockout mice. Mice were divided into saline control and heroin-dependent groups. Behavioral methods were used to record the withdrawal response, conditioned place preference (CPP) changes, and open field test results. Transmission electron microscopy (TEM) was used to observe the structure of autophagosomes in nucleus accumbens (NAc) neurons. The expression of SIRT1 and autophagy-related proteins and genes, such as LC3Ⅱ, ATG5 , and ATG7 , was detected in the NAc of each mouse group via western blot, real-time quantitative PCR (qPCR) analyzes, and immunofluorescence. The results of this experiment showed that compared with the saline group, mice in the wild-type heroin-dependent group showed marked withdrawal symptoms, with more autophagosomes observed in NAc via TEM. Compared with wild-type and Sirt1loxp/loxp D1-Cre(-) heroin-dependent groups, CPP formation was found to be reduced in the conditional knockout mouse group, with a significant decrease in spontaneous activity. Western blot, qPCR, and immunofluorescence results indicated that the expression of LC3Ⅱ, ATG-5, and ATG-7 was significantly reduced in the NAc of the Sirt1loxp/loxp D1-Cre(+) group. It was still, however, higher than that in the saline control group. These results suggest that inhibition of Sirt1 expression may prevent heroin-induced addiction-related behaviors via reducing D1 neuronal autophagy.

TRIM21 Promotes Oxidative Stress and Ferroptosis through the SQSTM1-NRF2-KEAP1 Axis to Increase the Titers of H5N1 Highly Pathogenic Avian Influenza Virus.

Tripartite motif-containing protein 21 (TRIM21) is involved in signal transduction and antiviral responses through the ubiquitination of protein targets. TRIM21 was reported to be related to the imbalance of host cell homeostasis caused by viral infection. Our studies indicated that H5N1 highly pathogenic avian influenza virus (HPAIV) infection up-regulated TRIM21 expression in A549 cells. Western blot and qPCR results showed that knockdown of TRIM21 alleviated oxidative stress and ferroptosis induced by H5N1 HPAIV and promoted the activation of antioxidant pathways. Co-IP results showed that TRIM21 promoted oxidative stress and ferroptosis by regulating the SQSTM1-NRF2-KEAP1 axis by increasing SQSTM1 K63-linked polyubiquitination under the condition of HPAIV infection. In addition, TRIM21 attenuated the inhibitory effect of antioxidant NAC on HPAIV titers and enhanced the promoting effect of ferroptosis agonist Erastin on HPAIV titers. Our findings provide new insight into the role of TRIM21 in oxidative stress and ferroptosis induced by viral infection.

Exploring the role of mitochondrial-associated and peripheral neuropathy genes in the pathogenesis of diabetic peripheral neuropathy

BackgroundDiabetic peripheral neuropathy (DPN) is a prevalent and serious complication of diabetes mellitus, impacting the nerves in the limbs and leading to symptoms like pain, numbness, and diminished function. While the exact molecular and immune mechanisms underlying DPN remain incompletely understood, recent findings indicate that mitochondrial dysfunction may play a role in the advancement of this diabetic condition.MethodsTwo RNA transcriptome datasets (codes: GSE185011 and GSE95849), comprising samples from diabetic peripheral neuropathy (DPN) patients and healthy controls (HC), were retrieved from the Gene Expression Omnibus (GEO) database hosted by the National Center for Biotechnology Information (NCBI). Subsequently, differential expression analysis and gene set enrichment analysis were performed. Protein–protein interaction (PPI) networks were constructed to pinpoint key hub genes associated with DPN, with a specific emphasis on genes related to mitochondria and peripheral neuropathy disease (PND) that displayed differential expression. Additionally, the study estimated the levels of immune cell infiltration in both the HC and DPN samples. To validate the findings, quantitative polymerase chain reaction (qPCR) was employed to confirm the differential expression of selected genes in the DPN samples.ResultsThis research identifies four hub genes associated mitochondria or PN. Furthermore, the analysis revealed increased immune cell infiltration in DPN tissues, particularly notable for macrophages and T cells. Additionally, our investigation identified potential drug candidates capable of regulating the expression of the four hub genes. These findings were corroborated by qPCR results, reinforcing the credibility of our bioinformatics analysis.ConclusionsThis study provides a comprehensive overview of the molecular and immunological characteristics of DPN, based on both bioinformatics and experimental methods.

Analysis and validation of hub genes in neutrophil extracellular traps for the long-term prognosis of myocardial infarction

IntroductionThe study focuses on the long-term prognosis of myocardial infarction (MI) influenced by neutrophil extracellular traps (NETs). It also aims to analyze and validate relative hub genes in this process, in order to further explore new therapeutic targets that can improve the prognosis of MI. Materials and methodsWe established a MI model in mice by ligating the left anterior descending branch (LAD) and conducted an 8-week continuous observation to study the dynamic changes in the structure and function of the heart in these mice. Meanwhile, we administered Apocynin, an inhibitor of NADPH Oxidase, which has also been shown to inhibit the formation of NETs, to mice undergoing MI surgery in order to compare. This study employed hematoxylin-eosin (HE) staining, echocardiography, immunofluorescence, and real-time quantitative PCR (RT-qPCR) to examine the impact of NETs on the long-term prognosis of MI. Next, datasets related to MI and NETs were downloaded from the GEO database, respectively. The Limma package of R software was used to identify differentially expressed genes (DEGs). After analyzing the “Robust Rank Aggregation (RRA)” package, we conducted a screening for robust differentially expressed genes (DEGs) and performed pathway enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to determine the functional roles of these robust DEGs. The protein–protein interaction (PPI) network was visualized and hub genes were filtered using Cytoscape. ResultsImmunofluorescence and qPCR results showed an increase in the expression of Myeloperoxidase (MPO) at week 1 and week 8 in the hearts of mice after MI. HE staining reveals a series of pathological manifestations in the heart of the MI group during 8 weeks, including enlarged size, disordered arrangement of cardiomyocytes, infiltration of inflammatory cells, and excessive deposition of collagen fibers, among others. The utilization of Apocynin could significantly improve these poor performances. The echocardiography displayed the cardiac function of the heart in mice. The MI group has a reduced range of heart movement and decreased ejection ability. Moreover, the ventricular systolic movement was found to be abnormal, and its wall thickening rate decreased over time, indicating a progressive worsening of myocardial ischemia. The Apocynin group, on the contrary, showed fewer abnormal changes in the aforementioned aspects. A total of 81 DEGs and 4 hub genes (FOS, EGR1, PTGS2, and HIST1H4H) were obtained. The results of RT-qPCR demonstrated abnormal expression of these four genes in the MI group, which could be reversed by treatment of Apocynin. ConclusionThe NETs formation could be highly related to MI and the long-term prognosis of MI can be significantly influenced by the NETs formation. Four hub genes, namely FOS, EGR1, PTGS2, and HIST1H4H, have the potential to be key genes related to this process. They could also serve as biomarkers for predicting MI prognosis and as targets for gene therapy.

Diagnostic, prognostic and treatment response of perilipin1 gene in breast cancer

BackgroundGenetic alterations in the perilipin (PLIN) family genes (PLIN1 to PLIN5) were infrequent in breast cancer (BC) where enhanced levels of PLIN1, PLIN3-5 were observed in the luminal A and luminal B subgroups, whereas increased PLIN2 expression was observed in the HER2-enriched and basal-like subgroups. However, the predictive value of PLIN1 for BC patient outcomes remains uncertain. In the present study, we aim to investigate the diagnostic, prognostic and treatment response roles of the PLIN1 gene expression in BC. MethodsWe obtained microarray BC trancriptomic data of 320 tumor (T) and 62 normal (N) breast samples from five GEO data-series; GSE7904 (38 T:7N), GSE42568 (101 T: 15 N), GSE26910 (6 T:6N), GSE45827 (144 T:7N), and GSE10810 (31 T:27 N). The Welch t test was used to analyze the significant differences in gene expression including PLIN1 with fold change > ±2 and p-value < 0.05. The expression of PLIN1 was confirmed by RTqPCR using clinical specimen samples from BC patients. The Kaplan-Meier Plotter was used to assess survival on large independent dataset (31 dataset for relapse-free survival and 14 datasets for overall survival) and significance was determined by calculating hazard ratios (>1) and log-rank p-values < 0.05. We also assessed the treatment outcomes of endocrine therapy (tamoxifen and aromatase-inhibitors), anti-HER2 therapy (trastuzumab and lapatinib), and chemotherapy (taxane, anthracycline, and ixabepilone) using robust statistical methods and correlated with PLIN1 gene expression. ResultsWe identified significantly reduced expression of PLIN1 (FC = −30.76, p value = 2.183e−24) in BC samples compared with normal controls. Our qPCR result confirmed the microarray expression pattern of PLIN1 in BC. Survival analysis revealed PLIN1 to be a moderately important prognostic biomarker. Our findings highlight the effectiveness of trastuzumab and anthracycline in classifying treatment responses, supported by Mann-Whitney tests indicating statistical significance in gene expression differences between responders and non-responders. ConclusionIn conclusion, our findings indicate that PLIN1 is one of the most down-regulated genes and a moderately important biomarker in BC for prognostic purposes. PLIN1 was a good indicator of trastuzumab and anthracycline treatment responses in BC.

A case report of Pallister-Killian syndrome with an unusual mosaic supernumerary marker chromosome 12 with interstitial 12p13.1-p12.1 duplication.

Pallister-Killian syndrome (PKS) is a rare inherited disease with multiple congenital anomalies, profound intellectual disability, and the presence in the karyotype of sSMC - i(12)(p10). The frequency of PKS may be underestimated due to problems with cytogenetic diagnosis caused by tissue-specific mosaicism and usually a low percentage of peripheral blood cells containing sSMC. Such tissue-specific mosaicism also complicates a detailed analysis of the sSMC, which, along with the assessment of mosaicism in different tissues, is an important part of cytogenetic diagnosis in PKS. Unfortunately, a full-fledged diagnosis in PKS is either practically impossible or complicated. On the one hand, this is due to problems with the biopsy of various tissues (skin biopsy with fibroblast culture is most often used in practice); on the other - a low percentage of dividing peripheral blood cells containing sSMC, which often significantly complicates the analysis of its composition and organization. In the present study, a detailed analysis of sSMC was carried out in a patient with a characteristic clinical picture of PKS. A relatively high percentage of peripheral blood cells with sSMC (50%) made it possible to perform a detailed molecular cytogenetic analysis of de novo sSMC using chromosomal in situ suppression hybridization (CISS-hybridization), multicolor FISH (mFISH), multicolor chromosome banding (MCB), array CGH (aCGH), and quantitative real-time PCR (qPCR), and short tandem repeat (STR) - analysis. As a result, it was found that the sSMC is not a typical PKS derivative of chromosome 12. In contrast to the classical i(12)(p10) for PKS, the patient's cells contained an acrocentric chromosome consisting of 12p material. Clusters of telomeric repeats were found at the both ends of the sSMC. Furthemore, the results of aCGH and qPCR indicate the presence of interstitial 8.9Mb duplication at 12p13.1-p12.1 within the sSMC, which leads to different representations of DNA from different segments of 12p within cells containing sSMC. The obtained data raise the question of the instability of the sSMC and, as a consequence, the possible presence of additional rearrangements, which, in traditional cytogenetic analysis of patients with PKS, are usually described as i(12)(p10).

Differential Symptomology, Susceptibility, and Titer Dynamics Manifested by Phytoplasma-Infected Periwinkle and Tomato Plants.

Phytoplasmas are intracellular pathogenic bacteria that infect a wide range of plant species, including agriculturally important crops and ornamental trees. However, our understanding of the relationship between symptom severity, disease progression, and phytoplasma concentration remains limited due to the inability to inoculate phytoplasmas mechanically into new plant hosts. The present study investigated phytoplasma titer dynamics and symptom development in periwinkle and tomato, both infected with the same potato purple top (PPT) phytoplasma strain using a small seedling grafting approach. Virescence, phyllody, and witches'-broom (WB) symptoms sequentially developed in periwinkle, while in tomato plants, big bud (BB, a form of phyllody), cauliflower-like inflorescence (CLI), and WB appeared in order. Results from quantitative polymerase chain reaction (qPCR) targeting the PPT phytoplasma's 16S rRNA gene revealed that in both host species, phytoplasma titers differed significantly at different infection stages. Notably, the highest phytoplasma concentration in periwinkles was observed in samples displaying phyllody symptoms, whereas in tomatoes, the titer peaked at the BB stage. Western blot analysis, utilizing an antibody specific to PPT phytoplasma, confirmed substantial phytoplasma presence in samples displaying phyllody and BB symptoms, consistent with the qPCR results. These findings challenge the conventional understanding that phytoplasma infection dynamics result in a higher titer at later stages, such as WB (excessive vegetative growth), rather than in the early stage, such as phyllody (abnormal reproductive growth). Furthermore, the PPT phytoplasma titer was markedly higher in periwinkles than in tomato plants, indicating differing susceptibilities between the hosts. This study reveals distinct host responses to PPT phytoplasma infection, providing valuable insights into phytoplasma titer dynamics and symptom development, with implications for the future management of agricultural disease.

Effect and mechanism of transient receptor potential canonical channel 3 on hyperoxic lung injury in neonatal rats

AbstractThe aim of this study is to research the expression of the transient receptor potential canonical channel 3 (TRPC3) in a neonatal hyperoxic lung injury model of bronchopulmonary dysplasia (BPD), and to further investigate the role of the TRPC3/nuclear factor‐κB (NF‐κB) signaling pathway in hyperoxia‐induced BPD by a TRPC3 agonist (GSK1702934A). The hyperoxic lung injury model of BPD was established in Sprague–Dawley neonatal rats. Hematoxylin and eosin (HE) staining and radial alveolar count (RAC) values showed that the hyperoxic lung injury model of BPD was successfully established in the neonatal rats, and pulmonary edema was found in the neonatal rats with BPD. The results of reference transcriptome sequencing, Quantitative real‐time PCR (qPCR), and western blot showed lower pulmonary expression of TRPC3 in the BPD group than in the control group. Immunofluorescence showed predominant expression of TRPC3 in airways and pulmonary vessels, and the fluorescence intensity of the BPD group was lower than that of the control group. Lung dry‐to‐wet weight ratio, HE staining, and RAC value showed that the lung histomorphology significantly improved in the BPD + TRPC3 agonist group compared with the BPD group on day 14 but did not revert to the level of the control group. According to qPCR results, compared with the control group, the expression of NF‐κB1 decreased and the expression of NF‐κBiz increased in the BPD group, whereas the expression of NF‐κBiz decreased in the BPD + TRPC3 agonist group. Therefore, we draw the conclusion that TRPC3 may activate NF‐κB by inhibiting NF‐κBiz to promote cell proliferation and lung growth and development.

Swimming exercise reverses transcriptomic changes in aging mouse lens

BackgroundThe benefits of physical activity for the overall well-being of elderly individuals are well-established, the precise mechanisms through which exercise improves pathological changes in the aging lens have yet to be fully understood.Methods3-month-old C57BL/6J mice comprised young sedentary (YS) group, while aging mice (18-month-old) were divided into aging sedentary (AS) group and aging exercising (AE) group. Mice in AE groups underwent sequential stages of swimming exercise. H&E staining was employed to observe alterations in lens morphology. RNA-seq analysis was utilized to examine transcriptomic changes. Furthermore, qPCR and immunohistochemistry were employed for validation of the results.ResultsAE group showed alleviation of histopathological aging changes in AS group. By GSEA analysis of the transcriptomic changes, swimming exercise significantly downregulated approximately half of the pathways that underwent alterations upon aging, where notable improvements were ‘calcium signaling pathway’, ‘neuroactive ligand receptor interaction’ and ‘cell adhesion molecules’. Furthermore, we revealed a total of 92 differentially expressed genes between the YS and AS groups, of which 10 genes were observed to be mitigated by swimming exercise. The result of qPCR was in consistent with the transcriptome data. We conducted immunohistochemical analysis on Ciart, which was of particular interest due to its dual association as a common aging gene and its significant responsiveness to exercise. The Protein-protein Interaction network of Ciart showed the involvement of the regulation of Rorb and Sptbn5 during the process.ConclusionThe known benefits of exercise could extend to the aging lens and support further investigation into the specific roles of Ciart-related pathways in aging lens.

Optimization of duplex digital PCR for the measurement of SARS-CoV-2 RNA

Quantitative PCR (qPCR) is the gold standard for detecting nucleic acid sequences specific to the target pathogen. For COVID-19 diagnosis, several molecular assays have been developed. In this study, we present an optimization strategy for the measurement of SARS-CoV-2 RNA via multiplex qPCR and digital PCR (dPCR). Compared to qPCR, both droplet and chip-based dPCR, which are known to be more sensitive and accurate, showed a better resilience to suboptimal assay compositions and cycling conditions following the proposed optimizations. In particular, the formation of heterodimers among assays greatly interfered with qPCR results, but only minimally with dPCR results. In dPCR, existing heterodimers lowered the PCR efficiency, producing a dampened fluorescent signal in positive partitions. This can be corrected by adjusting the PCR cycling conditions, after which dPCR shows the capability of measuring the expected copy number. In addition, we present a process to improve the existing RdRp assay by correcting the primer sequences and matching the melting temperature, ultimately producing highly sensitive and robust assays. The results of this study can reduce the cost and time of SARS-CoV-2 diagnosis while increasing accuracy. Furthermore, our results suggest that dPCR is a reliable method for the accurate measurement of nucleic acid targets.

Faster clinical decisions in B-cell acute lymphoblastic leukaemia: A single flow cytometric 12-colour tube improves diagnosis and minimal residual disease follow-up.

Assessing minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is essential for adjusting therapeutic strategies and predicting relapse. Quantitative polymerase chain reaction (qPCR) is the gold standard for MRD. Alternatively, flow cytometry is a quicker and cost-effective method that typically uses leukaemia-associated immunophenotype (LAIP) or different-from-normal (DFN) approaches for MRD assessment. This study describes an optimized 12-colour flow cytometry antibody panel designed for BCP-ALL diagnosis and MRD monitoring in a single tube. This method robustly differentiated hematogones and BCP-ALL cells using two specific markers: CD43 and CD81. These and other markers (e.g. CD73, CD66c and CD49f) enhanced the specificity of BCP-ALL cell detection. This innovative approach, based on a dual DFN/LAIP strategy with a principal component analysis method, can be used for all patients and enables MRD analysis even in the absence of a diagnostic sample. The robustness of our method for MRD monitoring was confirmed by the strong correlation (r = 0.87) with the qPCR results. Moreover, it simplifies and accelerates the preanalytical process through the use of a stain/lysis/wash method within a single tube (<2 h). Our flow cytometry-based methodology improves the BCP-ALL diagnosis efficiency and MRD management, offering a complementary method with considerable benefits for clinical laboratories.

Attenuation of neuronal ferroptosis in intracerebral hemorrhage by inhibiting HDAC1/2: Microglial heterogenization via the Nrf2/HO1 pathway.

The class I histone deacetylases (HDACs) implicate in microglial heterogenization and neuroinflammation following Intracerebral hemorrhage (ICH). Ferroptosis has also been reported in the ICH model. However, the relationship between HDAC1/2's role in microglial heterogenization and neuronal ferroptosis remains unclear. In both invivo and invitro models of ICH, we used Romidepsin (FK228), a selective HDAC1/2 inhibitor, to investigate its effects on microglial heterogenization and neuronal ferroptosis. In the invitro ICH model using Hemin, a transwell system was utilized to examine how microglia-driven inflammation and ICH-triggered neuronal ferroptosis interact. Immunostaining, Western blotting and RT-qPCR were used to evaluate the microglial heterogenization and neuronal ferroptosis. Microglial heterogenization, neuronal ferroptosis, and neurological dysfunctions were assessed invivo ICH mice model performed by autologous blood injection. HDAC1/2 inhibition altered microglial heterogenization after ICH, as showing the reducing neuroinflammation and shifting microglia towards an anti-inflammatory phenotype by immunostaining and qPCR results. HDAC1/2 inhibition reduced ferroptosis, characterized by high ROS and low GPx4 expression in HT22 cells, and reduced iron and lipid deposition post-ICH invivo. Additionally, the Nrf2/HO1 signaling pathway, especially acetyl-Nrf2, activated in the invivo ICH model due to HDAC1/2 inhibition, plays a role in regulating microglial heterogenization. Furthermore, HDAC1/2 inhibition improved sensorimotor and histological outcomes post-ICH, offering a potential mechanism against ICH. Inhibition of HDAC1/2 reduces neuro-ferroptosis by modifying the heterogeneity of microglia via the Nrf2/HO1 pathway, with a particular focus on acetyl-Nrf2. Additionally, this inhibition aids in the faster removal of hematomas and lessens prolonged neurological impairments, indicating novel approach for treating ICH.

Circulating cell-free DNA is elevated in postmenopausal compared with pre- and perimenopausal women.

With the rising use of circulating cell-free DNA (cirDNA) liquid biopsies for disease screening, it is important to understand biological differences that may impact the accuracy of cirDNA-based clinical tests. Although a number of biological factors have been researched, the relationship between menopause and cirDNA has not been thoroughly investigated. We aimed to compare plasma cirDNA concentration and DNA fragment integrity in healthy women pre- and postmenopause. Blood was collected from healthy female volunteers 40 years and older. cirDNA was extracted from plasma (n = 52) and quantified by quantitative polymerase chain reaction (n = 47; 26 premenopause, mean age-46 y; 21 postmenopause, mean age-59 y). cirDNA concentration was quantitated using an ALU repetitive sequence with a 115-base-pair (bp) product (ALU-115), and long cirDNA fragments were quantitated using an ALU repetitive sequence with a 247-bp product (ALU-247). cirDNA integrity was expressed as a ratio of ALU-247 over ALU-115. Mann-Whitney U test was used to compare pre- and postmenopause qPCR results, and a two-tailed, unpaired t test was undertaken to compare the integrity ratio between the two groups. Postmenopause plasma samples were found to have a significantly higher cirDNA concentration (P < 0.0001, premenopause: mean, 3.10 ± 1.84 ng/mL; median, 2.90 ng/mL; postmenopause: mean, 5.28 ± 2.76 ng/mL; median, 4.56 ng/mL) and significantly higher concentration of long-stranded cirDNA fragments (P = 0.0033, premenopause: mean, 1.06 ± 0.48 ng/mL; median, 0.96 ng/mL; postmenopause: mean, 1.69 ± 0.89 ng/mL; median, 1.48 ng/mL). There was no significant difference in the integrity ratio between the groups (P = 0.1788). Plasma cirDNA concentrations are higher in postmenopausal women. This has important implications in cirDNA liquid biopsy development and screening, especially for diseases such as cancer where the majority of cases are diagnosed postmenopause.

The life-history trait trade-offs mediated by reproduction and immunity in the brown planthopper, Nilaparvata lugens Stål

Reproduction and immune defense are costly functions, and they are expected to tradeoff with each other to drive evolution. The brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera, Delphacidae), is a global superpest that mostly damages rice crops. Yeast-like symbionts (YLS) exist in the abdominal fat body tissue and are tightly associated with the development, growth, and reproduction of BPH. Our previous research demonstrated that mating behavior promotes the release of YLS from the fat body into the hemolymph in the BPH, thereby triggering an immune response. Additionally, the fitness costs related to life-history traits of BPH (such as survival rate) have a strong dependence on the relative abundance of YLS. However, the possible relationship between reproduction and the immune response in BPH has not been identified. In this study, an omics-based approach was used to analyze the transcriptome of fat body tissues in mated and unmated BPH at 72 h post-eclosion, from which two antimicrobial peptide genes, NlDefensin A (NlDfA) and NlDefensin B (NlDfB), were selected since they were highly expressed in mated BPH. Subsequently, the full-length cDNA sequences of the NlDfA and NlDfB genes were cloned and analyzed. qPCR results showed up-regulation of the NlDfA and NlDfB genes in mated BPH when compared to unmated BPH. Spatial-temporal expression analysis indicated that the NlDfA and NlDfB genes were expressed in all tissues and developmental stages, and they were most highly expressed in the fat body at 24 h post-eclosion. Moreover, the symbionts in BPH were significantly inhibited by the in vitro expression of the NlDfA and NlDfB proteins. Furthermore, RNA interference (RNAi)-mediated suppression of NlDfA and NlDfB dramatically increased the relative abundance of YLS in the fat body, while YLS in the hemolymph decreased significantly. These BPHs also displayed some fitness disadvantages in survival, fecundity, hatchability, and possibly the vertical transmission of YLS from hemolymph to egg. Our results indicated that mating could heighten the immunity of BPH by up-regulating the expression of the NlDfA and NlDfB genes, which protect the host from pathogen challenges during reproduction. However, the reduced content of YLS may act as a fitness disadvantage in dictating the life-history traits of BPH. This work has significant theoretical and practical implications for the precise green control technology that involves crucial gene targeting, as well as for the “endosymbionts for pest control” strategy in insects.