Real-time PCR Analysis Research Articles

Selection of reference genes for quantitative real-time PCR analysis in exogenous hormone-treated Lycoris aurea.

Selection of reference genes for quantitative real-time PCR analysis in exogenous hormone-treated Lycoris aurea.

Casticin inhibits the hedgehog signaling and leads to apoptosis in AML stem-like KG1a and mature KG1 cells

Abstract Objectives Acute myeloid leukemia (AML) is a severe blood cancer with less than 50 % long-term survival. Despite advancements in treatment options, relapse is still the major obstacle. The main reason of this problem is ineffective targeting of leukemic stem cells (LSCs), which play an important role in tumor development and relapse. In our previous studies, we found that casticin, the major polyphenolic component of Vitex trifolia’s fruit, targets both leukemic cells and LSCs without affecting healthy tissues. Therefore, in this study, we aimed to investigate the effect of casticin-mediated cell death in relation to the LSCs-favored survival pathways at gene and protein expression levels using in vitro LSC-like and parental leukemic cell models. Methods We validated the LSC character of KG1a and KG1 cells (84.55 % CD34+, CD38- and 93.55 % CD34+, CD38+, respectively) by flow cytometry. For the investigation of casticin’s mechanism of action, we employed real time-PCR, western blotting and bioinformatics analyses. Results Our results showed an increase in cleaved PARP/β-actin ratio but no change in LC3BI/II and SQSTM/β-actin ratios. Our gene expression, bioinformatics and immunoblotting analyses represented significant decrease in Shh, Gli and Wnt levels. We also elucidated a possible crosstalk between Hedgehog and other oncogenic cascades via the Gli, Notch, YAP, p38, Mcl-1, and Myc proteins in casticin mediated anti-leukemic effect. Conclusions In conclusion, we found that casticin induces apoptosis in both LSC-like and parental leukemia cells mainly by suppressing Shh signaling, which is crucial for LSC survival and AML relapse.

Vacuolar phosphate efflux transporter ZmVPEs mediate phosphate homeostasis and remobilization in maize leaves.

Phosphorus (P) is an essential macronutrient for plant growth and development. Vacuoles play a crucial role in inorganic phosphate (Pi) storage and remobilization in plants. However, the physiological function of vacuolar phosphate efflux transporters in plant Pi remobilization remains obscure. Here, we identified three ZmVPE genes (ZmVPE1, ZmVPE2a, ZmVPE2b) by combining them with transcriptome and quantitative real-time polymerase chain reaction (PCR) analyses, showing a relatively higher expression in older leaves than in younger leaves in maize. Moreover, the expression of the ZmVPEs was triggered by Pi deficiency and abscisic acid. ZmVPEs were localized to the vacuolar membrane and responsible for vacuolar Pi efflux. Compared with the wild-type, Pi remobilization from older to younger leaves was enhanced in ZmVPE-overexpression lines. zmvpe2a mutants displayed an increase in the total P and Pi concentrations in older leaves, but a decrease in younger leaves. In rice, Pi remobilization was impaired in the osvpe1osvpe2 double mutant and enhanced in OsVPE-overexpression plants, suggesting conserved functions of VPEs in modulating Pi homeostasis and remobilization in crop plants. Taken together, our findings revealed a novel mechanism underlying Pi remobilization from older to younger leaves mediated by plant vacuolar Pi efflux transporters, facilitating the development of Pi-efficient crop plants.

Single-Nucleus RNA Sequencing Unravels Early Mechanisms of Human Becker Muscular Dystrophy.

The transcriptional heterogeneity at a single-nucleus level in human Becker muscular dystrophy (BMD) dystrophic muscle has not been explored. Here, we aimed to understand the transcriptional heterogeneity associated with myonuclei, as well as other mononucleated cell types that underly BMD pathogenesis by performing single-nucleus RNA sequencing. We profiled single-nucleus transcriptional profiles of skeletal muscle samples from 7 BMD patients and 3 normal controls. A total of 17,216 nuclei (12,879 from BMD patients and 4,337 from controls) were classified into 13 known cell types, including 9 myogenic lineages and 4 non-myogenic lineages, and 1 unclassified nuclear type according to their cell identities. Among them, type IIx myonuclei were the first to degenerate in response to dystrophin reduction. Differential expression analysis revealed that the fibro-adipogenic progenitors (FAPs) population had the largest transcriptional changes among all cell types. Sub-clustering analysis identified a significantly compositional increase in the activated FAPs (aFAPs) subpopulation in BMD muscles. Pseudotime analysis, regulon inference, and deconvolution analysis of bulk RNA-sequencing data derived from 29 BMD patients revealed that the aFAPs subpopulation, a distinctive and previously unrecognized mononuclear subtype, was profibrogenic and expanded in BMD patients. Muscle quantitative real-time polymerase chain reaction and immunofluorescence analysis confirmed that the mRNA and protein levels of the aFAPs markers including LUM, DCN, and COL1A1 in BMD patients were significantly higher than those in controls, respectively. Our results provide insights into the transcriptional diversity of human BMD muscle at a single-nucleus resolution and new potential targets for anti-fibrosis therapies in BMD. ANN NEUROL 2024;96:1070-1085.

Upregulated spinal histone deacetylases induce nociceptive sensitization by inhibiting the GABA system in chronic constriction injury-induced neuropathy in rats.

Neuropathic pain (NP) affects countless people worldwide; however, few effective treatments are currently available. Histone deacetylases (HDACs) participate in epigenetic modifications in neuropathy-induced nociceptive sensitization. Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter that can inhibit NP. The present study aimed to examine the role of spinal HDAC and its isoforms in neuropathy. Male Wistar Rat with chronic constriction injury (CCI)-induced peripheral neuropathy and HDAC inhibitor, panobinostat, was administrated intrathecally. We performed quantitative real-time polymerase chain reaction (RT-qPCR), western blot, and immunohistochemical analysis of lumbar spinal cord dorsal horn and nociceptive behaviors (thermal hyperalgesia and mechanical allodynia) measurements. Herein, RT-qPCR analysis revealed that spinal hdac3, hdac4, and hdac6 were upregulated in CCI rats. Western blotting and immunofluorescence staining further confirmed that HDAC3, HDAC4, and HDAC6 were significantly upregulated, whereas GABA and its synthesis key enzyme glutamic acid decarboxylase (GAD) 65 were dramatically downregulated. Intrathecal panobinostat attenuated nociceptive behavior and restored the downregulated spinal GAD65 and GABA expression in CCI rats. HDAC upregulation might induce nociception through GAD65 and GABA inhibition in CCI-induced neuropathy. These findings strongly suggest that HDACs negatively regulate inhibitory neurotransmitters, constituting a potential therapeutic strategy for an epigenetic approach to manage NP.

Genome-wide identification of HIPP and mechanism of SlHIPP4/7/9/21/26/32 mediated phytohormones response to Cd, osmotic, and salt stresses in tomato

Heavy-metal-associated isoprenylated plant proteins (HIPPs) contributed to abiotic tolerance in vascular plants. Up to now, the HIPP gene family of tomato (Solanum lycopersicum L.) had not been thoroughly understood. In the present study, 34 SlHIPP genes were identified from the tomato genome using the Hidden Markov Model (HMM). The phylogenetic analysis revealed that the evolution of SlHIPPs was highly conserved. The cis-acting element analysis indicated that SlHIPP genes might be involved in phytohormones and abiotic stresses. We constructed venn diagram with 17 genes containing stress-related motifs as well as 15 genes and 19 genes expressing in leaves and roots in RNA-seq data, suggesting that SlHIPP4/7/9/21/26/32 were selected as candidate genes for study. The quantitative real-time PCR (qRT-PCR) analysis showed that 6 candidate genes were indicated to be involved in osmotic and salt stress tolerance and SlHIPP7/21/26/32 responded to cadmium (Cd) tolerance. The virus-induced silencing of 6 candidate genes caused growth inhibition in stress conditions, further illustrating that 6 candidate genes played a positive role in abiotic conditions. Importantly, the phytohormone analysis implied that 6 candidate genes mediated abscisic acid (ABA), salicylic acid (SA), gibberellin (GA3), auxin (IAA), or methyl jasmonate (MeJA) responses to Cd, osmotic, or salt stress tolerance. These findings indicated that SlHIPP4/7/9/21/26/32 were key regulators of abiotic stress responses in tomato seedlings, functioning through multiple phytohormone pathways.

Hovenia dulcis (Guaizao) polysaccharide ameliorates hyperglycemia through multiple signaling pathways in rats with type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) poses a significant threat to human health, with its incidence and mortality rates increasing annually. This study investigated the hypoglycemic effects and underlying mechanisms of pure Hovenia dulcis (Guaizao) polysaccharide (HDPs-2A) in rats subjected to a high-fat and high-sugar diet combined with streptozotocin-induced T2DM. Oral administration of HDPs-2A resulted in significant increases in body weight and liver glycogen levels compared to untreated controls. Moreover, a reduction in fasting blood glucose levels, alleviation of hyperinsulinemia, enhanced glucose tolerance, and improved insulin resistance were observed in the HDPs-2A-treated group. HDPs-2A also effectively reversed diabetes-induced dyslipidemia, as evidenced by decreased total cholesterol and triglyceride levels, alongside increased high-density lipoprotein cholesterol levels. Histopathological analyses confirmed that HDPs-2A partially repaired liver tissue damage by mitigating oxidative stress responses in the liver. Additionally, treatment with HDPs-2A significantly elevated short-chain fatty acid levels in T2DM rats. Real-time quantitative PCR and Western blot analyses indicated that HDPs-2A significantly enhanced the expression of InsR, IRS2, PI3K, Akt, and GLUT4, suggesting that HDPs-2A regulates insulin resistance and glycometabolism through the activation of the PI3K/Akt signaling pathway. Furthermore, HDPs-2A appeared to modulate the expression of GS, GSK-3β, and FoxO1 to improve glucose metabolism and reduce insulin resistance. It also improved glucose metabolism by activating the AMPK pathway and modulating G6Pase and PEPCK expression. This study provides novel insights into the antidiabetic effects of HDPs, positioning them as promising nutritional agents for the management of T2DM.

Silver-Doped Zinc Ferrite Nanoparticles: Trigger ROS-Dependent Apoptosis and Inhibit Migration in MCF-7 Breast Cancer Cells

This study describes the fabrication of nanostructured silver-doped zinc ferrite nanoparticles (AgxZn1-xFe2O4, where x = 0.0 and 0.05) using a chemical co-precipitation method. Field-emission scanning electron microscopy (FE-SEM) analysis was performed to investigate the surface characteristics and particle size of the pure and Ag-doped nanoparticles. X-ray diffraction (XRD) patterns confirmed the formation of a single-phase cubic structure for the zinc ferrite nanoparticles. The anti-cancer potential of the nanoparticles was evaluated using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) against human breast cancer cells (MCF-7). The results showed that (AgxZn1-xFe2O4, where x = 0.05) nanoparticles with x = 0.05 exhibited significant cytotoxicity. At a concentration of 100 µg/mL, these nanoparticles reduced cell viability to 26.27%, with a calculated LC50 value (concentration lethal to 50% of cells) of 41.30 µg/mL after 24 hours of treatment. Notably, the LC50 value for (AgxZn1-xFe2O4, where x = 0.05) nanoparticles in normal L929 fibroblast cells was over four times higher compared to MCF-7 cancer cells, indicating a degree of selectivity for cancer cells. Further investigation revealed that (AgxZn1-xFe2O4, where x = 0.05) treatment significantly increased the generation of reactive oxygen species (ROS) within the cancer cells. This was confirmed by fluorescence intensity measurements, which showed a substantial increase from 1260.61 (control) to 15600 for cells treated with (AgxZn1-xFe2O4, where x = 0.05) nanoparticles. This included flow cytometry analysis of apoptosis (programmed cell death), migration assays to evaluate metastasis potential, measurement of antioxidant enzyme activity (SOD and catalase) to understand the impact on the cellular antioxidant system, indirect ELISA to detect caspase activity (a marker of apoptosis), cell cycle analysis, and finally, real-time PCR analysis to determine the mRNA expression of p53, a tumor suppressor gene. The apoptosis assay confirmed a significant increase in apoptotic cells upon treatment with (AgxZn1-xFe2O4, where x = 0.05) nanoparticles. This activity is likely mediated by the generation of ROS and subsequent oxidative stress within the cancer cells. These findings highlight the potential of these nanoparticles as a promising therapeutic strategy for cancer treatment.

Profiling mRNA encoding glucocorticoid receptor α in saliva: Relationship to hair cortisol levels in individuals aged 15-25 years

ObjectiveWe assessed levels of mRNA encoding two glucocorticoid receptor (GR) isoforms (GRα and GRβ) in saliva and examined their relationship with hair cortisol levels and dental caries experience. DesignAdolescents and young adults were assessed for dental caries experience, and hair cortisol was measured by ELISA. RNA was extracted from whole saliva using TRIzol, followed by quantitative real-time PCR analysis of GRα, GRβ, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ResultsGRβ mRNA was not detectable in most samples, whereas GRα mRNA was observed in all samples. There were significantly lower levels of GRα mRNA in individuals with elevated hair cortisol levels than in those with normal cortisol levels. Levels of GRα mRNA did not differ significantly in individuals with dental caries experience compared to individuals with no caries experience. ConclusionsWe identified and quantified mRNA encoding GRα in saliva. Its levels were inversely associated with hair cortisol (a marker of chronic stress). Although caries experience was associated with hair cortisol levels, there was no significant association between GRα levels and caries experience. Chronic stress has been proposed to be associated with reduced expression of GRα and this association appears to hold for GRα mRNA levels in saliva.

The effects of crocin on fear condition memory impairment caused by morphine in relation to gene expression of amygdala HSP70, HSP90 and NF-kB

BackgroundSome studies have shown that morphine causes memory impairment through different mechanisms. The roles of inflammation/NF-κB and BDNF/TrkB/CREB pathways are significant in the memory deficits caused by morphine. Aim: In this study, the effect of crocin on the impairment of fear memory caused by morphine was investigated, and the expression of HSP70, HSP90, and NF-kB genes that play an important role in memory in male Wistar rats was measured. Material and methodThe fear conditioning test was used to check the fear memory of rats, and total Freezing time was considered as an index of memory. Rats (96 Wistar rats) were randomly divided into 12 groups. The rats were injected with saline (1 ml/kg) or different doses of morphine (0.5, 2.5, and 5 mg/kg) 5 min after injection of saline (1 ml/kg) or subthreshold doses of crocin (60 mg/kg). Real-time PCR method was used for HSP70, HSP90, and NF-kB gene expression in the Amygdala. ResultAdministration of morphine 5 mg/kg decreased the total freezing and increased the latency freezing (P < 0.001). Also, the results showed that crocin 60 mg/kg reduced memory impairment induced by morphine. Real-time PCR analysis showed that morphine 5 mg/kg significantly increased the expression of the HSP70, HSP90, and NF-kB genes. However, the subthreshold dose of crocin (60 mg/kg) reduced the increase in gene expression. ConclusionThe study showed that morphine impacts memory and boosts gene expression of HSP70, HSP90, and NF-kB, while crocin could potentially reverse this effect by regulating these amygdala genes, suggesting avenues for future research.

Fluoxetine treatment reverses chronic stress-induced promotion on Fk506-binding protein 5 expression and multiple effects on glucocorticoid receptor phosphorylation in the paraventricular nucleus of mice

BackgroundFluoxetine is widely used as a first-line antidepressant. However, the molecular mechanisms for its antidepressant effects are still not fully understood. Hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis is a core pathogenic mechanism contributing to depression, and fluoxetine treatment prevents this dysfunction. The glucocorticoid receptor (GR) is a major negative feedback regulator of the HPA axis, while Fk506-binding protein 5 (Fkbp5) is a negative regulator of the GR signaling. Therefore, we examined the effects of fluoxetine on Fkbp5 and the GR signaling in the hypothalamic paraventricular nucleus (PVN) of depressed mice. MethodsMice were exposed to chronic social defeat stress (CSDS), chronic unpredictable mild stress (CUMS), or chronic restraint stress (CRS) with or without fluoxetine treatment (intraperitoneally injected, 20 mg/kg) and examined for changes in depression-like behaviors and HPA axis activity as well as Fkbp5 expression and GR phosphorylation in the PVN. We then examined if adeno-associated virus (AAV)-mediated Fkbp5 overexpression in the PVN affected the antidepressant actions of fluoxetine in mice. ResultsFluoxetine treatment significantly mitigated CSDS-, CUMS-, and CRS-induced depression-like behaviors and HPA axis hyperactivity in mice. Subsequent western blotting analyses showed that fluoxetine treatment fully reversed not only chronic stress-induced upregulation of Fkbp5 and CRH but also chronic stress-induced increase in Ser203 phosphorylation and decrease in Ser211 and Ser234 phosphorylation in GR in the PVN. Moreover, quantitative real-time reverse transcription PCR (qRT-PCR) analyses revealed that the enhanced mRNA levels of Fkbp5 and CRH in PVN neurons of mice subjected to CSDS/CUMS/CRS were also notably reversed by fluoxetine administration. Conversely, Fkbp5 overexpression in the PVN significantly eliminated the antidepressant effects of fluoxetine in mice without affecting their locomotor activity. ConclusionThese results together suggest that fluoxetine treatment reverses chronic stress-induced promotion on Fkbp5 expression and multiple effects on GR phosphorylation in the PVN of mice. Significance statementThe selective serotonin reuptake inhibitor fluoxetine (sold as Prozac) is a widely used treatment for depression, but the full spectrum of therapeutic mechanisms is still debated. Recent evidence suggests that these therapeutic mechanisms include suppression of chronic stress-activated hypothalamus–pituitary–adrenal (HPA) axis. The current study presents the first in vivo evidence showing that suppression of HPA axis hyperactivity by fluoxetine treatment involves reversal of glucocorticoid receptor (GR) phosphorylation via modulation of the GR negative regulator Fk506-binding protein 5 (Fkbp5) in the hypothalamic paraventricular nucleus (PVN). Fluoxetine treatment not only inhibited chronic stress-induced depression-like behaviors and HPA axis hyperactivity but also reversed Fkbp5 upregulation and GR phosphorylation changes in the PVN, while adeno-associated virus (AAV)-based Fkbp5 overexpression in the PVN eliminated the antidepressant effects of fluoxetine. These findings may expand our understanding of the pharmacological effects of fluoxetine, and further identify Fkbp5 as a possible target for novel antidepressants.

RNA binding protein RBM22 suppresses non-small cell lung cancer tumorigenesis by stabilizing LATS1 mRNA.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality worldwide. Despite advancements in diagnostics and therapeutics, the prognosis for NSCLC remains poor, highlighting the urgent need for novel treatment options. RNA binding proteins, particularly RBM22, have emerged as significant contributors to cancer progression by influencing RNA splicing and gene expression. This study investigates the role of RBM22 in NSCLC and its potential as a therapeutic target. We focus on the effects of RBM22 on cell proliferation, invasion, stemness, and its interaction with LATS1 mRNA. RBM22 expression was assessed in samples and cell lines of NSCLC through techniques such as real-time PCR and western blot analysis. To modify RBM22 levels, overexpression and knockdown methods were employed utilizing vectors and siRNAs. We conducted assays for cell proliferation, invasion, and stemness to evaluate the effects of altering RBM22. The interaction between RBM22 and LATS1 mRNA was investigated using RNA immunoprecipitation. In addition, in vivo studies involving subdermal tumor and lung metastasis models in athymic mice were carried out to evaluate how changes in RBM22 influence the tumorigenic and metastatic characteristics of NSCLC. Our analysis revealed a significant underexpression of RBM22 in NSCLC tissues compared to adjacent healthy tissues. Increasing RBM22 expression in NSCLC cell lines led to a marked decrease in cellular proliferation, invasiveness, and stemness, while silencing RBM22 produced opposing effects. Further investigations confirmed that RBM22 directly interacts with LATS1 mRNA, thereby stabilizing and enhancing its expression. In vivo studies validated that elevated RBM22 expression substantially reduced tumor formation and pulmonary metastases, as evidenced by decreased tumor size, mass, and Ki-67 proliferation marker expression, along with a significant reduction in the number of metastatic nodules in the lungs. Our study demonstrates that RBM22 suppresses NSCLC by stabilizing LATS1 mRNA, which in turn reduces tumor growth and metastasis. Consequently, RBM22 emerges as a valuable therapeutic target for NSCLC, offering new strategies for addressing this challenging condition.

Birch (Betula platyphylla) BES/BZR transcription factor BpBZR1-6 improves salt tolerance in transgenic Arabidopsis thaliana

BackgroundSalt stress is one of the major environmental factors affecting plant growth and productivity. BRI1-EMS suppressor 1/brassinazole-resistant 1 ((BES1/BZR1) plays an important role in responding to abiotic stress in plants. Although the impacts of BES1/BZR1 on plant growth and resistance have been documented, the potential mechanisms are not fully elucidated in Betula platyphylla. This work contributes to the understanding of how BES1/BZR1 promotes stress tolerance in woody plants.ResultsSix BES1/BZR1 family members were identified from Betula platyphylla. Cis-element analysis showed that the promoters of six genes were rich in ABA-responsive element (ABRE), MYB and MBS cis-acting elements, which are reported to be involved in abiotic stress responses. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that BpBZR1-6 (BPChr10G06000) could be induced by salt stress, ABA and BRs. BpBZR1-6 was localized in the nucleus and had transactivation activity. Ectopic expression of BpBZR1-6 enhanced Arabidopsis tolerance and decreased abscisic acid (ABA) sensitivity under salt treatment. Specifically, the seed germination rate, root length, fresh weight and chlorophyll content were significantly higher in BpBZR1-6-overexpressing (OE) transgenic plants than in wild-type (WT) plants after salt stress (P < 0.05). Additionally, BpBZR1-6 overexpression showed enhanced the reactive oxygen species (ROS) scavenging capability under salt stress, including increasing the activities of antioxidant enzyme, resulting in a decrease in O2− and H2O2 accumulation, and reducing malondialdehyde (MDA) content. Meanwhile, the expression levels of six antioxidant enzyme genes were higher in OE plants than in WT plants after stress.ConclusionBpBZR1-6 overexpression enhanced the salt tolerance of transgenic plants by modulating antioxidant enzyme gene expression and ROS scavenging, which may provide underlying strategy for breeding of salt-tolerant plants.

Receptor interacting serine/threonine kinase 2 promotes rheumatoid arthritis progression and partially regulates nuclear factor kappa B pathway

Objective: Rheumatoid arthritis (RA) is a disabling systemic autoimmune disease worldwide; however, its molecular pathway remains largely unknown. Thus, this study aimed to explore the effects of receptor-interacting serine/threonine kinase 2 (RIPK2) on RA progression and its underlying mechanism. Material and Methods: RIPK2 expression was analyzed using real-time quantitative polymerase chain reaction, immunohistochemical staining, and Western blot (WB) analysis in RA synovial tissues or cells. Cell viability or proliferation was determined using the cell counting kit-8 and 5-ethynyl-2'-deoxyuridine. Cell metastasis was analyzed using the transwell assay and wound healing assay. Flow cytometry was adopted to measure cell apoptosis. The level of inflammation-related factors was measured using the enzyme-linked immunosorbent assay. WB analysis was used to determine the expression level of nuclear factor kappa B (NF-κB) pathway-related genes. Results: RIPK2 was highly expressed in RA synovial tissues and cells. Transfection with RIPK2 short hairpin RNA plasmids reduced the gene expression level of RIPK2 in RA fibroblast-like synoviocytes (FLS) cells. Notably, RIPK2 silencing hindered the proliferation, invasion, and migration of tumor cells as well as accelerated the apoptosis of RA-FLS cells. Furthermore, RIPK2 silencing suppressed the RA-FLS cell inflammatory response and NF-κB pathway. Conclusion: RIPK2 silencing could retrain the malignant behavior and inflammatory response of RA-FLSs and partially modulate the NF-κB pathway.

Effect of Cow Bone Addition on the Humification, Heavy Metals Passivation and Fate of Resistance Genes During Swine Manure Composting

Bone meal has been used as economic and effective additive for heavy metals (HMs) pollution remediation due to the distinct components and structures that enable their favorable properties, such as its low cost, high adsorption capacity, acid-base adjustability, and ion-exchange capability. However, no attempt has been made to establish whether cow bone could promote the passivation of HMs and the removal of metal resistance genes (MRGs) and antibiotics resistance genes (ARGs) during the composting process. Two sizes of cow bone (meal (T2) and granule (T3)) were added to investigate their effects on humification, HMs passivation and the abundance of ARGs and MRGs during swine manure composting. Excitation-emission matrix (EEM)-parallel factor analysis showed that the percentage of maximum fluorescence intensity of humic-like substances were higher in T2 (91.82%) than in T3 (88.46%), implying that T2 could promote the humification process compared to T3. In comparison with control (T1), the addition of T2 and T3 could promote the change of exchangeable Cu and reducible Cu into oxidizable Cu, thus reducing the mobility factors (MF) of Cu in T2 and T3 treatments by 10.48% and 6.98%, respectively. In addition, T2 and T3 could increase exchangeable Zn into reducible Zn and oxidizable Zn, thereby reducing the MF of Zn in T2 and T3 treatments by 18.80% and 2.0%, respectively. Quantitative Real-time PCR (qPCR) analysis revealed that the total abundances of MRGs were decreased by 100% in T2 and T3 treatments, and T2 decreased the total relative abundance of ARGs. Furthermore, the relative abundance of ARGs and MRGs had significantly correlated with intI1 and bio-available of Cu and Zn, which was triggered by selective pressure of HMs and horizontal gene transfer. The present study suggested that cow bone meal as additives can be a feasible approach to promote the passivation of HMs and enhance the removal of MGRs and ARGs by decreasing horizontal gene transfer and selective pressure by bioavailable HMs.

Ershen Zhenwu Decoction Suppresses Myocardial Fibrosis of Chronic Heart Failure with Heart–Kidney Yang Deficiency by Down-Regulating the Ras Homolog Gene Family Member A/Rho-Associated Coiled-Coil Kinases Signaling Pathway

Ethnopharmacological significanceThe therapeutic efficacy of Ershen Zhenwu Decoction (ESZWD)—a famous formulation from Xin’an for patients with chronic heart failure heart–kidney Yang deficiency (CHF-HKYD)—is well established. Still, the underlying molecular mechanism is not clear. Aim of the studyThis study investigated mechanisms by which ESZWD suppresses cardiac pathology, including myocardial fibrosis, in CHF-HKYD model rats and Ang II-stimulated cardiac fibroblasts (CFs). Materials and methodsThe components in ESZWD were analyzed by ultra-high-performance liquid chromatography coupled with Quadrupole Time-Of-Flight mass spectrometry (UHPLC-Q-TOF-MS). CHF-HKYD model was established in the male Sprague–Dawley rats through bilateral thyroidectomy and intraperitoneal administration of 0.02% doxorubicin (DOX), twice weekly for 3 weeks. Subsequently, the CHF-HKYD model rats were randomly categorized into the Model, ESZWD-L (3.96 g/kg/d ESZWD), ESZWD-M (7.92 g/kg/d ESZWD), ESZWD-H (15.84 g/kg/d ESZWD), and Sac/Val (68 mg/kg/d sacubitril/valsartan) groups and treated daily for 4 weeks. As a control, the sham surgery group (Sham) was used. Primary cardiac fibroblasts (CFs) were categorized into Control, Model, ESZWD, and Sac/Val groups. Then, the CFs were stimulated with Ang-II. The ESZWD and Sac/Val groups were incubated with different concentrations of drug-containing sera and their effects on CF viability were assessed via the CCK-8 assay. The ESZWD and Sac/Val groups received drug-containing serum concentrations determined by CCK-8 assay results. The cardioprotective effects of ESZWD were determined using echocardiography, Hematoxylin & Eosin (H&E) staining, Masson staining, and Sirius red staining, and the Enzyme Linked Immunosorbent Assay (ELISA). ESZWD's effects on the Ras Homolog Gene Family Member A (RhoA) / Rho-Associated Coiled-Coil Kinases (ROCKs) signaling pathway and myocardial fibrosis were assessed by Western blotting and Quantitative Real-Time PCR (qRT-PCR) analyses. Immunofluorescence was used to observe fibrotic markers in CFs. ResultsESZWD treatment improved cardiac function in the CHF-HKYD rats by significantly reducing myocardial fibrosis and ventricular remodeling. ESZWD treatment increased the rats' body temperature (Tb) and 24-hour urine volume, left ventricular ejection fraction (LVEF) and LV fractional shortening (LVFS), and decreased LV internal systolic diameter (LVIDs), LV internal diastolic diameter (LVIDd), and heart weight/ body weight (HW/BW) compared to the Model group. In comparison to the model rats, ESZWD treatment decreased serum levels of B-type natriuretic peptide precursor (NT-proBNP), tumor necrosis factor-alpha (TNF-α), interleukin-11 (IL-11), and IL-17A. ESZWD treatment significantly down-regulated the protein and mRNA expression levels of collagen I A1, α-SMA, RhoA, ROCK1, and ROCK2 in the heart tissues of the CHF-HKYD rats and the Ang II-stimulated CFs. ConclusionESZWD significantly improved cardiac function and attenuated myocardial fibrosis and inflammation in the CHF-HKYD rats by inhibiting the RhoA/ROCKs signaling pathway.

Identification of STAT3 and MYC as critical ferroptosis-related biomarkers in septic cardiomyopathy: a bioinformatics and experimental study.

Ferroptosis is the well-known mechanism of septic cardiomyopathy (SCM). Bioinformatics analysis was employed to identify ferroptosis-related SCM differentially expressed genes (DEG). DEGs' functional enrichment was explored. Weighted gene co-expression network analysis (WGCNA) was employed to form gene clusters. The identified hub genes, signal transducer and activator of transcription 3 (STAT3) and myelocytomatosis (MYC) were further evaluated by generating receiver operator characteristic (ROC) curves and a nomogram prediction model. Additionally, survival rate, cardiac damage markers, and cardiac function and ferroptosis markers were evaluated in septic mouse model. STAT3 and MYC levels were measured in SCM heart tissue via immunohistochemical (IHC) staining, real-time polymerase chain reaction (qPCR) and western blot analysis. Analysis identified 225 DEGs and revealed 22 intersected genes. Of the 7 hub genes, STAT3 and MYC showed enrichment in septic heart tissue and a strong predicative ability based on AUC values. Cardiac damage, iron metabolism, and lipid peroxidation occurred in the SCM model. By experiments, STAT3 and MYC expression was increased in the SCM model. Impairment was reversed with a ferroptosis inhibitor, Fer-1. As conclusion, STAT3 and MYC are related with ferroptosis and may serve as potential SCM predictor indicators. KEY MESSAGES: Septic cardiomyopathy (SCM) often leads to high mortality in septic patients, and the diagnostic criteria still remains unclear. Ferroptosis as the pathogenic mechanism of SCM could help predict its progression and clinical outcomes. STAT3 and MYC are related with ferroptosis and may serve as potential SCM predictor biomarkers.

Down-regulation of ESRP2 inhibits breast cancer cell proliferation via inhibiting cyclinD1

Epithelial splicing regulatory protein 2 (ESRP2),an important alternative splicing protein of mRNA, is reported to have a dual role in tumors, which can promote or inhibit the occurrence and development of tumors. However, the function and mechanism of ESRP2 in breast cancer (BC) remain unclear. The distribution of ESRP2 expression in breast cancer and the correlation between ESRP2 expression and the overall survival rate were detected by The Cancer Genome Atlas (TCGA) database. Gene Ontology(GO)analysis, containing biological process, cellular components, and molecular function, was utilized to evaluate the potential mechanism of ESRP2 in breast cancer. The ESRP2 expression in breast cancer cell lines was detected by real-time quantitative PCR analysis (RT-qPCR) and western blotting. Cell clone was performed to examine the proliferation of ESRP2 knockdown in MCF-7 cells. The cell cycle was measured by flow cytometry assays. The role of ESRP2 knockdown in synergistic effect with chemotherapeutic agents was also determined by MTT assay. Bioinformatics analysis demonstrated that the ESRP2 gene was elevated in breast cancer cells and its overexpression was strongly correlated with shorter overall survival. GO analysis revealed that ESRP2 expression was related to cell proliferation. ESRP2 mRNA and protein expression were elevated in breast cancer cell lines, compared to the normal human breast cell line MCF-10 A. Dwon-regulation of ESRP2 inhibited cell proliferation and promoted the sensitivity of chemotherapy drug, Cisplatin(DDP) and Paclitaxel (TAXOL), in MCF-7 cells.Additionally, ESRP2 knockdown obstructed the cell cycle at the G1 phase and caused a decrease in cyclinD1 protein expression. These findings reveal that ESRP2 is highly expressed in breast cancer and is correlated with poor prognosis in breast cancer patients. ESRP2 knockdown can inhibit MCF-7 cell proliferation by arresting the cell cycle at the G1 phase and promoting the sensitivity of chemotherapy drugs (DDP and TAXOL)in MCF-7 cells. ESRP2 may be required for the regulation of breast cancer progression, as well as a critical target for the clinical treatment of breast cancer.

The LINC01094/miR-545-3p/SLC7A11 Signaling Axis Promotes the Development of Gastric Cancer by Regulating Cell Growth and Ferroptosis.

This study aimed to investigate the role and mechanism of action of LINC01094 in the development of gastric cancer (GC). The expression levels of LINC01094 in GC patients and healthy individuals were analyzed online using the Cancer Genome Atlas database. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to determine the expression of LINC01094/miR-545-3p/SLC7A11 in GC tissues and cells. Functional experiments (MTT assay, colony formation assay, and flow cytometry) were conducted to assess the effect of LINC01094 and miR-545-3p on cell proliferation, viability, apoptosis, cell cycle, and reactive oxygen species. Correlations between LINC01094 and miR-545-3p, as well as SLC7A11, were analyzed and validated using the dual-luciferase reporter assay and RNA immunoprecipitation. The levels of Fe2+, malondialdehyde, and glutathione in the cells were measured biochemically, and the protein expression levels of Bcl-2, cleaved caspase3, Cyclin D1, and p21 were detected by Western blotting. LINC01094 was significantly upregulated in the GC tissues and cells with a targeting relationship with miR-545-3p; the expression levels of LINC01094 and miR-545-3p were negatively correlated. Knockdown of LINC01094 notably inhibited the proliferation and viability of GC cells and promoted cell ferroptosis, which, however, was abrogated by the silencing of miR-545-3p. These findings indicate that miR-545-3p could target and positively correlate with SLC7A11 expression. Additionally, LINC01094 could promote GC cell progression and affect cellular ferroptosis by regulating the miR-545-3p/SLC7A11 signaling axis.

The role of the KEAP1-NRF2 signaling pathway in form deprivation myopia guinea pigs

In recent years, the global prevalence of myopia has reached an unprecedented level, especially‌ in East Asia. Multitude of studies has shown that the etiology of myopia is complex. Some researchers have suggested that oxidative stress (OS) may contribute to myopia, although there are limited reports on the alterations of related signaling pathways. Notably, the Kelch-like ECH-associated protein 1 (KEAP1) -nuclear factor erythroid 2-related factor 2 (NRF2), which plays a significant role in regulating OS and the mechanism, has not been explored in myopia. To investigate the modulation of KEAP1-NRF2 signaling pathway and its downstream superoxide dismutase (SOD) during the development of form-deprivation myopia, three-week-old guinea pigs were randomly assigned to four groups: negative control (NC), self-control (SC), form-deprivation myopia (FDM), and FDM group treated with tert-butylhydroquinone (TBHQ). Spherical equivalent (SE) and axial length (AL) were measured by retinoscopy and A-scan ultrasound, respectively. The results revealed that TBHQ treatment decelerated the progression in SE and AL changes. Immunohistochemistry (IHC) assessed the distribution and expression of KEAP1, NRF2, and SOD. The results shown that they located in the retinal ganglion cells (RGC). Subsequently, retinal mRNA and protein expression levels of KEAP1, NRF2, and SOD were quantified using real-time polymerase chain reaction (RT-PCR) and Western blot (WB) analysis. The RT-PCR and WB results demonstrated that TBHQ could activate NRF2, induce KEAP1 degradation, and enhance the expression of the antioxidant SOD. In summary, the modulation of KEAP1-NRF2 and it downstream SOD expression could alter the retinal antioxidant capacity and influence the development of myopia.