qPCR Analysis Research Articles

Integrated single-cell transcriptomic analyses identify a novel lineage plasticity-related cancer cell type involved in prostate cancer progression

Cancer cell plasticity is the ability of neoplastic cells to alter their identity and acquire new biological properties under microenvironmental pressures. In prostate cancer (PCa), lineage plasticity often results in therapy resistance and trans-differentiation to neuroendocrine (NE) lineage. However, identifying the cancer cells harboring lineage plasticity-related status remains challenging. Based on 13 multi-center human PCa bulk transcriptomic cohorts (samples=3314) and 9 bulk transcriptomic datasets derived from PCa experimental models, we established an integrated lineage plasticity-related gene signature, termed LPSig. Leveraging this gene signature, AUCell enrichment analysis was applied to identify the cell population with high lineage plasticity from a comprehensive single-cell RNA-sequencing (scRNA-seq) meta-atlas assembled by us, which consisted of 10 public human PCa scRNA-seq datasets (samples=93, cells=222,529). Moreover, additional scRNA-seq dataset of human PCa, multiplex immunohistochemistry staining for human PCa tissues, invitro and invivo functional experiments, as well as qPCR and Western blot analyses were employed to validate our findings. We found that LPSig could finely capture the dynamics of tumor lineage plasticity throughout the progression of PCa, accurately estimating the status of lineage plasticity. Based on LPSig, we identified a previously undefined minority population of lineage plasticity-related PCa cells (LPCs) from the human PCa scRNA-seq meta-atlas assembled by this study. Furthermore, in-depth dissection revealed pivotal roles of LPCs in trans-differentiation, tumor recurrence, and poor patient survival during PCa progression. Furthermore, we identified HMMR as a representative cell surface marker for LPCs, which was validated using additional scRNA-seq datasets and multiplexed immunohistochemistry. Moreover, HMMR was transcriptionally inhibited by androgen receptor (AR), and was required for the aggressive adenocarcinoma features and NE phenotype. Our study uncovers a novel population of lineage plasticity-related cells with low AR activity, stemness-like traits, and elevated HMMR expression, that may facilitate poor prognosis in PCa. This work was supported by National Key R&D Program of China (2022YFA0807000), National Natural Science Foundation of China (82160584), Advanced Prostate Cancer Diagnosis and Treatment Technology Innovation Team of Kunming Medical University (CXTD202216), and Reserve Talents of Young and Middle-aged Academic Leaders in Yunnan Province (202105AC160013).

Enhancing osteogenesis and mandibular defect repair with magnesium-modified acellular bovine bone matrix

An acellular bovine bone matrix modified to release Magnesium ions (Mg2+) (ABBM-Mg) was prepared and evaluated for its potential in osteogenesis and mandibular defect repair. Mg2+ was incorporated into ABBM using an ion exchange method. The microstructure and mechanical properties of both ABBM and ABBM-Mg were analyzed using SEM and a biomechanical testing machine. Cytocompatibility, cell adhesion, and osteogenic differentiation were assessed using various methods including CCK-8, Live/Dead staining, SEM, ALP staining, and qPCR analysis in MC3T3-E1 cells. Additionally, a mandibular defect model in rats was established. The bone defect repair outcomes were evaluated using Micro-CT, histological HE staining, and Masson staining. The study showed that mineralization containing magnesium was redeposited on the surface of the three-dimensional porous ABBM, and the ABBM-Mg scaffold promoted cell proliferation and osteogenic differentiation compared to the ABBM scaffold. In the rat mandibular defect model, the ABBM-Mg scaffold demonstrated superior bone repair ability. This study successfully incorporated Mg2+ into ABBM without significantly affecting its microstructure and compressive strength. Furthermore, ABBM-Mg showed sustained release of Mg2+ which enhanced cell proliferation, adhesion, and osteogenic differentiation in vitro, and promoted mandibular defect healing in rats. This research opens up new possibilities for the clinical application of functionalized acellular bone matrix.Graphical

NCOR1 orchestrates macrophage inflammation in heart failure with preserved ejection fraction: a translational study

Abstract Background/Introduction Inflammatory response is a key player in heart failure with preserved ejection fraction (HFpEF) but the underlying mechanisms are poorly understood. Recent studies have shown a critical involvement of cardiac macrophage activation in myocardial remodeling thus contributing to HFpEF. Purpose To investigate the role of cardiac macrophage inflammation in experimental and human HFpEF. Methods We employed a translational approach combining human and murine LV myocardial tissue, FACS analysis of cardiac immune cells, in vitro experiments in RAW 264.7 cells exposed to metabolic stress by palmitic acid (PA), bone marrow-derived macrophages from healthy and HFpEF mice, cardiomyocytes (H9c2), endothelial cells (HAECs) as well as ex vivo functional analyses in LV tissues. Human LV myocardial samples were used to evaluate the pro-inflammatory activity of macrophages in the HFpEF myocardium. Induction of HFpEF in mice was done through a validated mouse model combining metabolic (high-fat diet) and hemodynamic stress (L-NAME). Cardiac function was investigated by high resolution ultrasound imaging (Vevo3100) and the LV myocardial samples were used to study the macrophage polarization and their inflammatory activity. Based on interrogation of available RNA-seq datasets – showing the transcriptional repressor NCOR1 as a relevant signature in macrophages during cardiac inflammation, we generated myeloid cell-specific NCOR1 knockout (Ncor1-KO) transgenic mice to investigate the in vivo effects of NCOR1 deletion on the HFpEF phenotype. Results The expression of macrophage markers in human LV specimens showed an increase in pro-inflammatory macrophages (M1) and a decrease in regulatory macrophages (M2) as compared to age-matched control mice. HFpEF mice showed increased expression of vascular adhesion molecules (ICAM1, ICAM2, E-selectin) and a significant recruitment of M1 macrophages as proved by qPCR, WB, and flow cytometry analysis. Of interest, diastolic dysfunction - assessed by E/A ratio and isovolumic relaxation time (IVRT) – and lung congestion were significantly reduced in mice with macrophage NCOR1 deletion as compared to WT littermates, while exercise tolerance was significantly improved. These findings were associated with a reduction of myocardial inflammation (TNF-a, IL6, IL-1b) in Ncor1-KO mice. In vitro assays showed that stimulation of macrophages with PA induced M1 type switch and increased NCOR1, TNF-a, IL-6, and IL-1b levels, whereas NCOR1 depletion prevented detrimental changes of macrophage secretome (Fig.2). Conclusion NCOR1 is a crucial regulator of immune response and macrophage inflammation in the heart. Our findings highlight its potential as a target for therapies aimed at preventing myocardial damage in HFpEF.

Neutrophil extracellular traps, deoxyribonuclease and fibrosis in thoracic aortic aneurysm

Abstract Introduction Thoracic aortic aneurysm (TAA) is a rare but severe cardiovascular disease. The condition often goes undiagnosed, increasing the risk of acute aortic events. It is characterized by medial degeneration, caused by neutrophils infiltrating aortic tissue. Neutrophils can form proinflammatory and prothrombotic neutrophil extracellular traps (NETs), promoting disease progression. Purpose Recently, NETs and abdominal aortic aneurysm (AAA) were linked, while their effects on TAA development and progression remain unclear. We aimed to characterize NETs and NET-specific markers in tissue and plasma samples of TAA patients. Methods We included 171 patients with either ascending TAA (n=153) or Type A dissection (n=18). Healthy subjects were recruited as controls (n=135). Sample fiber composition was analyzed using trichrome; NET burden was quantified using immunofluorescence. Plasma NET surrogate markers including double-stranded (ds)DNA, myeloperoxidase (MPO), DNase activity and matrixmetalloproteinases (MMP) were measured using a fluorescence-based assay and ELISA, respectively. mRNA expression was assessed using qPCR. Results Median patient age was 65 years, and most were male (68.4%). Cardiovascular risk factors were common, including hypertension (77.8%), hyperlipidemia (57.9%), coronary artery disease (26.3%), and diabetes (18.7%). TAA patients were older with high BMI and impaired kidney function. Trichrome staining revealed abundant fibrotic material (median 47.99%), while collagen and elastic fiber distributions were 27.67% and 20.17%, respectively. TAA patients had increased amounts of aortic fibrotic material (47.99% vs 38.94%, p=0.001). Using immunofluorescence, we found higher NET expression in diseased tissue (0.87 [0.3, 1.85] vs 0.15 [0.03, 0.22]%, p=0.0017). This increase was primarily driven by the dissection patients (1.76 [0.89, 2.83]%, p=0.0037). Plasma MPO levels were increased in TAA compared to controls (36.93 [23.46, 57.55] vs 26.8 [19.7, 36.2]ng/mL, p<0.001), while DNase activity was decreased in patients (4.16 [2.66, 6.24] vs 6.06 [4.25, 8.11]mU/mL, p<0.001). Moreover, circulating plasma levels of MMP2 were elevated in TAA (250.36 [190.24, 351.32] vs 218.85 [171.32, 263.87]ng/mL, p=0.004). qPCR analysis of mRNA expressions in TAA cryospecimens revealed upregulations of TNFα (p=0.033) and CD45 (p=0.003), when compared to aortic disease-free controls. Conclusion Aortic fibrosis at the aneurysm site may be caused by inflammatory mechanisms involving TNFα and leucocytes (CD45+). High local NET-burden is indicative of activated neutrophils in the aortic media. TAA patients have suppressed DNase activity and higher MPO levels in venous blood, leading to dsDNA accumulation and increased neutrophil turnover. Elevated MMP2 suggests its involvement in TAA formation. Our findings could enable further research targeting NETs and inflammation in TAA, possibly finding novel therapies capable of mitigating aneurysm development.

Phytoplasma DNA Enrichment from Sugarcane White Leaves for Shotgun Sequencing Improvement

Sugarcane white leaf (SCWL) disease, caused by Candidatus Phytoplasma sacchari, poses a significant threat to sugarcane cultivation. An obligate parasite, phytoplasma is difficult to culture in laboratory conditions, making the isolation of its DNA from the massive amount of plant host DNA extremely challenging. Yet, the appropriate amount and quality of plant microbiome-derived DNA are key for high-quality DNA sequencing data. Here, a simple, cost-effective, alternative method for DNA isolation was applied using a guanidine-HCl-hydroxylated silica (GuHCl-Silica)-based method and microbiome DNA enrichment based on size-selective low-molecular-weight (LMW) DNA by PEG/NaCl precipitation. qPCR analysis revealed a significant enrichment of phytoplasma DNA in the LMW fraction. Additionally, the NEBNext Microbiome DNA enrichment kit was utilized to further enrich microbial DNA, demonstrating a remarkable increase in the relative abundance of phytoplasma DNA to host DNA. Shotgun sequencing of the isolated DNA gave high-quality data on the metagenome assembly genome (MAG) of Ca. Phytoplasma sacchari SCWL with completeness at 95.85 and 215× coverage. The results indicate that this combined approach of PEG/NaCl size selection and microbiome enrichment is effective for obtaining high-quality genomic data from phytoplasma, surpassing previous methods in efficiency and resource utilization. This low-cost method not only enhances the recovery of microbiome DNA from plant hosts but also provides a robust framework for studying plant pathogens in complex plant models.

Identification and Functional Analysis of Git3 G Protein-Coupled Receptors in Ganoderma boninense PER71

G protein-coupled receptors (GPCRs) are integral components of eukaryotic heterotrimeric G proteins, playing crucial roles in detecting extracellular signals and initiating the activity of signaling proteins within cells to activate cellular responses to these signals. The objectives of this study are to identify and characterize the function of Git3, a Class III GPCR protein, in the oil palm pathogen Ganoderma boninense. To identify the potential genes encoded for GPCR in this fungus, intensive data mining on the genome and transcriptome data has been carried out. A total of six classes of GPCRs have been identified. These include Class II pheromone detectors, Class III carbon detectors, Class IV nitrogen detectors, Class VII proteins similar to glycosyltransferase, Class VIII proteins similar to hemolysin, and Class X protein receptors. Among these, the Class III protein Git3, postulated to be involved in glucose sensing and fungal pathogenicity, was selected for gene knockdown using RNA interference (RNAi). A plasmid, designated pUChph-GIT3, was constructed, to target git3 silencing by incorporating a hygromycin resistance gene cassette and antisense sequences of git3. Transformation of G. boninense PER71 with pUChph-GIT3 produced five potential Δgit3 gene-silenced mutants. PCR analysis confirmed the integration of the RNAi expression cassette into the fungal genome. Quantitative PCR (qPCR) analysis revealed significant reductions in git3 expression in three G. boninense mutants, M42, M66, and M5 by 47%, 23%, and 13%, respectively. The Disease Severity Index (DSI) indicated slower disease progression in oil palm plantlets infected with Δgit3 mutants compared to those infected with wild-type G. boninense PER71. In conclusion, this study successfully isolated and characterized the git3 GPCR from G. boninense and demonstrated that it might play a role during the early stages of infection, as the mutants were able to slow the progression of infection in oil palm plantlets.

Concomitant gut dysbiosis and defective gut barrier serve as the bridges between hypercortisolism and chronic systemic inflammation in Cushing disease.

The aim of this study was to investigate the gut microbial signatures and related pathophysiological implications in patients with Cushing's disease (CD). 27 patients with CD and 45 healthy controls were enrolled. Based on obtained metagenomics data, we performed correlation, network study and genome interaction group (GIG) analysis. Fecal metabolomics and serum ELISA analysis were conducted in dichotomized CD patients. Caco-2 cells were incubated with gradient concentrations of cortisol for subsequent transepithelial electrical resistance (TEER) measurement, FITC-dextran transwell permeability assay, qPCR and western blot analysis. Gut microbial composition in patients with CD was notably different from that in healthy controls. Network analysis revealed that Eubacterium siraeum might serve as the core specie in the gut microbial system of CD patients. Subsequent GIG analysis identified the positive correlations between GIG9 and UFC. Further serum ELISA and fecal metabolomics uncovered that CD patients with elevated UFC levels were characterized with increased lipopolysaccharide binding protein (LBP). Moreover, remarkable positive association was found between LBP level and relative abundance of Eubacterium siraeum. TEER and FITC-Dextran transwell assays demonstrated that hypercortisolism induced increased gut permeability. Further qPCR and western blot analysis suggested that dysregulated AhR/Claudin 2 axis might be involved in the development of hypercortisolism-induced defective gut barrier function. Disease activity associated dysbiosis and defective gut barrier might jointly facilitate the development of systemic inflammation in patients with CD.

Dysregulation of the 3β-hydroxysteroid dehydrogenase type 2 enzyme and steroid hormone biosynthesis in chronic kidney disease

IntroductionChronic kidney disease (CKD) presents a critical global health challenge, marked by the progressive decline of renal function. This study explores the role of the 3β-hydroxysteroid dehydrogenase type 2 enzyme (HSD3B2) and the steroid hormone biosynthesis pathway in CKD pathogenesis and progression.MethodsUsing an adenine-induced CKD mouse model, we conducted an untargeted metabolomic analysis of plasma samples to identify key metabolite alterations associated with CKD. Immunohistochemistry, Western blotting, and qPCR analyses were performed to confirm HSD3B2 expression in both human and mouse tissues. Additionally, Nephroseq and Human Protein Atlas data were utilized to assess the correlation between HSD3B2 and kidney function. Functional studies were conducted on HK2 cells with HSD3B2 knockdown to evaluate the impact on cell proliferation and apoptosis.ResultsMetabolic characteristics revealed significant shifts in CKD, with 61 metabolites increased and 65 metabolites decreased, highlighting the disruption in steroid hormone biosynthesis pathways influenced by HSD3B2. A detailed examination of seven key metabolites underscored the enzyme's central role. HSD3B2 exhibited a strong correlation with kidney function, supported by data from Nephroseq and the Human Protein Atlas. Immunohistochemistry, Western blotting, and qPCR analyses confirmed a drastic reduction in HSD3B2 expression in CKD-affected kidneys. Suppressed proliferation and increased apoptosis rates in HSD3B2 knocked down HK2 cells further demonstrated the enzyme's significance in regulating renal pathophysiology.DiscussionThese findings underscore the potential of HSD3B2 as a clinical diagnostic and therapeutic target in CKD. While further studies are warranted to fully elucidate the mechanisms, our results provide valuable insights into the intricate interplay between steroid hormone biosynthesis and CKD. This offers a promising avenue for precision medicine approaches and personalized treatment strategies.

Genome-Wide Analysis of the MAPKKK Gene Family Under Abiotic Stresses in Moso Bamboo (Phyllostachys edulis)

Mitogen-activated protein kinase kinase kinases (MAPKKKs) are the upstream components of MAPK cascades and are involved in mediating stress responses and developmental processes. Although MAPKKK genes have been investigated in many plants, the identification and characterization of MAPKKKs in moso bamboo were still limited. Here, 134 MAPKKKs were identified as unevenly distributed on 23 chromosomes (except for chromosome 1) of moso bamboo and divided into three subfamilies by phylogenetic analysis. The gene structure and conserved motif of PeMAPKKKs were investigated. The expansions of PeMAPKKKs were driven by whole-genome duplication (WGD) or segmental duplication events. The duplicated gene pairs were under purifying selection based on the Ka/Ks ratios, suggesting they underwent functional conservation. Most PeMAPKKKs contained cis-elements related to development, hormones, and stress responses. Tissue expression patterns showed that PeMAPKKKs had multiple expression patterns. The qPCR analysis showed distinct expression patterns of PeMAPKKKs under drought, salt, and cold stress conditions. Taken together, this study provides a solid foundation for future functional characterizations of MAPKKKs and identifies candidate stress-responsive genes for further study in moso bamboo.

Drug-Free Mesoporous Silica Nanoparticles Enable Suppression of Cancer Metastasis and Confer Survival Advantages to Mice with Tumor Xenografts.

Despite advancements in nanomedicine for drug delivery, many drug-loaded nanoparticles reduce tumor sizes but often fail to prevent metastasis. Mesoporous silica nanoparticles (MSNs) have attracted attention as promising nanocarriers. Here, we demonstrated that MSN-PEG/TA 25, with proper surface modifications, exhibited unique antimetastatic properties. In vivo studies showed that overall tumor metastasis decreased in 4T1 xenografts mice treated with MSN-PEG/TA 25 with a notable reduction in lung tumor metastasis. In vitro assays, including wound-healing, Boyden chamber, tube-formation, and real-time cell analyses, showed that MSN-PEG/TA 25 could modulate cell migration of 4T1 breast cancer cells and interrupt tube formation by human umbilical vein endothelial cells (HUVECs), key factors in suppressing cancer metastasis. The synergistic effect of MSN-PEG/TA 25 combined with liposomal-encapsulated doxorubicin (Lipo-Dox) significantly boosted mouse survival rates, outperforming Lipo-Dox monotherapy. We attributed the improved survival to the antimetastatic capabilities of MSN-PEG/TA 25. Moreover, Dox-loaded MSN-PEG/TA 25 suppressed primary tumors while retaining the antimetastatic effect, thereby enhancing therapeutic outcomes and overall survival. Western blot and qPCR analyses revealed that MSN-PEG/TA 25 interfered with the phosphorylation of ERK, FAK, and paxillin, thus impacting focal adhesion turnover and inhibiting cell motility. Our findings suggest that drug-free MSN-PEG/TA 25 is highly efficient for cancer treatment via suppressing metastatic activity and angiogenesis.

Genetic analysis of preaxial polydactyly: identification of novel variants and the role of ZRS duplications in a Chinese cohort of 102 cases.

Preaxial polydactyly (PPD) is a congenital limb malformation, previously reported to be caused primarily by variants in the ZRS and upstream preZRS regions. This study investigated genetic variations associated with PPD, focusing on point variants and copy number variations (CNVs) in the ZRS and preZRS regions. Comprehensive genetic analyses were conducted on 102 patients with PPD, including detailed clinical examinations and Sanger sequencing of the ZRS and preZRS regions. Additionally, real-time quantitative PCR (qPCR) was used to detect CNVs in the ZRS region. The evolutionary conservation and population frequencies of identified variants were also evaluated. Six point variants were identified, among which four are likely pathogenic novel variants: 93G > T (g.156584477G > T), 106G > A (g.156584464G > A), 278G > A (g.156584292G > A), and 409A > C (g.156585378A > C). Additionally, qPCR analysis revealed that 66.67% of patients exhibited ZRS duplications. Notably, these duplications were also present in cases with newly identified potential pathogenic point variants. These findings suggest the possible interaction of point variants in ZRS and preZRS through a common pathogenic mechanism, leading jointly to PPD. The findings expand the variant spectrum associated with non-syndromic polydactyly and highlight that, despite different classifications, anterior polydactyly caused by variants in ZRS and nearby regions may share common pathogenic mechanisms. The incorporation of various variant types in genetic screening can effectively enhance the rate of pathogenic variant detection and contribute to the cost-effectiveness of genetic testing for limb developmental defects, thereby promoting healthy births.

Drug-Induced Differential Gene Expression Analysis on Nanoliter Droplet Microarrays: Enabling Tool for Functional Precision Oncology.

Drug-induced differential gene expression analysis (DGEA) is essential for uncovering the molecular basis of cell phenotypic changes and understanding individual tumor responses to anticancer drugs. Performing high throughput DGEA is challenging due to the high cost and labor-intensive multi-step sample preparation protocols. In particular, performing drug-induced DGEA on cancer cells derived from patient biopsies is even more challenging due to the scarcity of available cells. A novel, miniaturized, nanoliter-scale method for drug-induced DGEA is introduced, enabling high-throughput and parallel analysis of patient-derived cell drug responses, overcoming the limitations and laborious nature of traditional protocols. The method is based on the Droplet Microarray (DMA), a microscope glass slide with hydrophilic spots on a superhydrophobic background, facilitating droplet formation for cell testing. DMA allows microscopy-based phenotypic analysis, cDNA extraction, and DGEA. The procedure includes cell lysis for mRNA isolation and cDNA conversion followed by droplet pooling for qPCR analysis. In this study, the drug-induced DGEA protocol on the DMA platform is demonstrated using patient-derived chronic lymphocytic leukemia (CLL) cells. This methodology is critical for DGEA with limited cell numbers and promise for applications in functional precision oncology. This method enables molecular profiling of patient-derived samples after drug treatment, crucial for understanding individual tumor responses to anticancer drugs.

Gentiopicroside Attenuates Lithium/Pilocarpine-Induced Epilepsy Seizures by Down-Regulating NR2B/CaMKII/CREB and TLR4/NF-κB Signaling Pathways in the Hippocampus of Mice

Background: Epilepsy is a prevalent and disabling neurological condition characterized by recurrent seizures. Approximately 50% of adults with active epilepsy have at least one comorbidity and they are at a greater risk of premature death than the general population. Gentiopicroside (Gent) is a primary component of Gentiana macrophylla Pall. that has been shown to have diverse pharmacological properties. However, its role in epileptic seizures in adult mice and its underlying mechanism of action remain obscure. We aimed to explore the anti-epileptic effect and mechanism of Gent on lithium/pilocarpine (Pilo)-induced epilepsy seizures in mice. Methods: In this study, we established a lithium/Pilo-induced epilepsy model, and Gent was first given to mice 30 min before Pilo administration. Then, we detected behavioral and histopathological changes through electrocorticographic (ECoG) measurements, Nissl staining, Fluoro-Jade B (FJB) staining, and immunohistochemical staining. We then used molecular biology techniques, such as Western blotting, quantitative polymerase chain reaction (qPCR) analysis, and the enzyme-linked immunosorbent assay (ELISA) to investigate the mechanisms of Gent in lithium/Pilo-induced epileptic seizures in mice and lipopolysaccharide (LPS)-induced inflammatory astrocytes. Results: We confirmed that Gent could prevent abnormal ECoG activity, behavioral changes, and neurodegeneration. Subsequently, we found Gent could downregulate the factors that could promote apoptosis (i.e., the NR2B/CaMKII/CREB signaling cascade) and neuroinflammatory-related factors (i.e., the TLR4/NF-κB signaling cascade). Conclusions: Gent could be a potential therapeutic agent for epilepsy, offering possibilities for both prevention and treatment. Our research establishes a preliminary experimental framework for ongoing studies into Gent’s efficacy as a treatment for epilepsy.

Gene expression responses of CF airway epithelial cells exposed to elexacaftor/tezacaftor/ivacaftor (ETI) suggest benefits beyond improved CFTR channel function.

The combination of elexacaftor/tezacaftor/ivacaftor (ETI, Trikafta) reverses the primary defect in Cystic Fibrosis (CF) by improving CFTR mediated Cl- and HCO3- secretion by airway epithelial cells (AEC), leading to improved lung function and less frequent exacerbations and hospitalizations. However, studies have shown that CFTR modulators like ivacaftor, a component of ETI, has numerous effects on CF cells beyond improved CFTR channel function. Because little is known about the effect of ETI on CF AEC gene expression we exposed primary human AEC to ETI for 48 hours and interrogated the transcriptome by RNA-seq and qPCR. ETI increased CFTR Cl- secretion, and defensin gene expression (DEFB1) an observation consistent with reports of decreased bacterial burden in the lungs of people with CF (pwCF). ETI decreased MMP10 and MMP12 gene expression, suggesting that ETI may reduce proteolytic induced lung destruction in CF. ETI also reduced the expression of the stress response gene heme oxygenase (HMOX1). qPCR analysis confirmed DEFB1, HMOX1, MMP10 and MMP12 gene expression results observed by RNA-seq. Gene pathway analysis revealed that ETI decreased inflammatory signaling, cellular proliferation and MHC Class II antigen presentation. Collectively, these findings suggest that the clinical observation that ETI reduces lung infections in pwCF is related in part to drug induced increases in DEFB1, and that ETI may reduce lung damage by reducing MMP10 and MMP12 gene expression. Moreover, pathway analysis also identified several other genes responsible for the ETI induced reduction in inflammation observed in pwCF.

Ferulic acid alleviates cardiac injury by inhibiting avermectin-induced oxidative stress, inflammation and apoptosis

Avermectin (AVM) is a broad-spectrum antibiotic from the macrolide class, extensively employed in fisheries and aquaculture. Nevertheless, its indiscriminate utilisation has resulted in a substantial accumulation of remnants in the aquatic ecosystem, potentially inflicting significant harm to the cardiovascular system of aquatic species. Ferulic acid (FA) is a naturally occurring compound in wheat grain husks. It possesses potent anti-inflammatory and antioxidant properties, which can help reduce cardiovascular damage. Additionally, its affordability makes it an excellent option for aquaculture usage as a feed additive. This article explored the potential of FA as a feed additive to protect against AVM-induced heart damage in carp. We subjected carp to AVM for 30 days and provided them with a diet of 400 mg/kg of FA. FA substantially reduced the pathogenic damage to heart tissue caused by AVM, as shown through hematoxylin-eosin staining. The biochemical analysis revealed that FA markedly enhanced the activity of antioxidant enzymes catalase (CAT), glutathione (GSH), and total antioxidant capacity (T-AOC) while reducing the malondialdehyde (MDA) content. Furthermore, qPCR analysis demonstrated a substantial increase in the mRNA levels of transforming growth factor-β1 (tgf-β1) and interleukin-10 (il-10) simultaneously, significantly reducing the expression levels of interleukin-10 (il-6), interleukin-1β (il-1β), tumor necrosis factor-α (tnf-α) and inductible nitric oxide synthase (inos). Through the mitochondrial apoptotic route, FA reduced AVM-induced cell death in carp heart cells by upregulating bcl-2 while downregulating the mRNA expression levels of bax, fas, caspase8 and caspase9. In summary, FA alleviated cardiac injury by inhibiting AVM-induced oxidative stress, inflammatory response, and apoptosis in carp heart tissue.

Ethanolic Extract of Bergenia purpurascens Exhibits Antimelanogenic Effects in B16F10 Cells through Multiple Mechanisms That Suppress Tyrosinase.

Bergenia purpurascens, renowned for its antibacterial and antioxidant properties, remains relatively unexplored in its impact on melanogenesis. This study delves into the antimelanogenic potential of the ethanol extract derived from B. purpurascens (BPE). Our investigations reveal the robust antioxidant capabilities of the BPE, along with its effective inhibition of mushroom tyrosinase activity. Remarkably, these effects were significantly stronger than those observed with arbutin, the positive control. In vitro assays demonstrate the BPE's efficacy in reducing the melanin content and tyrosinase activity in α-MSH-stimulated B16F10 cells. Immunofluorescence and qPCR analyses further reveal the BPE's ability to inhibit MITF-mediated gene expression levels associated with melanogenesis, including Trp-1, Trp-2, and tyrosinase. These findings suggest that the extract operates through dual mechanisms, suppressing both tyrosinase activity and key transcription factor-mediated downstream signaling. Utilizing UPLC-MS/MS analysis, we identified six key compounds implicated in tyrosinase activity inhibition and melanogenesis suppression. Docking simulations confirm moderate binding affinities between these compounds and tyrosinase. This study highlights the potential of B. purpurascens as a novel natural agent for depigmentation in medicinal and cosmetic applications, elucidating its dual mechanism of action in melanogenesis inhibition.

Rectal Epithelial Stem Cell Kinetics in Acute Radiation Proctitis

The intestinal tract is a typical radiosensitive tissue, and radiation rectal injury is a severe side effect that limits the prescribed dose in radiotherapy of the abdominal and pelvic region. Understanding the post-irradiation kinetics of Lgr5-positive stem cells is crucial in comprehending this adverse process. In this study, we utilized Lgr5-EGFP knock-in mice expressing EGFP and LGR5 antibody fluorescence staining of wild-type mice. At the state of radiation injury, the qPCR analysis showed a significant decrease in the expression level of Lgr5 in the rectal epithelial tissue. The dose-response relationship analysis showed that at low to moderate doses up to 10 gray (Gy), Lgr5-clustered populations were observed at the base of the crypt, whereas at sublethal doses (20 Gy and 29 Gy), the cells exhibited a dot-like scatter pattern, termed Lgr5-dotted populations. During recovery, 30 days post-irradiation, Lgr5-clustered populations gradually re-emerged while Lgr5-dotted populations declined, implying that some of the Lgr5-dotted stem cell populations re-clustered, aiding regenerations. Based on statistical analysis of the dose-response relationship using wild-type mice, the threshold dose for destroying these stem cell structures is 18 Gy. These findings may help set doses in mouse abdominal irradiation experiments for radiation intestinal injury and for understanding the histological process of injury development.

Sequence characteristics, expression and subcellular localization of PtCYP721A57 gene from cytochrome P450 family in Polygala tenuifolia willd.

The Cytochrome P450 (CYP450) family is the largest enzyme protein family in plants, distributed across various organs and involved in significant catalytic activities in primary and secondary metabolic processes. In this study, we cloned the PtCYP721A57 gene, characterized its open reading frame (ORF), and conducted comprehensive analyses including physicochemical properties, evolutionary relationships, subcellular localization, prokaryotic expression, and correlation between the relative expression of different parts and the content of tenuifolin, hormones, and abiotic stress response associated with the encoded protein. The ORF of PtCYP721A57 was 1,521 bp, with a secondary structure predominantly composed of α-helices and random coils. Subcellular localization experiments confirmed the presence of PtCYP721A57 in the endoplasmic reticulum. For prokaryotic expression, we constructed the recombinant plasmid pET28a-PtCYP721A57 using pET28a as the vector, which was then transformed into BL21(DE3). Induction with Isopropyl β-D-1-thiogalactopyranoside (IPTG) at temperatures of 16 and 25 °C and varying concentrations (0.1, 0.2, 0.5, 1, 2 mM) resulted in the formation of inclusion bodies, with higher expression observed at 25 °C. Our qPCR analyses revealed that PtCYP721A57 exhibited the highest expression in the cortex of Polygala tenuifolia, followed by roots and xylem, correlating with the observed tenuifolin content distribution. Induction with abscisic acid (ABA) and chitosan (CHT) initially decreased PtCYP721A57 expression followed by a subsequent increase, peaking at 48 h. Similarly, drought stress induced a gradual increase in PtCYP721A57 expression, also peaking at 48 h. NaCl treatment for 6 h significantly upregulated PtCYP721A57 expression. In conclusion, our study provides foundational insights into the PtCYP721A57 gene in Polygala tenuifolia, laying the groundwork for further exploration of its role in the biosynthesis pathway of triterpenoid saponins.

Transcriptomic Analysis of the Effect of Glabridin on Biofilm Formation in Staphylococcus Aureus.

Staphylococcus aureus (S. aureus) is among the major skin infection-causing pathogens in animals and humans. Its ability to form biofilms has become a foremost cause of bacterial infections and the extensive spread of drug resistance, which poses a great difficulty in clinical treatment. Glabridin (Glb), an extract of licorice with antibacterial and anti-infective properties, has a partially understood biofilm-inhibitory mechanism. This study investigated the inhibitory and antibiofilm activities of subinhibitory concentrations of Glb against S. aureus. The crystal violet assay revealed that Glb significantly suppressed biofilm expression. Scanning electron microscopy observations unveiled that Glb reduced S. aureus adhesion and accumulation by disrupting the spatial structure of the biofilm. In vitro extracellular DNA (eDNA) inhibition assays demonstrated that Glb inhibited biofilm formation by S. aureus by suppressing eDNA secretion. In total, 184 differentially expressed genes were obtained through transcriptomic (RNA-seq) sequencing, of which 81 and 103 genes were upregulated and downregulated, respectively. Glb regulated the transcript levels of biofilm-related genes through the phosphatase transfer system, two-component regulatory system, and nitrogen metabolism. The qPCR analysis was performed to confirm whether Glb interfered with the expression of regulatory genes involved in S. aureus biofilm formation (SarA, ArlR, FnbA, ClfA, icaD, and icaR) as well as the virulence gene Hla. In conclusion, this study demonstrates that Glb has a significant inhibitory effect on biofilm activity and is expected to be a good antibiofilm drug.

Selenium participates in the formation of kidney stones by alleviating endoplasmic reticulum stress and apoptosis of renal tubular epithelial cells

ABSTRACT Objectives: To investigate the role of selenium and selenium-containing proteins in the etiology and pathogenesis of kidney stones. Methods: The HK-2 cell line was subjected to supersaturation oxalate treatment to establish an in vitro model of calcium oxalate kidney stones, while SD rats were administered with ethylene glycol to establish an in vivo model of calcium oxalate kidney stones. qPCR analysis was employed to investigate the alterations in selenoproteins within the models, and subsequently, genes exhibiting significant changes were identified. Subsequently, based on the functions of these genes, their regulatory effects on endoplasmic reticulum stress (ERS) and apoptosis during the disease progression were examined both in HK-2 cells and rat kidneys. Finally, Selenomethionine (SeMet) supplementation was introduced to explore its therapeutic potential for kidney stone management. Results: The involvement of Selenoprotein K in the pathogenesis of calcium oxalate kidney stone disease has been confirmed, exhibiting significant alterations. Manipulation of its expression levels through overexpression and knockdown techniques resulted in a corresponding reduction or increase in oxidative stress, ERS, and apoptosis within renal tubular epithelial cells. SelK regulates ERS and apoptosis by controlling the IRE1-ASK1-JNK pathway. In addition, SeMet treatment, which contains selenium, effectively reduced the levels of oxidative stress, ERS, and apoptosis in vivo and in vitro models, thereby alleviating tubular epithelial cell damage and reducing the formation of kidney stones in experimental rats. Discussion: Selenium is involved in the occurrence and development of kidney stones by regulating oxidative damage to renal tubular epithelial cells. The results suggest that dietary selenium supplementation in daily life may be of great significance for the prevention and treatment of kidney stones.