Immunofluorescence Research Articles

Optimization of pan-lyssavirus LN34 assay for streamlined rabies diagnostics by real-time RT-PCR

Reliable, validated diagnostic tests are critical for rabies control in animals and prevention in people. We present a performance assessment and updates to the LN34 real-time RT-PCR assay for rabies diagnosis in postmortem animal brain samples. In two U.S. laboratories during 2017 to 2022, routine used of the LN34 assay produced excellent diagnostic sensitivity (99.72% to 100%) and specificity (99.99% to 100%) compared to the direct fluorescence antibody test (DFA). Almost all (>90%) DFA indeterminate results caused by non-specific or atypical fluorescence were negative by LN34 testing, representing up to 111 cases where unnecessary post-exposure prophylaxis could be avoided. LN34 assay original primer sequences showed low sensitivity for some rare lyssaviruses. Increased primer concentration combined with new primer formulation showed improved performance for impacted lyssaviruses with no loss in performance across diverse rabies virus variants from clinical samples. The updated LN34 and internal control assays were combined into a single-well LN34 multiplexed (LN34M) format, run at half reagent volumes. The LN34M assay showed similar detection of rabies virus to the singleplexed assay with simplified assay set-up, lower cost, and improved quality controls.

Development of a new platform for testing antiviral drugs using coronavirus-infected human nasal mucosa organoids

To establish a coronavirus (CoV) infection model using human nasal mucosa organoids for testing antiviral drugs and evaluate the feasibility of using human nasal mucosa organoids with viral infection as platforms for viral research and antiviral drug development. Human nasal mucosa organoids were tested for susceptibility to SARS-CoV-2 and HCoV-OC43 pseudoviruses. In a P3 laboratory, nasal mucosa organoids were infected with the original strain of SARS-CoV-2 and 4 variant strains, and the infection conditions were optimized. The viral loads in the culture supernatants were measured at different time points using RT-qPCR, and immunofluorescence assay was employed to localize SARS-CoV-2 nucleocapsid protein to determine the type of the infected cells. In the optimized nasal mucosa viral infection model, the antiviral effects of camostat and bergamot extract (which were known to inhibit SARS-CoV-2) were tested and the underlying molecular mechanisms were explored. In the optimized nasal mucosa organoid models infected with SARS-CoV-2 and HCoV-OC43 pseudoviruses, the viral load in the culture supernatants increased significantly during the period of 2 to 24 h following the infection, which confirmed infection of the organoids by both of the pseudoviruses. The nasal mucosa organoids could be stably infected by the original SARS-CoV-2 strain and its 4 variant strains, validating successful establishment of the viral infection model, in which both camostat and bergamot extract exhibited dose-dependent antiviral effects. Human nasal mucosa organoids with SARS-CoV-2 infection can serve as platforms for screening and testing antiviral drugs, particularly those intended for nasal administration.

Multiomics and experimental approaches reveal the anti-acute lung injury effects of Fallopia aubertii (L. Henry) Holub extract via IL-17/NF-κB pathway inhibition

Ethnopharmacological relevanceFallopia aubertii (L. Henry) Holub (F. aubertii), a traditional Tibetan medicine, is used in China for treating respiratory inflammatory diseases, including acute lung injury (ALI). However, the chemical constituents of F. aubertii and its anti-inflammatory mechanisms in the lungs remain poorly understood. Aim of the studyThis study aimed to identify the chemical constituents of the F. aubertii extract (FAE), evaluate its effectiveness in reducing ALI in mice, and elucidate the underlying mechanisms of its action. Materials and methodsThe chemical composition of FAE was determined using UPLC-LTQ Velos Pro-Orbitrap Elite. Network pharmacology was employed to predict the mechanisms by which FAE might mitigate ALI. Mice were administered FAE orally for seven days, followed by intratracheal instillation of lipopolysaccharide (LPS) to induce ALI. On the final day, the mice were euthanized, and their lungs were collected for transcriptome analysis, proteomics, pharmacodynamic evaluation, and mechanistic studies. Hematoxylin and eosin (H&E) staining assessed lung pathology. Transcriptome and proteomic analyses, along with real-time quantitative PCR (RT-qPCR) and western blotting, were used to investigate FAE's effects on lung inflammation and related signaling pathways. In vitro experiments further explored the anti-ALI mechanisms of FAE. Immunofluorescence assays in RAW264.7 cells examined the nuclear translocation of NF-κB. ResultsFifty-one compounds were identified in FAE, predominantly flavonoid glycosides. Network pharmacology suggested that FAE may inhibit ALI by modulating the NF-κB pathway and Th17 differentiation. RNA-seq analysis indicated that FAE might suppress inflammation through the IL-17 signaling pathway, with these findings corroborated by mRNA level measurements in vivo and in vitro. FAE alleviated LPS-induced ALI by modulating the IL-17A signaling pathway, which was confirmed through proteomic analysis. Western blotting revealed that FAE reduced the expression of IL-17A, Act1, TRAF6, and p-NF-κB, while immunofluorescence assays showed FAE inhibited LPS-induced NF-κB nuclear translocation. ConclusionFAE attenuates inflammation-mediated ALI by inhibiting the IL-17A/NF-κB signaling pathway. This study highlights the anti-ALI effects of FAE and provides a theoretical foundation for its potential use in ALI treatment.

Expression profiles of TNF-Alpha and HERV-K Env proteins in multiple types of colon and lung disease.

Human endogenous retroviruses (HERVs) were integrated into the human genome millions of years ago and have since proliferated to comprise about 8% of the human genome. For a long time, HERVs were thought to be remnants of ancient viruses, rendered inactive over the ages. However, recent studies have revealed that HERVs are involved in various diseases, including cancer. Notably, HERVs have been found to play a crucial role in immune responses and inflammatory processes, indicating their significant influence on the regulation of immune-related diseases. We reported in previous reports that HERV-K119 env Knockout (KO) and inflammatory response were associated. In this study, we identified the correlation between inflammatory disease and HERV-K Env and TNF-Alpha protein expression in multiple types of colon disease tissue and lung disease spectrum tissue. We performed Immunofluorescence (IF) using multiple types of colon disease and lung disease spectrum tissue microarray (TMAs) and compared and analyzed the patient clinical data provided. As a result, we identified that the expression of HERV-K Env and TNF-Alpha proteins in certain colorectal inflammatory diseases and certain lung inflammatory diseases showed specific expression. And through the analysis of the clinical data provided, environmental factors could be identified. Our study demonstrates that the relationship between TNF-Alpha and HERV-K Env expression in inflammation disease and clinical significance of disease tissues.

Dual inhibition of HERs and PD-1 counteract resistance in KRASG12C-mutant head and neck cancer

BackgroundBasket clinical trials targeting the KRASG12C-mutation in solid tumors have shown initial promise, including in orphan KRASG12C head and neck cancer (HNC). However, development of resistance to KRASG12C-mutant-specific inhibitors (KRASG12Ci) remains a major obstacle. Here, we investigated the intrinsic (tumor-cell autonomus) and tumor-microenvironment (TME) mechanisms of resistance to the KRASG12Ci—MRTX849 and AMG510 in a unique syngenic murine KRASG12C-mutated HNC cell line.MethodsWestern-blotting was used for protein abundance and activation, overexpression, and ligand activation studies to verify the intrinsic mechanism of resistance to KRASG12Ci in KRASG12C-mutated HNC cell line, 4NQO-L. In vitro KRASG12C-acquired-resistant cells were developed from 4NQO-L (4NQO-L-AcR). MRTX849/lapatinib combination efficacy, and CD8+ T-cells depletion, were assessed in C57BL/6 J mice and supplementation of anti-PD-1 (αPD-1) to MRTX849/lapatinib was also performed in 4NQO-L– KRASG12Ci-senisitve and 4NQO-L-AcR tumors. Immunohistochemistry (IHC) and Immunoflourescence (IF) analyses were performed to profile the TME and programmed death-ligand 1 (PD-L1) expression in tumors.ResultsActivation and upregulation of EGFR and HER2/3 (pan-HERs) are the intrinsic mechanism of resistance to KRASG12Ci in 4NQO-L cells, and blocking pan-HERs signaling with lapatinib enhanced MRTX849 efficacy in vitro by inhibiting the MAPK and AKT/mTOR pathways. 4NQO-L-AcR upregulated the expression of pan-HERs, and lapatinib treatment re-sensitized 4NQO-L-AcR to MRTX849. In mice, MRTX849 showed a slight anti-tumor effect, but in combination with lapatinib a significant tumor growth delay was observed, but all tumors progressed over time. Histopathology analysis of the TME revealed infiltration of CD8+ T-cells after treatment combination, and these CD8+ T-cells play a key role in MRTX849/lapatinib efficacy. MRTX849/lapatinib treatment upregulated PD-L1 overexpression in both stromal and tumor cells, which presumably suppressed CD8+ T-cells and enabled immune escape and tumor progression. Supplementation of αPD-1 prolonged the progression-free survival of 4NQO-L-bearing mice treated with MRTX849/lapatinib. MRTX849/lapatinib treatment delayed tumor growth of 4NQO-L-AcR in mice; however, the percentages of CD8+ T-cells in 4NQO-L-AcR were low, and supplementation of MRTX849/lapatinib with αPD-1 did not improve the outcome.ConclusionsOur study highlights the critical need for blocking both intrinsic and extrinsic mechanisms of resistance for the prolonged response and shows that such treatment is ineffective in KRASG12Ci-AcR tumors.

A Multi-task learning U-Net model for end-to-end HEp-2 cell image analysis

Antinuclear Antibody (ANA) testing is pivotal to help diagnose patients with a suspected autoimmune disease. The Indirect Immunofluorescence (IIF) microscopy performed with human epithelial type 2 (HEp-2) cells as the substrate is the reference method for ANA screening. It allows for the detection of antibodies binding to specific intracellular targets, resulting in various staining patterns that should be identified for diagnosis purposes. In recent years, there has been an increasing interest in devising deep learning methods for automated cell segmentation and classification of staining patterns, as well as for other tasks related to this diagnostic technique (such as intensity classification). However, little attention has been devoted to architectures aimed at simultaneously managing multiple interrelated tasks, via a shared representation.In this paper, we propose a deep neural network model that extends U-Net in a Multi-Task Learning (MTL) fashion, thus offering an end-to-end approach to tackle three fundamental tasks of the diagnostic procedure, i.e., HEp-2 cell specimen intensity classification, specimen segmentation, and pattern classification. The experiments were conducted on one of the largest publicly available datasets of HEp-2 images. The results showed that the proposed approach significantly outperformed the competing state-of-the-art methods for all the considered tasks.

Epidemiological characteristics of five non-COVID respiratory viruses among 37,139 all-age patients during 2018 − 2023 in Weifang, China: a cross-sectional study

BackgroundCommon non-COVID respiratory viruses, such as influenza virus (IFVA/IFVB), parainfluenza virus (PIV), respiratory syncytial virus (RSV), and adenovirus (ADV), often cause acute respiratory infections (ARIs). This study aimed to explore the epidemiological characteristics of these five viruses in patients with ARIs before, during, and after the COVID-19 pandemic from 2018 to 2023.MethodsA total of 37,139 serum specimens and epidemiological data from all-aged patients who presented with ARIs were collected from January 2018 to December 2023. The IgM antibodies of five non-COVID respiratory viruses were tested by an IgM kit with indirect immunofluorescent assay (lFA).Results12,806 specimens were screened as positive for any one of the targeted viruses, with an overall positivity rate of 34.48%. Among all age groups, the most prevalent respiratory viruses were PIV (21.30%) and influenza virus (17.30% of IFVB and 9.91% of IFVA). Children aged 1–14 years were most vulnerable to lower respiratory viruses, and children aged 4–6 years have the highest prevalence no matter the positivity rate for overall viruses (53.06%) or for each virus. From 2018 to 2023, the annual percentage change (APC) revealed that the prevalence of total viruses have a 13.53% rise (p < 0.05), which increased with statistically significant for all age groups. In addition, both the infection rate and the number of samples detected have decreased significantly in the “first-level response” stage of the COVID-19 pandemic and in the “first three months” after fully lifted. Compared to those in the previous five years, the total infection rate (44.64%) and infection rate (26.93%) of the older adults (> 60 years) were all the highest in 2023, and the number of samples collected in 2023 sharply increased, increasing by 77.10% compared to the average of the number of detected in 2018–2022.ConclusionsThe data from this study indicate that the epidemiological characteristics of five non-COVID respiratory viruses are vulnerability to the environment, age, sex, and epidemics status among AIR patients, and that the detected number and positivity rate of these viruses have increased in the “post-pandemic era”, which is critical for the late or retrospective diagnosis and can serve as a useful surveillance tool to inform local public policy in Weifang, China.

DYRK2 regulates epithelial-mesenchymal transition restriction in pancreatic cancer liver metastasis by inhibiting Twist.

To explore the underlying variables and molecular pathways leading to pancreatic cancer liver metastasis. Hs766T and Hs766T-L3 cells were used to create in vitro and in vivo pancreatic cancer liver metastasis models. DYRK2 involvement in pancreatic cancer metastasis was investigated using cell adhesion assays, wound healing assays, and migration and invasion assays. To examine the link between DYRK2 expression and epithelial-mesenchymal transition, Western blot, quantitative real-time PCR, immunofluorescence assays, and immunoprecipitation (IP) were utilized. We found that mice with DYRK2 overexpression had a lower incidence of liver metastasis compared to controls. DYRK2 expression decreased pancreatic cancer tumorigenic activities, including proliferation, migration, and invasion. By analyzing the expression levels of epithelial-mesenchymal transition markers and IP results after overexpressing DYRK2, we found that DYRK2 decreased Twist levels by increasing Twist ubiquitination, thereby inhibiting epithelial-mesenchymal transition. Our findings provide theoretical and experimental support for the ongoing development of DYRK2-based targeted therapies for pancreatic cancer liver metastases.

Advancing Rabies Diagnosis: Time for a New "Gold Standard"?

Rabies, a neglected zoonosis, claims approximately 60,000 lives globally each year. One of the significant challenges in rabies control efforts is the lack of surveillance data and underreporting, stemming from inadequate diagnostic facilities, particularly in low- and middle-income countries. At present, the World Health Organization recognizes the fluorescent antibody test (FAT) on postmortem brain specimens as the "gold standard" for confirming rabies in humans and animals. In this opinion article, we highlight several limitations of FAT and advocate for superior alternatives to replace it as the reference diagnostic technique for rabies. We argue that molecular techniques, specifically PCR-based methods, offer rapid, accurate, and convenient means of laboratory confirmation for rabies. Their implementation is now feasible due to the expanded technical and logistical capabilities achieved during the COVID-19 pandemic.

Integrative transcriptome analysis reveals Serpine2 promotes glomerular mesangial cell proliferation and extracellular matrix accumulation via activating ERK1/2 signalling pathway in diabetic nephropathy.

Diabetic nephropathy (DN) is one of the main causes of end-stage renal disease (ESRD), but its mechanism has not been clearly studied. We utilized integrative transcriptome analysis to explore the pathogenesis of DN. We conducted an analysis by combining bulk dataset and single-cell transcriptome dataset. Through this approach, we identified that Serpine2 may regulate the 'collagen-containing extracellular matrix' pathway involved in DN. Subsequently, we established DN animal and cell models using db/db mice and mesangial cells (MCs) to validate the role of Serpine2 in DN. In the animal model, we detected the expression level of Serpine2 in DN using western blotting (WB) and immunofluorescence (IF) assays. To further clarify the molecular mechanism of Serpine2 in DN, we knocked down Serpine2 and observed its effects on MCs proliferation and extracellular matrix (ECM) accumulation. Our single-cell analysis of DN models highlighted a pivotal role for MCs in the disease's initiation. Next, through Cytoscape analysis of differentially expressed genes (DEGs) in MCs, we identified the following 10 hub genes: Acta2, Angpt2, Ccn1, Col4a1, Col4a2, Col8a1, Kdr, Thbs1, Tpm4 and Serpine2. Subsequently, we identified that Serpine2 and Kdr were also significantly DEGs in the bulk analysis of glomeruli. Additionally, our integrated gene set enrichment analysis of bulk dataset and single-cell RNA dataset revealed that the 'collagen-containing extracellular matrix' was a key pathway in DN progression. Serpine2 was one of the crucial genes involved in regulating this pathway. Therefore, we speculated that the regulation of the 'collagen-containing extracellular matrix' pathway by Serpine2 was an important mechanism. Importantly, WB and IF staining confirmed that Serpine2 expression was upregulated in the MCs of diabetic mice. Knockdown of Serpine2 in cultured MCs alleviated high-glucose-induced excessive MCs proliferation and ECM accumulation. Finally, we found that ERK agonist Ro 67-7476 eliminated the effect of Serpine2 siRNA. In summary, Serpine2 regulates MCs proliferation and ECM synthesis through activation of the ERK1/2 pathway, which is an important pathogenesis mechanism of DN. These findings offer fresh perspectives on the mechanisms of glomerulosclerosis in DN pathogenesis and may provide new targets for treating DN.

Antibody selection and automated quantification of TRPV1 immunofluorescence on human skin

Assessing localization of the transient receptor potential vanilloid-1 (TRPV1) in skin nerve fibers is crucial for understanding its role in peripheral neuropathy and pain. However, information on the specificity and sensitivity of TRPV1 antibodies used for immunofluorescence (IF) on human skin is currently lacking. To find a reliable TRPV1 antibody and IF protocol, we explored antibody candidates from different manufacturers, used rat DRG sections and human skin samples for screening and human TRPV1-expressing HEK293 cells for further validation. Final specificity assessment was done on human skin samples. Additionally, we developed two automated image analysis methods: a Python-based deep-learning approach and a Fiji-based machine-learning approach. These methods involve training a model or classifier for nerve fibers based on pre-annotations and utilize a nerve fiber mask to filter and count TRPV1 immunoreactive puncta and TRPV1 fluorescence intensity on nerve fibers. Both automated analysis methods effectively distinguished TRPV1 signals on nerve fibers from those in keratinocytes, demonstrating high reliability as evidenced by excellent intraclass correlation coefficient (ICC) values exceeding 0.75. This method holds the potential to uncover alterations in TRPV1 associated with neuropathic pain conditions, using a minimally invasive approach.

Self-replicating recombinant virus-like particles of lentivirus proliferating in glioblastoma cells and normal human macrophages

Introduction. Both attenuated and inactivated vaccines are used in disease control. Inactivated vaccines are very diverse and include whole cell and acellular vaccines containing protein target antigens or nucleic acids encoding target antigens. Immunity induced by inactivated vaccines is not believed to be long-lasting. It is very problematic to develop a vaccine against viruses that integrate into the genome of the host cell, as well as against persistent viruses that penetrate the central nervous system (CNS), which is typical for the human immunodeficiency virus type 1 (HIV-1). Aim of the study: to evaluate the possibility of forming HIV-1 recombinant virus-like particles (recVLPs) and HIV-1B recombinant VLPs and simian immunodeficiency virus (SIV) — SHIV89.6P based on self-replicating RNAs (srRNAs) producing target lentivirus antigens on the alphavirus replicon platform (Sindbis virus or Venezuelan equine encephalomyelitis virus (VEEV)), and also to evaluate the ability of HIV-1B and SHIV89.6P VLPs to infect glioblastoma cells and normal human macrophages. Materials and methods. BHK-21 cells were transfected with the srRNA mixture by electroporation. Recombinant virus-like particles (recVLP’s) in recVLP’s-infected cells were detected using the immunofluorescence assay (ELISA) and electron microscopy. recVLP’s were used to infect glioblastoma cells and normal macrophages from a healthy donor. Results. Based on the genomic RNA of the alphavirus, the plasmids were created, transcription from which makes it possible to obtain RNA that expresses lentiviral gene products in cells in quantities sufficient for the formation of mature VLPs. In BHK-21 cells infected with recVLP’s, virus-specific antigens are detected only in the cytoplasm, but not in the nucleus. Both glioblastoma cells (U87) and normal human macrophages containing CD4 receptor and SSR5 and CXR4 co-receptors give infectious progeny of HIV-1B and SHIV89.6P recVLP’s when infected with supernatant obtained after transfection of BHK-21 cells with srRNA. Discussion. The results obtained show the possibility of expressing lentivirus structural proteins in glioblastoma cells (U87) and in normal human macrophages and can be used in the future to study the presentation of antigens in native and functional conformations in appropriate model systems to study the possibility of suppressing HIV infection in viral reservoirs in the CNS.

An Insulin Upstream Open Reading Frame (INSU) Is Present in Skeletal Muscle Satellite Cells: Changes with Age.

Insulin resistance, stem cell dysfunction, and muscle fiber dystrophy are all age-related events in skeletal muscle (SKM). However, age-related changes in insulin isoforms and insulin receptors in myogenic progenitor satellite cells have not been studied. Since SKM is an extra-pancreatic tissue that does not express mature insulin, we investigated the levels of insulin receptors (INSRs) and a novel human insulin upstream open reading frame (INSU) at the mRNA, protein, and anatomical levels in Baltimore Longitudinal Study of Aging (BLSA) biopsied SKM samples of 27-89-year-old (yrs) participants. Using RT-qPCR and the MS-based selected reaction monitoring (SRM) assay, we found that the levels of INSR and INSU mRNAs and the proteins were positively correlated with the age of human SKM biopsies. We applied RNAscope fluorescence in situ hybridization (FISH) and immunofluorescence (IF) to SKM cryosections and found that INSR and INSU were co-localized with PAX7-labeled satellite cells, with enhanced expression in SKM sections from an 89 yrs old compared to a 27 yrs old. We hypothesized that the SKM aging process might induce compensatory upregulation of INSR and re-expression of INSU, which might be beneficial in early embryogenesis and have deleterious effects on proliferative and myogenic satellite cells with advanced age.

Characterization of primary cilia in different epithelial cells of thyroid gland.

The primary cilium (PC) is a biosensor with diverse functions, depending on cellular type. In the thyroid, where it was first described, PCs are located at the apical pole of the follicular epithelium, sensing the lumen's environment. They probably contribute to follicular homeostasis, although their presence in other thyroid epithelial cells remains unclear. Thyroglobulin, stored in the lumen as colloid, is the primary regulator of thyroid-specific gene expression under constant TSH levels. The mechanism by which thyroglobulin signal is transduced remains unresolved. This study investigates the evolution of PCs in different types of thyroid follicles, based on their functional activity, using both normal human thyroids and functional thyroid pathologies as models. It also compares PC morphology between human and rat thyrocytes and explores their presence in other thyroid epithelial components such as C cells and ultimobranchial remnants. Human and Wistar rat thyroid tissues were analyzed using histological, immunohistochemical, immunofluorescence, and electron microscopy techniques. Morphometric analyses quantified PC changes (frequency and length) in various follicular patterns, and statistical analyses were performed. Four types of thyroid follicles were identified: active, hyperactive, hypoactive, and empty follicles. PCs were most abundant and longest in active and significantly reduced in empty follicles. PCs were more prominent in human than in rat thyrocytes, present in both normal and neoplastic C cells, but sporadic in ultimobranchial remnants. PCs vary according to follicular activity and likely act as mechanosensors in thyroid hormone regulation, detecting colloid density and contributing to the regulation of thyroid hormone biosynthesis.

USP11 promotes lipogenesis and tumorigenesis by regulating SREBF1 stability in hepatocellular carcinoma

BackgroundThe relationship between hepatocellular carcinoma (HCC) metastasis and cancer metabolism reprogramming is becoming increasingly evident. Ubiquitin-specific protease 11 (USP11), a member of the deubiquitinating enzyme family, has been linked to various cancer-related processes. While USP11 is known to promote HCC metastasis and proliferation, the precise mechanisms, especially those related to cancer metabolism, remain unclear.MethodsThrough mass spectrometry, co-immunoprecipitation, immunofluorescence, and ubiquitination assays, we identified USP11 as the key deubiquitinase for SREBF1.Lipogenesis was evaluated using Oil Red O and Nile Red staining, along with the detection of triglycerides and cholesterol. To assess HCC cell proliferation, migration, and invasion in vitro, Transwell assays, EDU, colony formation, and CCK-8 were conducted. Xenograft models in nude mice were developed to verify the role of the USP11/SREBF1 axis in lipogenesis and tumor growth in vivo.ResultsUSP11 directly interacts with SREBF1, and its silencing leads to the disruption of SREBF1 stabilization through K48-linked deubiquitination and degradation. Importantly, the truncated mutant USP11 (503–938 aa) interacts with the truncated mutant SREBF1 (569–1147aa), with K1151 playing a crucial role in this interaction. Higher levels of USP11 enhance lipogenesis, proliferation, and metastasis in HCC cells. Importantly, the knockdown of SREBF1 weakened the effects of USP11 in enhancing lipogenesis and tumorigenesis. Futhermore, the elevated expression of USP11 and SREBF1 in HCC tissue serves as an indicator of poor prognosis in HCC patients.ConclusionsIn summary, our study reveals that USP11 promotes HCC proliferation and metastasis through SREBF1-induced lipogenesis. These findings provide a foundation for novel therapies targeting lipid metabolism in HCC.

Ethanol inhibited growth hormone receptor-mediated endocytosis in primary mouse hepatocytes

Abstract Objectives Growth hormone (GH) exhibits various essential physiological functions, which are exerted by its binding to growth hormone receptor (GHR). Ethanol has been demonstrated to have an impact on GH’s biological activity. Nevertheless, mechanism underlying the regulation of the biological activity of GH by ethanol have yet to be fully elucidated. Methods This study utilized an indirect immunofluorescence assay to identify GHR expression in mouse hepatocytes. Western blot was used to determine the impact of ethanol on GH-induced intracellular signalling. Indirect immunofluorescence and colocalization experiments were used to determine the effect of ethanol on GH-GHR’s nuclear localization and endocytosis. Results GHR was primarily localized in the cell membrane and cytoplasm. The phosphorylation levels of JAK2 and STAT1/3/5 were markedly lowered after treatment with ethanol. On this basis, we further explored the mechanism underlying the regulation of GH biological activity by ethanol from the perspective of cell internalization. We found that the nuclear translocation of GH-GHR was inhibited when treated with ethanol. In addition, the results of colocalization analyses revealed that ethanol inhibited GHR-mediated nuclear translocation may mainly by inhibiting caveolin-dependent endocytosis. Conclusions Our study showed that ethanol inhibits GH signaling ability in a time-dependent manner. Ethanol could inhibit the nuclear localization of GH-GHR, which may be linked to the inhibition of the interaction between GHR and caveolin. The combined effect of these factors downregulated the GH-GHR signal. This study laid a foundation for further exploring the mechanism that the effects of ethanol on GH biological activity.

Sennoside A represses the malignant phenotype and tumor immune microenvironment of non-small cell lung cancer cells by inhibiting the TRAF6/NF-κB pathway.

Non-small cell lung cancer (NSCLC) is a prominent cause of cancer death worldwide. Sennoside A (SA) is the primary anthraquinone active component from Rheum officinale Baill and exerts antitumor functions in multiple human tumors. This research aimed to elucidate the function and mechanism of SA in NSCLC. SA functions in NSCLC were determined using Cell Counting Kit-8 (CCK-8) assay, Terminal deoxynucleotidyl transferase dUTP nick-end labeling analysis, Transwell assay, Enzyme-Linked Immunosorbent Assay (ELISA), and Western blot. Meanwhile, the SA mechanism in NSCLC was examined with Western blot, immunofluorescence assay, CCK-8 assay, Transwell analysis, and ELISA. Furthermore, SA functions in NSCLC growth in vivo were assessed by the establishment of a tumor xenograft model, hematoxylin-eosin staining, analysis of NSCLC tissue apoptosis, and immunohistochemistry assays. Functionally, less than 200µM SA had no significant effect on normal human bronchial epithelial cell BEAS-2B cell viability. Furthermore, H460 cell viability was decreased when the SA dose was greater than or equal to 25µM (IC50 = 53.34µM). A549 cell viability was reduced when the SA dose was greater than or equal to 12.5µM (IC50 = 48.21µM). Also, SA repressed NSCLC cell proliferation and boosted cell apoptosis. SA restrained NSCLC cell invasion and tumor microenvironment. Mechanically, SA weakened NSCLC cell proliferation, invasion, and tumor microenvironment, yet this impact was abolished after transfecting pcDNA3.1-TRAF6, which indicated that SA repressed NSCLC cell proliferation, invasion, and tumor microenvironment through inactivating TRAF6/NF-κB. Moreover, SA reduced subcutaneous tumor volume and promoted NSCLC tissue apoptosis in vivo. SA repressed NSCLC cell proliferation, invasion, and tumor microenvironment through inactivating TRAF6/NF-κB.

Exploring the Endothelin-1 pathway in chronic thromboembolic pulmonary hypertension microvasculopathy.

Targeted vasopeptide therapies have significantly advanced the management of pulmonary arterial hypertension (PAH). However, due to insufficient preclinical evidence regarding the involvement of the endothelin-1 (ET-1) pathway in chronic thromboembolic pulmonary hypertension (CTEPH) pathophysiology, the potential of ET-1 receptor antagonism in treating CTEPH remains uncertain. In this study, we investigated the role of the ET-1 pathway in CTEPH microvasculopathy using a multifaceted approach. Plasma ET-1 levels were measured in a cohort of 59 CTEPH patients and 41 healthy controls. Additionally, we evaluated the expression of key ET-1 pathway members in pulmonary explants from CTEPH, idiopathic PAH, and control patients. We used an in vitro system to test the hypothesis that the turbulent flow, observed near the vascular obstructions pathognomonic of CTEPH, enhances ET-1 expression. Our findings were further validated in vivo using a CTEPH piglet model that contains distinct regions representing pre- and post-thrombus lung territories. We found a twofold increase in circulating ET-1 levels in CTEPH patients compared to healthy subjects. Pulmonary explants from CTEPH patients exhibited pronounced overexpression of ET-1, endothelin receptor A (ETA), and phosphorylated myosin light chain (p-MLC) in muscularized pulmonary microvessels, suggesting heightened vascular contraction. In vitro experiments showed that turbulent flow facilitates ET-1 secretion compared to laminar flow regions. Additionally, in the CTEPH piglet model, elevated plasma ET-1 levels were observed compared to controls. Immunofluorescence and confocal microscopy analyses confirmed increased ETA and p-MLC in remodeled arteries from both pulmonary territories. However, ET-1 protein elevation was exclusively observed in the obstructed territory. These findings collectively indicate impaired vascular tone in microvessels of CTEPH patients and the CTEPH piglet model. Furthermore, our data implicates the ET-1 pathway in microvasculopathy, with turbulent flow playing a pathological role. These insights underscore the potential utility of ET-1 receptor antagonists as a promising therapeutic approach for CTEPH treatment.

CKS2 induces autophagy-mediated glutathione metabolic reprogramming to facilitate ferroptosis resistance in colon cancer

BackgroundFerroptosis, a form of cell death characterized by lipid peroxidation, plays a crucial role in tumor suppression, offering novel avenues for cancer therapy. Previous studies have indicated that high levels of cyclin-dependent kinase subunit 2 (CKS2) promote the progression of various cancers. However, the potential interplay between CKS2 and ferroptosis in colon cancer (CC) remains unclear.MethodsBioinformatics and RNA-seq analyses were employed to study genes associated with the ferroptosis signaling pathway. CKS2 expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot (WB). The in vitro and in vivo effects of CKS2 on CC cells were assessed through the CCK-8 assay, colony formation assay, propidium iodide (PI) staining, BODIPY staining, DCFH-DA staining, and animal experiments. Additionally, the impact of CKS2 on autophagy and glutathione (GSH) metabolism was investigated using a transmission electron microscope (TEM), immunofluorescence (IF) assays, WB experiments, and relevant assay kits.ResultsCKS2 expression was elevated in CC, indicating a poor clinical outcome. Knockdown of CKS2 significantly enhanced Erastin-induced ferroptosis in CC cells, leading to reduced GSH metabolism. Conversely, CKS2 overexpression produced opposite effects. Mechanistically, CKS2-induced autophagy reinforced GSH metabolism, thereby increasing resistance to ferroptosis in CC cells. Furthermore, inhibiting CKS2 promoted tumor ferroptosis by downregulating GPX4 expression. Additionally, CKS2 knockdown effectively increased sorafenib-induced ferroptosis both in vitro and in vivo.ConclusionCKS2 suppresses ferroptosis in CC by modulating GSH metabolism in both in vitro and in vivo settings. These findings offer new insights into targeting CKS2 for CC treatment and shed light on the mechanism of ferroptosis in CC.

No detectable loss of myonuclei from human muscle fibers after six weeks of immobilization following an Achilles tendon rupture.

Muscle disuse has rapid and debilitating effects on muscle mass and overall health, making it an important issue from both scientific and clinical perspectives. However, the myocellular adaptations to muscle disuse are not yet fully understood, particularly those related to the myonuclear permanence hypothesis. Therefore, in this study, we assessed fiber size, number of myonuclei, satellite cells, and capillaries in human gastrocnemius muscle after a period of immobilization following an Achilles tendon rupture. Six physically active patients (5M/1F, 43 {plus minus} 15 years) were recruited to participate after sustaining an acute unilateral Achilles tendon rupture. Muscle biopsies were obtained from the lateral part of the gastrocnemius before and after six weeks of immobilization using a plaster cast and orthosis. Muscle fiber characteristics were analyzed in tissue cross-sections and isolated single fibers using immunofluorescence and high-resolution microscopy. Immobilization did not change muscle fiber type composition nor cross-sectional area of type I or type II fibers, but muscle fiber volume tended to decline by 13% (p=0.077). After immobilization, the volume per myonucleus was significantly reduced by 20% (p=0.008). Myonuclei were not lost in response to immobilization but tended to increase in single fibers and type II fibers. No significant changes were observed for satellite cells or capillaries. Myonuclei were not lost in the gastrocnemius muscle after a prolonged period of immobilization, which may provide support to the myonuclear permanence hypothesis in human muscle. Capillaries remained stable throughout the immobilization period, whereas the response was variable for satellite cells, particularly in type II fibers.